Industrial Microbiology and Biotechnology: Emerging concepts in Microbial Technology 981992815X, 9789819928156

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Pradeep Verma   Editor

Industrial Microbiology and Biotechnology Emerging concepts in Microbial Technology

Industrial Microbiology and Biotechnology

Pradeep Verma Editor

Industrial Microbiology and Biotechnology Emerging concepts in Microbial Technology

Editor Pradeep Verma Bioprocess and Bioenergy Laboratory Department of Microbiology Central University of Rajasthan Kishangarh, Rajasthan, India

ISBN 978-981-99-2816-3 ISBN 978-981-99-2815-6 https://doi.org/10.1007/978-981-99-2816-3

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Dedicated to My Beloved Mother

Preface

The first book entitled Industrial Microbiology and Biotechnology provided its readers with an updated information on basics of microbiology, and how it can be explored in various dimensions at an industrial scale. The success of the previous book encouraged us to edit this book that focuses on the role of biotechnological advances that directly impact industrial production using microbial biotechnological approaches. These approaches have been developed to harness the potential of microbes for the production of industrially important products through greener approaches rather than traditional chemical and energy-intensive processes. The microbes-based products have widespread applications from agriculture, food processing, paper and pulp industries, biorefinery, bioremediation, pharmaceutical, and medical sectors. Hence, looking up the trends, speed of developments, and recent technological updates that have been incorporated in the book, the newly revised Industrial Microbiology and Biotechnology—Emerging Concepts in Microbial Technology, meets the needs of the current scenario. This book focuses on the recent cutting-edge research advances which include various emerging concepts in microbial technology such as the basics of omics, microbial genome structure, organization of major bioinformatics tools, and resources for functional genomics and different strain improvement strategies of industrially important microorganisms. It also includes high-throughput sequencing or next-generation sequencing along with strain improvement strategies of industrially important microorganisms. Various aspects of agriculture microbiology, role of bacteria, cyanobacteria, and microalgae in biofertilizer production, plant growth-promoting rhizobacteria (PGPR), applied aspects of environmental microbiology exploring fungal endophytes association with medicinal orchids, and the role of microbes in environmental pollution control and bioremediation have also been included. Additionally, techno-economics analysis and life cycle assessment, for example, biowaste treatment, microalgal production, various industrially relevant bioactive compounds, new products from microbial sources, and biomedical application of advanced microbial approaches in nutraceuticals, biomedicine, and vaccine development have also been elaborated. Further, this book is an attempt to focus on recent advances and emerging topics such as advanced CRISPR technology, exploring plant–microbe interaction via genome editing, molecular docking in drug designing and metabolism, an overview of genomics (genomics, metagenomics, metabolomics, etc.), functional genomics, vii

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and structural and system biology approaches for enhanced production of industrially relevant products. Thus, this book complied is an attempt to incorporate the much-anticipated section using advanced microbial approaches in nutraceuticals, biomedicine and vaccine development, amino acids, vitamins and metabolites from microbial sources, and various aspects of the biomedical application via microbial technology, for example, neurological disorders and development of new molecules through molecular docking. A variety of 25 chapters bring together an overview of current advancements and trends in emerging concepts of microbial technology. The various chapters are dedicated to exploring the ongoing state-of-the-art concepts and tools directed toward the fruitful production, modifications/tailoring, and applications of microbial technology in both academic and industrial sectors. The discussion in each chapter is comprehensive, commanding, and extremely informative. Thus, this book was developed with the motive to benefit students, academicians, as well as researchers. I sincerely thank all the contributors for their generous and timely responses and their excellent contributions on the recent outstanding and emerging area of microbial technology. Their participation and my efforts made the organization of the book possible. I would also like to express my deep sense of appreciation to all of the editorial and publishing staff members associated with Springer-Nature, for their all-round help to ensure that the highest standards of publication have been maintained to develop this book. Kishangarh, Rajasthan, India

Pradeep Verma

Acknowledgment

First of all, I would like to convey my gratitude to the Publisher-Springer Nature for considering my thoughts to develop this book entitled Industrial Microbiology and Biotechnology- Emerging concepts in Microbial Technology which is the first of its kind book covering various emerging areas of Microbial Technology. This book is only possible because of the support from all the researchers and academicians who contributed to the book; therefore, the editor is thankful for their contribution. I would also like to thank my research scholars for providing me with all the necessary technical support and editorial assistance during the developmental stage of book. I am also thankful to the Central University of Rajasthan (CURAJ), Ajmer, India for providing infrastructural support and a suitable teaching and research environment. The teaching and research experience at CURAJ has allowed me to make all possible attempts and develop a book that can cater to the needs of academicians, students, and researcher. I am also thankful to the Department of Biotechnology for providing me funds through sponsored projects (Grant No. BT/304/ NE/TBP/2012 and BT/PR7333/PBD/26/373/2012), for setting up my laboratory “Bioprocess and Bioenergy Laboratory.” I am always thankful to God and my parents for their blessings. This book is affectionately dedicated to the two most caring and loving women in my life, i.e., my mother and my wife and my two wonderful kids. Their unconditional love, care, trust, and encouragement gives me the strength and motivation to always do well and contribute to the scientific world.

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Contents

1

Basic of Omics and Its Applications . . . . . . . . . . . . . . . . . . . . . . . . Divyanshu Darshna and Sachin S. Tiwari

2

An Introduction to Omics in Relevance to Industrial Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Madhumita Priyadarsini, Jyoti Rani, Jeetesh Kushwaha, Kailash Pati Pandey, Yashpal Singh, and Abhishek S. Dhoble

3

4

1

23

Databases and Tools for Microbial Genome and Human Microbiome Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sibasree Hojaisa and Anupam Nath Jha

41

CRISPR/Cas9 System: An Advanced Approach for the Improvement of Industrially Important Microorganisms . . . . . Sharmilee Sarkar, Mohit Yadav, and Aditya Kumar

69

5

Biomedical Application of Industrial Microbiology . . . . . . . . . . . . . Komal Bana and Sachin S. Tiwari

99

6

The Role of Whole-Genome Methods in the Industrial Production of Value-Added Compounds . . . . . . . . . . . . . . . . . . . . . 121 Kaushika Olymon, Upalabdha Dey, Eshan Abbas, and Aditya Kumar

7

New Developments in the Production and Recovery of Amino Acids, Vitamins, and Metabolites from Microbial Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Priya Shukla, Pradeep Srivastava, and Abha Mishra

8

Exploring Plant-Microbe Interaction Through the Lens of Genome Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Upasna Chettry, Sunita Upadhaya, Amilia Nongbet, Nikhil Kumar Chrungoo, and S. R. Joshi

9

Biomedical Application of Advanced Microbial Approaches: Nutraceuticals, Biomedicine, and Vaccine Development . . . . . . . . . 273 Neha Namdeo, Ragini Arora, Harit Jha, Neha Namdeo, and Ragini Arora xi

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10

Microbial Technology for Neurological Disorders . . . . . . . . . . . . . . 299 Asmita Dasgupta

11

Frontiers in Fungal Endophytes Associated with Medicinal Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Bishal Pun and S. R. Joshi

12

Nutraceuticals: Advancement in Microbial Production and Biomedical Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Dixita Chettri, Manswama Boro, Shahil Ansari, and Anil Kumar Verma

13

Hyaluronic Acid Microbial Synthesis and Its Explicit Uses in the Development of Nutraceuticals, Biomedicine, and Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Priya Shukla, Pradeep Srivastava, and Abha Mishra

14

Molecular Docking in Drug Designing and Metabolism . . . . . . . . . . 403 Shyamalima Saikia, Minakshi Puzari, and Pankaj Chetia

15

Recent Advances in PGPRs and Their Application in Imparting Biotic and Abiotic Stress Tolerance in Plants . . . . . . . . . . . . . . . . . 431 Babita Joshi, Satya Narayan Jena, S. R. Joshi, and Brijmohan Singh Bhau

16

Microbial Hyaluronidase: Its Production, Purification and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 Sandip P. Patil, Kiran S. Dalal, Leena P. Shirsath, and Bhushan L. Chaudhari

17

Strain Improvement Strategies of Industrially Important Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Sayani Ghosh, Pooja, and Supratim Datta

18

Microbial Diversity for Agricultural Productivity . . . . . . . . . . . . . . 519 Pompee Chanda, Bishal Pun, and S. R. Joshi

19

Role of Microbes in Bioremediation . . . . . . . . . . . . . . . . . . . . . . . . 549 Devargya Ganguly, K. L. V. Prasanna, Swaroopa Neelapu, and Gargi Goswami

20

Reuterin: A Broad Spectrum Antimicrobial Agent and Its Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 Kiran S. Dalal, Sandip P. Patil, Girish B. Pendharkar, Dipak S. Dalal, and Bhushan L. Chaudhari

21

Seaweed Farming: An Environmental and Societal Perspective . . . 605 Meenakshi Singh, Sahil Kapoor, Trisha Bagchi, Sanchita Paul, and Surojit Kar

Contents

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22

Development of New Molecules Through Molecular Docking . . . . . 643 Charu Jaiswal, Kushal Kant Pant, Ravi Kiran Sriniwas Behera, Renu Bhatt, and Vikas Chandra

23

Strategies for Improved Production of Microalgae-Derived Carotenoids and Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661 Sahil Kapoor, Arup Giri, Pushpender Bhardwaj, Meenakshi Singh, Murthy Chavali, and Pradeep Verma

24

Strategies for Strain Improvement of Economically Important Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695 Ashutosh Kosariya, Dharmendra Kumar, Kushal Kant Pant, Renu Bhatt, and Vikas Chandra

25

Techno-Economic Analysis and Life Cycle Assessment of Bio-Based Waste Materials for Biogas Production: An Indian Perspective . . . 729 Rubia Kouser, Anu Bharti, Rifat Azam, Deepak Pathania, and Richa Kothari

Editor and Contributors

About the Editor Pradeep Verma Prof. Verma completed his Ph.D. from Sardar Patel University Gujarat, India, in 2002. In the same year, he was selected as a UNESCO fellow and joined the Czech Academy of Sciences Prague, Czech Republic. He later moved to Charles University, Prague to work as Post-Doctoral Fellow. In 2004, he joined as a visiting scientist at UFZ Centre for Environmental Research, Halle, Germany. He was awarded a DFG fellowship which provided him with another opportunity to work as a Post-Doctoral Fellow at Gottingen University, Germany. Later in 2007, he moved to India and joined Reliance Life Sciences, Mumbai, and worked extensively on biobutanol production which attributed a few patents to his name. Later, he was awarded JSPS Post-Doctoral Fellowship Programme and joined the laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Japan. Prof. Verma has also been the recipient of various prestigious awards such as the Ron-Cockcroft Award by Swedish society, and UNESCO Fellow ASCR Prague. Recently for his contribution to the area of Fungal Microbiology, Industrial Biotechnology, and Environmental Bioremediations, he has been awarded the prestigious Fellow Award from Mycological Society of India (2020), P.C. Jain Memorial Award (MSI), and Biotech Research Society of India (2021). He is also awarded Fellow Award 2020 of Biotechnology Research Society of India (BRSI) and Fellow of Academy of Sciences of AMI India 2021 (FAMSc). Further, he has also been awarded JSPS Bridge Fellow Award in 2022 and a xv

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short-term visit to Kyoto University, Kyoto, Japan, to strengthen types between two laboratories. Prof. Verma in 2009 began his independent academic career as a Reader and Founder Head at the Department of Microbiology, Assam University. In 2011, he moved to the Department of Biotechnology at Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, and served as an Associate Professor till 2013. He is currently working as a Professor at the Department of Microbiology, CURAJ, and was also the former Head and Dean, School of Life Sciences. He is a member of various National and International societies/academies and has also completed two collaborated projects worth 150 million INR in the area of microbial diversity and bioenergy. Prof. Verma is a Group Leader of the Bioprocess and Bioenergy laboratory at the Department of Microbiology, School of Life Sciences, CURAJ. His area of expertise involves Microbial Diversity, Bioremediation, Bioprocess Development, Lignocellulosic, and Algal Biomass-based Biorefinery. He holds 12 international patents in the field of microwave-assisted biomass pretreatment and biobutanol production. He has more than 65 research articles in peer-reviewed international journals and contributed to several book chapters (32 published; 11 in press) in different edited books. He has also edited 11 books in international publishers such as Springer. He is a Guest Editor to several journals such as Biomass Conversion and Biorefinery (Springer). He is acting as a reviewer for more than 60 journals in different publication houses.

Contributors Eshan Abbas Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Shahil Ansari Department of Microbiology, Sikkim University, Gangtok, Sikkim, India Ragini Arora Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Rifat Azam Department of Environmental Sciences, Central University of Jammu, Rahya-Suchani, (Bagla) Samba, Jammu, Jammu and Kashmir, India

Editor and Contributors

xvii

Trisha Bagchi Department of Botany, West Bengal State University, Barasat, West Bengal, India Department of Botany, Goswami Ganesh Dutta Sanatan Dharma College, Chandigarh, India Komal Bana Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India Ravi Kiran Sriniwas Behera Department of Biotechnology, Gur Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Pushpender Bhardwaj Medicinal Plant Division, Defence Institute of High Altitude Research (DRDO), Leh-Ladakh, Jammu and Kashmir, India Anu Bharti Department of Environmental Sciences, Central University of Jammu, Rahya-Suchani, (Bagla) Samba, Jammu, Jammu and Kashmir, India Renu Bhatt Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Brijmohan Singh Bhau Department of Botany, Central University of Jammu, Rahya-Suchani (Bagla), Jammu, Jammu and Kashmir, India Manswama Boro Department of Microbiology, Sikkim University, Gangtok, Sikkim, India Pompee Chanda Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India Vikas Chandra Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Bhushan L. Chaudhari Department of Microbiology, School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, India Murthy Chavali Office of the Dean (Research) and Division of Chemistry, Department of Sciences, Faculty of Sciences and Technology, Alliance University, Bengaluru, Karnataka, India Pankaj Chetia Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India Dixita Chettri Department of Microbiology, Sikkim University, Gangtok, Sikkim, India Upasna Chettry Department of Botany, School of Biological Sciences, NorthEastern Hill University, Shillong, Meghalaya, India Nikhil Kumar Chrungoo Royal Global University, Guwahati, Assam, India Dipak S. Dalal School of Chemical Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India

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Editor and Contributors

Kiran S. Dalal Department of Microbiology, School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India Divyanshu Darshna Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Roorkee, Uttarakhand, India Asmita Dasgupta Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry, India Supratim Datta Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Center for the Advanced Functional Materials, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Center for the Climate and Environmental Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Upalabdha Dey Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Abhishek S. Dhoble School of Biochemical Engineering, IIT (BHU), Varanasi, Uttar Pradesh, India Devargya Ganguly Department of Biotechnology, GITAM School of Science, Gandhi Institute of Technology and Management (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Sayani Ghosh Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Arup Giri Department of Zoology, Faculty of Sciences, Baba Mastnath University, Rohtak, Haryana, India Gargi Goswami Department of Biotechnology, GITAM School of Science, Gandhi Institute of Technology and Management (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Sibasree Hojaisa Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Department of Zoology, Biswanath College, Biswanath Chariali, Assam, India Charu Jaiswal Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India Satya Narayan Jena Plant Molecular Genetics Laboratory, CSIR-National Botanical Research Institute, Lucknow, Uttarakhand, India Anupam Nath Jha Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India

Editor and Contributors

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Harit Jha Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Babita Joshi Plant Molecular Genetics Laboratory, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India S. R. Joshi Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India Sahil Kapoor Department of Botany, Goswami Ganesh Dutta Sanatan Dharma College, Chandigarh, Punjab, India Department of Ecology and Biodiversity, Terracon Ecotech Private Ltd., Mumbai, Maharashtra, India Surojit Kar Department of Botany, West Bengal State University, Barasat, West Bengal, India Ashutosh Kosariya Department of Biotechnology, Vishwavidyalaya, Bilaspur, Chhattisgarh, India

Guru

Ghasidas

Richa Kothari Department of Environmental Sciences, Central University of Jammu, Rahya-Suchani, (Bagla) Samba, Jammu, Jammu and Kashmir, India Rubia Kouser Department of Environmental Sciences, Central University of Jammu, Rahya-Suchani, (Bagla) Samba, Jammu, Jammu and Kashmir, India Aditya Kumar Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Dharmendra Kumar Department of Biotechnology, Vishwavidyalaya, Bilaspur, Chhattisgarh, India

Guru

Ghasidas

Jeetesh Kushwaha School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India Abha Mishra School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, Uttar Pradesh, India Neha Namdeo Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India Swaroopa Neelapu Department of Biotechnology, GITAM School of Science, Gandhi Institute of Technology and Management (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Amilia Nongbet Department of Botany, School of Biological Sciences, University of Science and Technology Meghalaya (USTM), Baridua, Meghalaya, India Kaushika Olymon Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India

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Editor and Contributors

Kailash Pati Pandey School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India Kushal Kant Pant Department of Biotechnology, Vishwavidyalaya, Bilaspur, Chhattisgarh, India

Guru

Ghasidas

Deepak Pathania Department of Environmental Sciences, Central University of Jammu, Rahya-Suchani, (Bagla) Samba, Jammu, Jammu and Kashmir, India Sandip P. Patil Department of Microbiology and Biotechnology, R. C. Patel Arts, Commerce and Science College, Shirpur, Maharashtra, India Sanchita Paul Department of Botany, West Bengal State University, Barasat, West Bengal, India Girish B. Pendharkarc Department of Microbiology, Sadguru Gadage Maharaj College, Karad, Maharashtra, India Pooja Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India K. L. V. Prasanna Department of Biotechnology, GITAM School of Science, Gandhi Institute of Technology and Management (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Madhumita Priyadarsini School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India Bishal Pun Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India Minakshi Puzari Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India Jyoti Rani School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India Shyamalima Saikia Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India Sharmilee Sarkar Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Leena P. Shirsath Department of Microbiology and Biotechnology, R. C. Patel Arts, Commerce and Science College, Maharashtra, India Priya Shukla School of Biochemical Engineering, Indian Institute of Technology (BHU), Varanasi, Uttar Pradesh, India Meenakshi Singh Department of Ecology and Biodiversity, Terracon Ecotech Pvt. Ltd., Mumbai, Maharashtra, India

Editor and Contributors

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Yashpal Singh School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India Pradeep Srivastava School of Biochemical Engineering, Indian Institute of Technology (BHU), Varanasi, Uttar Pradesh, India Sachin S. Tiwari Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Roorkee, Uttarakhand, India Sunita Upadhaya Department of Botany, School of Biological Sciences, NorthEastern Hill University, Shillong, Meghalaya, India Anil Kumar Verma Department of Microbiology, Sikkim University, Gangtok, Sikkim, India Pradeep Verma Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Kishangarh, Rajasthan, India Mohit Yadav Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India

1

Basic of Omics and Its Applications Divyanshu Darshna and Sachin S. Tiwari

Abstract

“Omics” is a suffix derived from the Greek word “ome” which means “all” or “every.” In the biological perspective, it is the global or universal characterization of molecular system. When we study genes or their product (RNA or protein), we are focusing on a single gene or single gene product at a time, but with omics technologies we are taking all the genes or entire genome in consideration instead of a single gene. So, the study of whole genome of an organism is called “genomics.” The study of complete set of RNA that is encoded by the genome is called “transcriptomics.” The study of all the proteins encoded by a genome is called “proteomics.” Other cellular units like lipids, metabolites, etc., are studied using various large dataset approaches falling under the umbrella term of “omics technologies.” This chapter will help to understand the basics of mentioned highthroughput omics technologies and their applications in microbial biotechnology. Keyword

Genomics · Proteomics · Transcriptomics · Metabolomics · Lipidomics

Abbreviation cDNA PCR

Complementary DNA Polymerase chain reaction

D. Darshna · S. S. Tiwari (✉) Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Roorkee, Uttarakhand, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_1

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D. Darshna and S. S. Tiwari

1.1

Introduction

1.1.1

What Is Genome?

A genome is a complete collection of genetic instructions for an organism. Each genome includes all the information required to construct an organism and enable an organism to develop and function properly. Genetic instructions are made up of deoxyribonucleic acid (DNA), which contains the instruction in the form of chemical code (this chemical code is composed of nucleotides, i.e., adenine (A), guanine (G), cytosine (C), and thymine (T)) for our genetic machinery to transcribe and translate the required information needed for the cell. DNA has a double-helix structure, with two opposite strand base pairs specifically. Adenine pairs with thymine and two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds. The permutation–combination and the order in which these nucleotides are arranged in helix make every DNA arrangement unique, and thus the translated code is also unique (Fig. 1.1).

1.2

Genome to Genomics

As we now know, all the DNA encoded by an organism is called genome, while the study of the genome is called genomics. But what do we exactly study in genomics? Genomics essentially studies the DNA code contained in the nucleus of a fungus, plant, or animal cell, within the cytoplasm of bacterial cell, and inside the viruses. And how does this DNA encoded by these organisms lead to growth and development of these organisms? How are bacteria, fungus, and viruses able to cause disease? What are the potential ways in which the disease caused by these organisms can be treated? How can changes in DNA encoded by an organism lead to change in a phenotype of an organism? It can tell us about disease resistance or susceptibility of an organism or about the evolutionary history of an organism and relative relatedness (history of an organism with other species). It can tell us about the genome size and complexity of an organism, as well as how two are both related and unrelated. The study of genomics and advances in it can bring the personalized medicine era. The field of genomics has a tremendous potential for various applications in biomedical advances. So, the very next question that must come in our mind is: how do we start studying genomics?

1.2.1

DNA Sequencing

To start the study of genomics, we need a genome of an organism. But how do we get this genome? The very first thing we need is a cell of an organism. Cell can be of any organism that you wish to study, starting from bacterial to plant or animal cell. Once we have the cell, we need to break it open. Cell disruption can be achieved in

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Basic of Omics and Its Applications

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Fig. 1.1 Molecular structure of DNA showing base pairing of guanine with cytosine and adenine with thymine. (Self-made figure created from the portal Biorender (www.biorender.com/))

enzymatic/biochemical way or through mechanical pressures. In both scenarios, we are disrupting the cell membrane. Once the membrane is disrupted, all the cell content is released, i.e., DNA (genome), RNA, proteins, and metabolites. After the cell content is released, we need to isolate the DNA from rest of the things. Various physical and biochemical methods are used to isolate the DNA. After the DNA is isolated, we need to sequence it. What exactly does the “sequencing” refer to here? Sequencing here refers to the order determination of nucleotides in the strand of DNA. We don’t need to sequence both the strands of DNA; sequencing only one strand of DNA can give us the information needed for the other strand of DNA. Okay! So, how do we start sequencing? There are a couple of methods that we can use for sequencing. A very first method for DNA sequencing was the MaxamGilbert method where chemical breakdown of nucleotide occurs to determine the

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D. Darshna and S. S. Tiwari

Fig. 1.2 DNA sequencing steps. (Source of figure: Bioicons portal)

nucleotide sequence. But the most prominent one that was used in the Human Genome Project (2003) was the Sanger sequencing, where sequencing by synthesis method is used. Others include next-generation sequencing (NGS) (Fig. 1.2).

1.2.1.1 Sanger Sequencing The Sanger sequencing gives us a high-quality sequence for relative long stretches of DNA and is accurate up to 800–900 base pairs. Mostly, it is used to sequence plasmids, PCR obtained DNA product and fragments of DNA. For larger DNA, this method is bit inefficient as accuracy gets reduced. So, how did scientists use this method for the Human Genome Project? For the Human Genome Project, human genome was cut into the small fragments and was sequenced using the Sanger sequencing method and was reassembled using overlap regions of the DNA; this process was called the shotgun method of sequencing. Before we start with the Sanger sequencing, we need a couple of ingredients to start with. These ingredients include a short stretch of primer, four nucleotides (dATP, dGTP, dTTP, dCTP), DNA polymerase, template DNA, and a unique version of the nucleotides which is not deoxynucleotide but rather dideoxynucleotide. Dideoxynucleotides are identical to deoxynucleotides; the only difference remains is the 3′ carbon of the sugar ring in dideoxynucleotides (ddNTP) does not have a hydroxyl group. In addition, every nucleotide is fluorescently tagged with the different dye for the detection. Since hydroxyl group is not available for further addition of the nucleotides, DNA synthesis gets terminated and is detected by the fluorescent marker of the nucleotide. Cycle is repeated several times so that every dideoxynucleotide is incorporated in the target DNA. At the end of these cycles, the product will have fragments of different lengths which will be run in thin tube containing gel matrix in the presence of electric field; this process is also called

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Fig. 1.3 Sanger sequencing. Sequence is determined by relative length of the DNA fragment in a gel. (Self-drawn figure)

capillary gel electrophoresis. DNA is negatively charged, so it will move toward anode in the presence of electric field. The short fragment will run faster, while the longer fragment will move slowly. As the fragment will reach at the end of the tube, fragments will be illuminated with laser, which will detect the nucleotide with the help of attached fluorescent dye. Since we know the primer length and sequence that we used, the smallest fragment will have only one ddNTP incorporated just after the primer. So, the next fragment will have the dNTP and ddNTP incorporated, and the cycle will go on until the last nucleotide is incorporated. Since each nucleotide is having a different dye, fluorescence peaks of dye will be recorded, and we will get a series of peaks due to different dyes, and the chromatogram that we will obtain can be translated to the nucleotides that are incorporated in the DNA sequence (Fig. 1.3).

1.2.1.2 Next-Generation Sequencing NGS is a large number of the Sanger sequencing in parallel. NGS is a collective term used to describe all the modern technologies used for the DNA sequencing. Due to its methodologies, which include library construction, parallel sequencing, and simple data analytics, it enables speedy and affordable sequencing of DNA and RNA. It can generate many million short reads in parallel and speed up the sequencing process compared to the Sanger sequence alone. NGS incorporates four main DNA sequencing methods: (1) sequencing by synthesis, (2) pyrosequencing, (3) sequencing by ligation, and (4) ion semiconductor sequencing. Some of the examples of NGS include Illumina sequencing, SoLid systems, Roche 454 sequencing, Ion torrent: proton/PGM sequencing, nanopore sequencing, and pacific bio-sequencing. Workflow of NGS includes the selection of genomic DNA and creating a library of fragmented nucleotides which is usually done by amplification or ligation with custom adaptor sequences. Fragments are usually 100–500 bp in length. Template

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Fig. 1.4 Next-generation sequencing platform (Illumina). (Self-made figure created from the portal Biorender (www.biorender.com/))

for detection of DNA will be prepared, and automated sequencer would perform parallel sequencing. Analysis of data is usually performed using software (Fig. 1.4). Pyrosequencing It is a method of DNA sequencing where partial principle of “sequence by synthesis” is used by the SoLid system device. Pyrophosphate is released at each nucleotide incorporation, and this allows us to track the sequencing procedure. Pyrophosphate that is released in the reaction is used in certain chemical reactions by the sulfurylase and luciferase enzyme, resulting in the production of light at the end of the reaction. The camera captures the proper sequence of the cluster after detecting the light produced by the reaction. The Detection is based on the fact that only one nucleotide at a time is incorporated in the reaction and if only right nucleotide gives the light emission. For example, if the complementary sequence of the DNA has a nucleotide “G” and reaction is incubated with different nucleotides “T,” “A,” “C,” and “G.” For the reaction 1, if we supply “A” nucleotide, then the reaction won’t proceed and there is no generation of light and all the remaining nucleotides are degraded by the machines. But for the reaction 2, if the base added is “C,” then the reaction proceeds and there is ultimately generation of light and device detects which nucleotide was added and thus sequencing occurs. The limitation of pyrosequencing is the high reagent costs and high error rates resulting due to homopolymers (continuous same base incorporation). Sequence by Synthesis The Illumina NGS platforms use the principle of “sequencing by synthesis,” which involves the sequential incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing. Each nucleotide is reversibly attached to a different but single fluorescent molecule. Also, the terminator group is attached to the nucleotides which is reversible and can be removed later on the reaction. The terminator group ensures that only one nucleotide is added at a time. So, in a chip each of nucleotides, i.e., adenine, guanine, cytosine, and thymine are added together at the same time, and after the nucleotide incorporation, the remaining nucleotides are washed away. After the incorporation of nucleotides, the fluorescent signals are recorded, the terminator group and the fluorescent group are cleaved and are washed away, and the process continues for the next nucleotide incorporation. This method

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overcomes the issue of homopolymer error rate faced by the pyro-sequencer. The device’s shortcoming is the ineffective removal of the fluorescent molecule, which produces background noise and increases the error rate on increased read lengths. Sequence by Ligation The ligation sequencing differs from the other two methods. Nucleotide incorporation using this technique does not require the usage of a DNA polymerase. Instead, small oligonucleotide probes that have been ligated together are used. Oligonucleotides used are made up of eight bases (3′–5′): two probe-specific bases and six degenerate bases; at the 5′ end of the probe, fluorescent dye is attached which is specific to the nucleotide. The sequencing reaction begins with the primer binding to the adapter sequence, followed by the hybridization of the appropriate probe. The two bases unique to the probe serve as a guide for its hybridization, which is then annealed and ligated to the primer sequence by a ligase. The process starts with the removal of unbound oligonucleotides, followed by the detection and recording of the signal and cleaving of the fluorescent signal (removal of the last three bases), and the cycle starts again. The DNA strand is denatured after approximately seven rounds of ligation, and a new sequencing primer is utilized, offset by one base from the previous primer, to repeat these procedures—a total of five sequencing primers are employed. Ion Semiconductor Sequencing To detect the sequence of DNA, this method makes use of the hydrogen ion release during the sequencing reaction. Directly above each cluster is a semiconductor transistor that can detect variations in solution pH. A single hydrogen ion is released into the solution during nucleotide incorporation, in which the semiconductor detects. It is very similar to pyrosequencing, but instead of using light, we measure the change in pH of the solution when nucleotides are added. This method is both cost-effective and time-efficient. Since, the whole genome sequencing requires creation of the small fragments. After creating such a short fragments and sequencing of such short fragment. Using sequence assembly software, the original sequence is extracted from the readings. First, contigs—larger composite sequences—are created by combining overlapping reads. Tracing links between mate pairs allows contigs to be joined to create scaffolds. The distance between contigs may be calculated using mate pair sites if the average library fragment length is known and has a small margin of error. Depending on the magnitude of the gap between contigs, several strategies may be employed to discover the sequence in the gaps. NGS is revolutionizing fields such as genetic diseases, personalized medicines, and clinical diagnosis by offering high-throughput results and allows users to sequence multiple genomes of individuals at the same time.

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1.3

Coverage

Coverage is the measure of how many reads on average in the reconstructed sequence correspond to a given nucleotide. It may be calculated as N * L/G, where G is the length of the original genome, N is the number of reads, and L is the average read length. For example if the size of 10,000 nucleotides is reconstructed from the number of reads equals 20 and average number of nucleotides in every read is 1000 will get two times of redundancy. This is an important parameter while assembly of the fragments and also allows us with the information that how much of the genome is covered by the reads. Since we have a DNA sequence now, what do we do now with this information? The things that can be done with the newly sequenced DNA are: 1. Compare the sequence with the existing databases. 2. Look for unknown things such as: • Protein-coding regions • Evolutionary history of the DNA • Open reading frames (ORFs) • Stop codons • GC content • Mutations • Diseases associated with the specific genes (if related sequences are available) • Polymorphism, etc.

But after the sequencing, we need to annotate the genome.

1.4

Genome Mapping

A given gene is assigned to a specific area of a chromosome, and the position and relative distances between genes on the chromosome are determined. Genome mapping consists of identifying the portion of genome that codes for proteins, identifying regions that do not code for proteins, identifying genes which may be coded by the DNA, and attaching a biological information to those gene. Structural annotation consists of identification of gene elements such as open reading frames (ORFs), coding regions, stop codons, etc., while functional annotation attaches biological functions such as biochemical function of the gene, regulations and interaction of the gene, expression, etc. Computational methods have been designed to automatically annotate the gene using previous records in the database.

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Proteomics

Proteomics is the study of all the proteins that are expressed by a genome. All the proteins that are expressed by an organism are called proteome. But before dwelling into the proteomics, we must know what proteins are and what are they made up of.

1.5.1

Amino Acids

The building components of proteins are amino acids. Amino acids are white, crystalline, high-melting solids in their purest form. Chemically, amino acids are defined as “any organic molecule with at least one Carboxyl group (organic acid) and at least one Amino group (Organic base).” They are considered as a weak base because they cannot dissociate completely in the water, and thus in physiology they also act as buffers. Amino acids act as catalysts in many biochemical reactions and also are the metabolic intermediates in certain biochemical cycles. They act as carrier of energy and waste products. Some amino acids also display hormones like properties. There are 20 naturally occurring amino acids. These include glycine, proline, alanine, methionine, valine, leucine, isoleucine, serine, threonine, cysteine, asparagine, glutamine, tyrosine, phenylalanine, tryptophan, aspartate, glutamate, histidine, lysine, and arginine. These amino acids are further classified into different groups because of the functional groups present on them. Mainly functional groups that are present in amino acids are hydrophobic, polar, charged, and aromatic. Hydrophobic amino acids: alanine, valine, isoleucine, leucine, methionine, glycine, tryptophan, and phenylalanine Polar amino acids: serine, threonine, tyrosine, asparagine, and glutamine Charged amino acids: 1. Positively charged amino acids: lysine and histidine 2. Negatively charged amino acids: aspartate and glutamate Hydrophobic amino acids: tryptophan, tyrosine, and phenylalanine (Fig. 1.5). Amino acids are linked with peptide bond (-CONH-). Peptide bond creation is a condensation process because elimination of water occurs when two amino acids are linked. Different permutation and combination of the 20 amino acids lead to different kinds of proteins. Since we now have some understanding of the amino acids, let’s dive into the proteins (Fig. 1.6).

1.5.2

Proteins

Proteins are the polymers of amino acids (monomer). It contains four levels of protein structure: primary, secondary, tertiary, and quaternary structure. The primary structure (1°) of a protein includes a linear sequence of amino acids. The

Fig. 1.5 Amino acid structure. (Self-made figure created from the portal Biorender (www.biorender.com/))

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Fig. 1.6 Amino acids linked with each other through peptide bond. (Self-made figure created from the portal Biorender (www.biorender.com/))

secondary structure (2°) includes a local three-dimensional structure of the peptide backbone (which includes C-N-C-C bond between the amino acids). The tertiary structure (3°) is the global arrangement of all the secondary structure, functional groups, prosthetic groups, side chains, etc., while the quaternary structure (4°) is the arrangement of multiple proteins into one or many different complexes. Proteins regulate many different functions of an organism. They regulate many vital processes in an organism. They are part of cytoskeletal function; provide structural support to cell and organism, as it gives strength to bones, skins, and tendons; and serve as biological catalysts in certain biochemical reaction. They act as carrier for small molecules, electrons, etc., help in contraction of muscle and movement, are also used as immune-protective agents, and have regulatory functions. Any misfunctioning of the protein results in a diseased state.

1.5.3

Why Proteomics?

Proteomics is important because in diseased conditions, drugs we administered into the body either alter the cell receptors or have an effect on a protein. Unlike genome, proteome is dynamic, not static. An organism phenotype is dependent on the proteome that an organism expresses. For example, caterpillar and butterfly share the same genome, but their phenotype is completely different just because of the proteome that these two organisms express. Also, a single gene is capable of giving rise to multiple proteins because of alternative splicing, thus having dynamic and diverse proteome. Proteomics studies the extent of expression, how co- and posttranslational modifications are altering the protein functionality and ultimately the phenotype, how enzymatic regulations are regulated, and how protein-protein expression leads to changes in the health or diseased conditions of an organism.

1.5.4

How Do We Start Studying Proteomics?

To start studying the proteomics, we need to have proteins. Again, how do we get this protein? Just like in genomics studies, we need cells which are expressing

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proteins. Steps involved in proteomic studies are (1) protein extraction, (2) separation, (3) identification, and (4) characterization. Protein extraction is done by rupturing the cell membrane of the cells, after which various biophysical methods are applied for the separation of proteins from other macromolecules such as DNA, RNA, lipids, and other metabolites. Once the proteins are separated, they are identified and characterized using different methods. One of the most implied methods of protein separation is electrophoresis. Two types of protein electrophoresis are used, namely, one-dimensional gel electrophoresis (1DE) and two-dimensional gel electrophoresis (2DE). 1DE is used to resolve the protein complexes in electric field basis on the mass. Proteins are first administered with sodium dodecyl sulfate (SDS) and are resolved in gel. Usually, proteins of molecular mass of 10–300 kDa are resolved. But it has a limitation; when complex protein mixture is introduced, its capability to resolve gets compromised. This problem is solved by 2DE to some extent, because 2DE resolves the proteins according to the protein net charge or protein isoelectric point (pI) in the first dimension. The total net charge on protein molecule depends on pH of the surrounding environment. As the pH changes, the total charge on the protein also changes, and there comes a point where protein acquires a net zero charge; that point is called isoelectric point. Once the proteins are separated based on pI, they are transferred to a different gel where they are separated using molecular weight of the protein in the second dimension. After the proteins are run in the gel, they are stained by different dyes such as Coomassie Brilliant Blue (CBB), silver stain, SYPRO Ruby Red, SYPRO Orange, cyanine dyes, lightning fast, Pro-Q Diamond, etc., in order to visualize. So, the end result of 2DE is usually spots and smears of the proteins which can both qualitatively and quantitatively give us the information about the protein differential expression and the intent to which the proteins are being expressed in the cells (high or low expression). One of the biggest advantages of using 2DE is that it is able to resolve some form of posttranslationally modified proteins such as phosphorylated proteins. Phosphorylation introduces negative charge to the protein. So, the same protein in 2DE with non-phosphorylated form and phosphorylated form will appear at different positions in the gel. The disadvantage of a 2DE remains the poor reproducibility due to gel-to-gel variation, identification and quantification of the spot sometime is very poor and is troublesome for the experiments (Fig. 1.7).

1.5.4.1 Spot Detection After the gel is stained by different dyes, we get dot and smears of the proteins. CBB and silver staining are used in most laboratories because they are sensitive enough to detect most proteins. But no single method is sensitive enough to detect every protein that may be present. Different methodologies are applied to detect every possible protein that may be present in our pool of protein complex. Radioactivity is one of the methods by which detection can be done, but it has its own drawbacks and cannot be used routinely. Recently, fluorescent dyes are created which can bind to the proteins without altering the protein and can be detected using fluorescence. This method also overcomes the drawback of 2DE. Since, the two spots can be very close

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Fig. 1.7 Steps involved in proteomic studies

to each other in gel, but detection method is entirely based on fluorescence thus it makes simple for a user to detect and analyze the protein. Another method by which proteins can be detected is by the use of Western blot. Although it is a powerful technique to identify the proteins, it is limited by the availability of antibodies of the proteins.

1.5.4.2 Fluorescence-Based Difference in Gel Electrophoresis (DIGE) Since, many drawbacks are associated with the 2DE such as gel-to-gel variation, spot detection and quantification problem, etc. An advanced gel-based technique was devised by a group of scientists (Unlu et al. 1997) to overcome these issues and enhance the reproducibility of the experiments. In this method, protein are labeled with two different dyes such as cyanine dyes—Cy3, Cy5 and internal control (pool of control + test sample) with Cy2. All of these can be detected on a single gel and quantified based on the intensity of the samples. 1.5.4.3 Identification Since, proteins are separated using gel-based method and identification of protein still is in question. The identification approach used by the scientists was to create 2D fingerprints of the proteins, but this approach failed as gel-to-gel variation was a major problem. Another approach was to cut out the spot and sequence the protein using the Edman degradation which was quite labor-intensive work and expensive. But as protein sequencing technology and database improved, the approach of

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sequencing also changed. Mass spectrometry is the technique used for the quantitative approach of proteomics and identification of the proteins.

1.5.4.4 Mass Spectrometry One of the quantitative and detective-based approaches for the proteomic studies is to use the mass spectrometer. Mass spectrometers distinguish the mass of the protein. As the proteins can be separated on mass-to-charge ratio. The mass of the protein gives us information about the protein, posttranslational modifications, and structure. Mass spectrometer requires ionization of the protein, separation, fragmentation, and mass determination. Ionization of the protein is either carried out by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). In ESI, the protein must be in solvent phase. For ionization of the protein, high voltage is applied to highly conductive coated needle. The strong electric field created by ESI atomizes the liquid pouring out of the capillary into small, charged droplets. The charge intensity on the droplet’s surface gradually rises as the solvent evaporates, until the droplet divides into one or more charged ions, allowing the analyte to reach the nanoscale. As the analytes the nanoscale. The phase transition of the analyte occurs from liquid to gas phase, as a single charge or several charges and become a gas phase ion. In MALDI, a laser illuminates the protein mixture, and by energy absorption, the protein is ionized. MALDI is an ionization technology that creates ions from big molecules with little fragmentation by using a laser energy absorption matrix. MALDI approach consists of three steps. The sample is first combined with a suitable matrix material (a-cyano-4-hydroxcinnamic acid (CHCA), 2,5 dihydroxybenzoic acid (DHB), 3,5-dimethoxycinnamic acid (sinapinic acid)) before being put to a metal plate. The sample is then irradiated with a pulsed laser, causing ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized in the hot plume of ablated gases by being protonated or deprotonated and then are propelled into mass spectrometer. Note: Ionized proteins are referred to as “ions.” 1.5.4.5 Separation After the proteins have been ionized, their mass and charge must be calculated. The ions are collected first; after collecting the ions, they are passed through mass analyzers. Different types of mass analyzers exist including time of flight (TOF), ion trap, quadrupole, etc. The commonly used is the TOF; here, ions are collected and are transmitted to mass analyzer. The time it takes to transmit particular groups of identical protein to the detector is measured. This measure of the time gives us mass (m)-to-charge (z) ratio. Mass discrimination by flight time is applied for the separation, as ions of lower masses get accelerated to higher velocities compared to the ions with heavy mass. As the m/z ratio is determined for the protein, the computer selects only identical proteins from a single sample and sends them to the activation chamber at regular intervals (Fig. 1.8).

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Fig. 1.8 Time-of-flight mass spectrometer

1.5.4.6 Activation Activation is done by the collision of the proteins with the inert gas. So, for activation, proteins must be broken down into little pieces after being picked by the m/z ratio. Each protein is fragmented by being placed in an argon-filled room. This inert gas collides with the ionized protein, and the gas vibrational energy forces it to split into two pieces. As protein breaks down randomly into two pieces, N-terminal of the protein is labeled as b ions, and the other containing carboxyl or C-terminal of the protein is labeled as y ions. 1.5.4.7 Mass Determination and Characterization After the proteins are activated, another mass determination is done. This is called tandem MS. The m/z ratio of the fragmented protein peptides is determined. Computer algorithms determine this pool of peptide pairs with existing database of the proteins. The spectrum generated by the existing pool is compared with the spectrum of the database until the ideal spectrum is matched. We’ll have the information about protein, its sequence, and probable structure by the database. If the existing database does not have any information about the protein, characterization can be done by using computer-generated models of the proteins (Fig. 1.9).

1.6

Transcriptomics

Transcriptomics is the study of an organism’s transcriptome, which is the total of all of its RNA transcripts. An organism’s information content is stored in its genome and via the principle of central dogma gets expressed through transcription. In this case, mRNA acts as a temporary intermediate molecule in the information network, whereas noncoding RNAs perform a variety of other activities. To start studying, we need the RNA. To get the RNA, similarly like the study of genomics and proteomics, we need to disrupt the cell mechanically or enzymatically. But there are RNAs present in the cell which degrades the RNA. To remove them,

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Fig. 1.9 Proteomic workflow

chaotropic salts are added. In addition, we need to separate the RNA from other biomolecules present in the cell-free extract. Different biophysical methods are applied in order to isolate the RNA. And once we get the RNA, it is sometimes additionally treated with DNAs to degrade any residual DNA present. In a cell, the total RNA content is 98% ribosomal RNA, and the remaining are the messenger RNAs and noncoding RNAs. Thus, the purification required for the isolation of RNA is most necessary. Enrichment of mRNAs can be performed by depleting the ribosomal RNA content by using sequence-specific probes or using poly-A affinity methods. If care is not taken, RNA may get degraded.

1.6.1

Expressed Sequence Tags (ESTs)

It refers to a brief nucleotide sequence made from a single RNA transcript. A reverse transcriptase enzyme first copies RNA as complementary DNA (cDNA). ESTs can be obtained from organism mixtures or environmental samples. Despite the fact that higher-throughput technologies are being employed, for early microarray designs, it was frequently provided by EST libraries.

1.6.1.1 Serial Analysis of Gene Expression (SAGE) An EST methodological advancement called SAGE increased the throughput of the generated tags and permitted some estimation of transcript abundance. After cDNA is synthesized from RNA, it is digested into short base pair tag fragments. This is done using restriction enzymes that cut this cDNA at a specific sequence of 8–10 bp length along that sequence. These cDNA tags are then connected head to tail to

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generate lengthy strands, which are subsequently sequenced using the Sanger sequencing. In a procedure known as deconvolution, the sequences are reversed back into original tags using computational algorithms. These tags generated by the computational algorithms can be linked to the genome and which gene expresses it by comparing it to a database. In case of unavailability of reference genome, these tags can be utilized as biomarkers as some markers are readily expressed in diseased state.

1.6.1.2 Cap Analysis of Gene Expression (CAGE) A SAGE variant called cap analysis gene expression (CAGE) only sequences tags from an mRNA transcript’s 5′ end. The transcriptional start positions of genes can be identified when the tags are compared to a reference present in the databases. For promoter research and full-length cDNA cloning, determining gene start sites is helpful.

1.6.2

Microarray

Microarray is a technique used to detect the expression of multiple genes at the same time by the hybridization of complementary sequence. Microarray is often made up of a grid of small nucleotide oligomers which are called probes. These probes are then arrayed on a glass slide. The amount of transcripts is measured by hybridizing fluorescently labeled transcripts to these probes. The abundance of transcripts for that probe sequence is measured by the fluorescence intensity.

1.6.3

RNA-Seq

RNA-Seq is a technique that combines high-throughput sequencing with computational approaches to collect and quantify transcripts in an RNA extract. After the RNA isolation, the next step is to break the RNA into small fragments. Usually, the length of the resulting nucleotide sequences is normally approximately 200–300 bp. Based on the sequencing technique employed, this length of RNA fragment can vary. After the RNA is fragmented, it is converted into double-stranded DNA (dsDNA). This conversion is necessary because DNA is more stable than the RNA. Depending on the sequencing machine usage, adapter can be added to this DNA. Adaptors help the sequencing machines to identify the fragments and allow sequence of different samples at the same time. Next, the dsDNA is amplified. After this quality control is done for the fragments, it uses deep transcriptome sampling with numerous short fragments from a transcriptome to enable computational reconstruction of the initial RNA transcript by matching reads to a reference genome or with each other. An RNA-Seq experiment can quantify both low- and highabundance RNAs, which is a significant advantage over microarray transcriptomes. In addition, quantity level required for RNA-Seq experiments and analysis is lower when compared to microarrays, allowing for better investigation of cellular

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Fig. 1.10 Transcriptomic overview. (Self-made figure created from the portal Biorender (www. biorender.com/))

structures down to the single-cell level when paired with linear amplification of cDNA. In addition to being able to identify genes, read counts may be used to assess relative gene expression levels precisely or determine a number of genes that are active at any given time. The RNA-Seq approach has improved through time, owing mostly to advancements in DNA sequencing technology, which have boosted throughput, accuracy, and read length (Fig. 1.10).

1.7

Metabolomics

It is the study involving the metabolites or small molecules generated through biochemical process inside the cells. Metabolome includes a complete set of small molecules or metabolites that are present inside the cell. Metabolome includes hormones, signaling molecules, secondary metabolites, etc., and includes the study of the substrates and products of the metabolism.

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But What Are Metabolites?

Metabolites are the low molecular weight organic compounds including substrates, intermediates, and products that are formed in biological processes. The mass range of these molecules is generally 50–1500 Daltons (Da) or < 1.5 kDa. In plants, metabolites are commonly referred to as primary or secondary depending on the function. Primary metabolites are directly involved in plant growth and function, while secondary metabolites act as ecologically relevant or protective element for the plants such as pigmentation and antibiotic formation, respectively. Whereas in a human-based metabolomics these metabolites are referred to as endogenous (produced by host itself) or exogenous (or taken exogenously such as drugs, chemicals, etc.).

1.7.2

Metabolome and Metabolic Reactions

“Metabolome is complete set of metabolites within a cell, tissues, or biological sample at any given time point.” Metabolomes are very dynamic in nature; they undergo binding, dissociation, degradation, modifications, etc., in classic biochemical pathways that are occurring inside the cells. Many reactions undergo at the same time inside the cells. Thus, the concentration of the metabolites also changes. And the concentrations of metabolites are governed by how fast the reaction is taking place. Reactions in turn are dependent on the catalytic efficiency of enzymes. Where product of a one reaction can be the substrate of another reaction. These metabolic reactions are classified as anabolic and catabolic. Anabolic means the formation of the product by using the energy, and catabolic means the breakdown of the substance/product to make energy.

1.7.3

But What Are the Analytical Techniques That We Need to Study Metabolomics?

Workflow includes sample collection, preparation, data acquisition and analysis. The samples are first collected from the cells, tissues, saliva, etc., of the diseased and control group. The problem occurs with the highly complex mixture of the cell. Separation techniques are used to separate the analytes from other by-products. Gas chromatography (GC) with mass spectrometry (MS) (GS-MS) is widely used as separation technique for metabolic analysis. Gas chromatography offers high resolution and can be used to detect the volatile compounds present in the biological systems. GC is used with flame ionization detector (GC-FID) or mass spectrometer (GC-MS) for volatile compounds. Limitation remains the requirement of chemical derivatization of biomolecules. Another separation technique is high-performance liquid chromatography (HPLC). HPLC coupled with the MS (HPLC-MS). Although, this is a lower

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resolution technique, but provides with a higher sensitivity for range of molecules and it overcomes the limitation posed by the GC-MS. Capillary electrophoresis is a rarely used separation technique and is only suitable for charged analytes. It has a higher separation efficiency for the molecules. An only limitation remains is the time required for separating the analytes.

1.7.4

Detection Methods

Mass spectrometry is used for identifying and quantifying the metabolite after separation by GC, GC-MS, HPLC, HPLC-MS, or CE. The identification of the molecules is distinguished by analyte fragmentation patterns. Analyte fragmentation pattern libraries exist that allows the identification of these biomolecules. Mass spectrometry techniques explained in proteomic parts are used as such. Another detection technique that does not require separation of the analytes is the use of NMR spectroscopy. All kinds of small molecules can be measured simultaneously. The sample preparation and detection is also user-friendly. The advantages it provides are the high reproducibility and sample recovery and preparation. An only limitation is the relative insensitive compared to the MS. Machine learning and computational methods including statistics are used for the data analysis once we get the desired results/data from the techniques (Fig. 1.11).

Fig. 1.11 Metabolomic overview. (Self-made figure created from the portal Biorender (www. biorender.com/))

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Lipidomics

It is the study of lipids in biological system settings such as cells, tissues, biochemical pathways, etc. Lipids are the commonly known fat molecules. They are hydrophobic in nature and are made up of long carbon chain acid molecules. Lipidome is the complete lipid profile within a cell, tissue, or an organism. It is also considered as the subset of the metabolome. Lipids are components of cell membranes and are also associated with proteins such as lipoproteins. They have many roles including the integrity of the cell membrane, signaling, acting as barrier, being an energy storehouse, etc. Lipid research involves the identification and quantification of cellular lipids and their interactions among other molecules or lipids. Research in lipidomics mainly involves the deciphering structure of the lipid, its functions, interaction among lipids and other molecules, and dynamics.

1.8.1

Experimental Techniques

The workflow is sample acquisition, from the cells or tissues. Then the extraction of the lipids. After the extraction, lipid separation and detection.

1.8.2

Lipid Extraction

Majority of the lipid extraction and isolation techniques uses lipid’s ability to solubilize in organic compound. Conventional method used is the chloroform/ methanol-based protocol that incorporates phase partitioning. And the extraction of lipids in organic layer.

1.8.3

Lipid Separation

Thin-layer chromatography (TLC) is one of the methods used for separation of lipids. It is a quick and thorough screening tool. Although the method is not sensitive enough, it provides a general overview before advancing to more sensitive techniques. Solid-phase extraction (SPE) chromatography is used to separate the crude lipid mixture into several lipid classes such as glycerophospholipids, cholesteryl esters, fatty acids, glycerolipids, and sterols. HPLC. It is a frequently employed technique to separate lipids before mass spectrometry. Using normal phase HPLC or reverse phase HPLC, different types of lipids can be separated. Using length chains, degree of unsaturation, substitution, etc., different lipid types can be extracted.

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Lipid Detection

Detection method used here is also dependent on the mass spectrometry. General and soft ionization techniques (ESI, desorption electrospray ionization (DESI), MALDI) are employed so that fragmentation of lipids does not occur.

1.8.5

Lipid Profiling

Targeted metabolic platform offers a high-throughput examination of the lipid species present in cells or tissues. This profiling is based on mass spectrometry using ESI and tandem MS and can give us quantitative data. And it is analyzed using computational algorithm and machine learning.

Reference Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077

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An Introduction to Omics in Relevance to Industrial Microbiology Madhumita Priyadarsini, Jyoti Rani, Jeetesh Kushwaha, Kailash Pati Pandey, Yashpal Singh, and Abhishek S. Dhoble

Abstract

The notion of genetics has been permanently imprinted in all living organisms by the basic central dogma of molecular biology (DNA, RNA, protein, and metabolite). Although the idea has been accepted for many years, the development of high-throughput technologies, particularly omics (genomics [study of genome], cytomics [study of cytology], transcriptomics [study of transcriptome], proteomics [study of proteome], and metabolomics [study of metabolome]), has revolutionized the field and allowed it to incorporate extensive data analysis, including bioinformatics and systems biology, as well as the area of synthetic biology and industrial microbiology. Omics technologies, often known as highdimensional biology, incorporate information about cells, tissues, and organisms and assist in its interpretation across various platforms. The method offers the possibility for unveiling the intricate details in diverse aspects of biology and quickens the pace of scientific innovation. This chapter elaborates the fundamentals of omics and their applications in industrial microbiology. Keywords

Genomics · Transcriptomics · Cytomics · Proteomics · Metabolomics · Industrial microbiology

M. Priyadarsini · J. Rani · J. Kushwaha · K. P. Pandey · Y. Singh · A. S. Dhoble (✉) School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, Uttar Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_2

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Introduction

Omics describes the field of life sciences ending with “omics,” such as proteomics, transcriptomics, genomics, or metabolomics. The field of omics has advanced via the development of genomes, transcriptomics, and proteomics. The term omics was first used by Marc Wilkins in 1994. This was made possible by technological advancements in fields like high-resolution two-dimensional electrophoresis. With the rise in omics-based analysis, it has become possible to quantitatively and qualitatively analyze cellular systems at depth. Now with the help of omics, our knowledge about systems biology is deepening. The advantage of the omics study is that it produces precise data that advance comprehension. Omics can decrypt the whole organism completely, from genes to metabolites. Omics includes cytomics (study of cytome), genomics (study of the genome), metagenomics (the complete genetic material of a community), transcriptomics (study of RNAs), proteomics (study of proteins), metabolomics (study of metabolites), and fluxomics (complete ensemble of fluxes in a cell). Metabolic engineering is primarily driven by the sequencing of complete genomes and the measurement of cellular components and their global interactions (transcripts, proteins, metabolites, and fluxes) (Chae et al. 2017; Hansen et al. 2017). Genome mining (Du and van Wezel 2018; Ohnishi et al. 2002), genome breeding (Ohnishi et al. 2002), and genome-scale metabolic shaping (Matsuda et al. 2017) are based on the unraveling of an organism’s genetic repository by genome sequencing and metagenome analysis (Becker et al. 2015; Hug et al. 2018). Systems-wide profiling of the cytome, proteome, transcriptome, metabolome, and fluxome has been shown to be helpful in post-genomic research to understand network operation and globalscale regulation (Chae et al. 2017; Hansen et al. 2017). Although microbes conduct many desired biochemical conversions required for the production of important compounds, naturally, it occurs at a very low rate. Therefore, it is crucial to engineer cells to make effective cell factories with high yield and productivity, which exhibit good process robustness and can utilize broad substrate spectrum. A microbial cell is normally optimized by incorporating one or more features into its genome, which then mediate the desired phenotype. Omics technologies open the way to make customized cell for the production of various products with economic significance and generating unique, alluring industrial processes (Becker et al. 2015). The omics technique helps in choosing the most promising genetic trait combinations for desirable phenotypic characters from a wide range of options or in carrying out precise modifications to the industrial process environment. With the aid of knowledge, strains with outstanding performance were created in a comparatively short amount of time not only for the production of traditional compounds such as L-arginine, L-lysine, and L-valine but also new ones like artemisinin (Paddon et al. 2013; Westfall et al. 2012), diaminopentane (Kind et al. 2014), etc. Because of the enormous potential of omics technology, it has been studied in a number of areas of medical and health science. Omics is also very helpful in drug discovery and toxicity evaluation processes. In order to personalize

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and optimize drug therapy, pharmacogenomics connects genomics and pharmacology to look at how inheritance affects individual variance in drug response. This chapter gives a brief summary of every omics technique, their workflow, and methods of data analysis. This chapter also covers their application from the perspective of industrial microbiology.

2.2

Different Omics Techniques

Omics techniques are widely used to study microbial communities, including bacteria, archaea, viruses, and other microorganisms. Here are some omics techniques specifically applied to microbial research as represented in Fig. 2.1.

2.2.1

Metagenomics

The word “metagenomics” was initially used by Handelsman et al. (1998) to describe the technique of determining the overall genetic content of a microbial community in order to study the genetic and microbiological diversity as well as the metabolic processes of bacteria in a given environment (Méndez-García et al. 2018). Third-generation sequencing (TGS) and next-generation sequencing (NGS) technologies have significantly altered metagenomics research since the sequencing field first underwent research in 2005. Metagenomics directly extracts a number of nucleotide fragments from environmental samples and uses DNA sequencing to obtain the target information, in contrast to conventional cultivation-dependent techniques for microbiological research. This method is more effective at identifying the state of microbes in a particular environment. To obtain metagenomes from environmental data, methodologies are being used: sequence- and functional-based metagenomics (Chen et al. 2022). In order to evaluate enzymatic functions and activities associated with microorganisms and metabolism, functional metagenomics

Fig. 2.1 Representation of different omics techniques

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primarily use an expression library constructed by heterologous production of a number of cloned DNA fragments in insert-holding vectors (Felczykowska et al. 2015) (Allen et al. 2009) (Liebl et al. 2014). To find functional genes, such as heavy metal resistance genes, antibiotic resistance genes (ARGs), genes involved in nitrogen/phosphorus metabolism, and genes involved in pollutant degradation, functional metagenomics is frequently used (Allen et al. 2009; Guo et al. 2017). Furthermore, functional metagenomics can identify genes that code for enzymes with a specific activity, enabling the identification of novel enzymes whose activities might not be deduced from DNA sequences (Ferrer et al. 2007). It is possible to create metabolic processes and anticipate the function of prospective genes using sequence-based metagenomics, which can give genetic data for the whole community (Chong et al. 2020).

2.2.2

Cytomics

To examine and alter cells at the single cell level and to determine their distribution, functionality, and physiological condition within cohorts of cells, tissues, cell populations, and organisms, a study known as cytomics is used (Valet and Tárnok 2003). The microbial community analyses are well suited to this concept, even though it was developed for therapeutic objectives. As many cell parameters as possible could be gathered using the cytomics technique to provide a detailed picture of the complicated dynamics of microbial communities. In addition, subclusters of cells can be distinguished in community patterns based on DAPI-DNA fluorescence and forward light scatter attributed to the DNA content, size, and corresponding cell density properties of the individual cells. In comparison to other omics technologies, cytomics techniques involve quantification of the observed community structure changes. Microbial cytomics could potentially provide a new approach for investigation into how communities interact and the fundamental ecological principles that govern natural or manipulated communities. It will aid in resolving tangible problems that are now arising in a variety of fields, including biotechnological, bioenergetic, biomedical, and environmental issues.

2.2.3

Metatranscriptomics

Analyzing a gene’s expression throughout a population or an entire community is referred to as metatranscriptomics (Aransay and Trueba 2016). With the use of metatranscriptomics, we can better understand how the microbial community responds to changes in the environment through its gene expression, regulatory mechanisms, and metabolic pathways. Massive metatranscriptomics assessments have been used in a number of studies over the past 10 years for diverse environmental samples, including the investigation of soil fertility, biofertilizers, secondgeneration biofuel, and agricultural pathogen.

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Metaproteomics

Characterization of all the proteins expressed at a specific time in an environment is known as metaproteomics (Wilmes and Bond 2004). It is a crucial component of system methods and is vital in understanding how effectively microorganisms function. A metaproteomic strategy uses a total of seven major steps, including collection of samples, retrieval of the targeted fraction, protein harvesting, protein isolation and/or separation and purification, mass spectrometry analyzation, data analysis, and ultimately interpretation of data, where the recognized expressed proteins and pathways are used to access knowledge about operation of the system (Wilmes and Bond 2006; Mahowald et al. 2009). Each scenario presents particular difficulties and constraints for this procedure.

2.2.5

Metabolomics

Characterizing the metabolic precursors and products is done via metabolomics. Since metabolites often have low molecular weights and are largely in flux, their concentrations and compositions within an environment can change considerably over time. An effective method for assessing environmental phenotypic features emerging from the web of interconnections between the components of microbial communities is provided by metabolomics. Because of its importance in identifying ecosystem emergent attributes, this methodology is frequently employed in the search for and diagnosis of biomarkers. In metabolomics, there are mainly two experimental workflows: a non-targeted approach that aims to characterize complete metabolomes and a targeted approach that quantifies known compounds. The discovery of significant portions of unidentified metabolites is a typical feature of non-targeted metabolomics, which is caused by the diverse range in metabolite chemical structures. Additionally, metabolite databases may not be accurate and are not appropriate for identifying isomers (Chen et al. 2008). Comparatively to other omics technologies, metabolomics has less success in the scenario of diverse microbial populations, and, more crucially, the detection of metabolites does not provide much information on microbial community.

2.2.6

Fluxomics

The word “fluxomics” is used to refer to a broad range of methods that all share the same objective: metabolic flux analysis (MFA). The concentration change of intracellular metabolite pools can be expressed mathematically as a function of the reaction stoichiometry and the fluxes to and from the metabolite pool. A metabolic reaction’s stoichiometric coefficients from each participant are represented in a matrix known as the stoichiometric matrix. In-depth descriptions of the formation

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and characteristics of the stoichiometric matrix can be found in the literature (Joyce and Palsson 2006). Ultimately, the mass balance constrains a metabolic network, preventing the creation or disappearance of new mass. Orth et al. review the metabolic network topology for flux analysis in significant detail.

2.3

Advancement in Omics in Profiling and Characterization of Industrially Relevant Microbial Consortia

Since classical era, human societies have used microbial population to yield a variety of products, including food and beverages. Recently, a number of companies focused on microbiological activities have started to emerge. Nowadays, from industrial enzymes production to the development of new pharmaceuticals, there are many technologies based on microbes. The development of novel and more effective production techniques is made possible by the implementation of omics methodologies to microbial populations regulating dairy fermentation, cheese making (Yeluri Jonnala et al. 2018), natural fiber manufacturing, or nutrient accession from diets of goat, sheep, swine, and other livestock. The microbiological, biotechnological, and biopharmaceutical sectors stand to benefit much from the assessment of secondary microbial metabolites made possible by omics and meta-omics methods (Cuadrat et al. 2018). Shi et al. (2017) discovered the roles of numerous unique transcriptional regulators associated in responses to various environmental stresses (heat, heavy metals), resistance to ethanol, carbohydrate transport, and metabolism using omics technique (metabolomics) to analyze variants of Synechocystis sp. For instance, Córdova et al. (2018) optimized the generation of biogas during the anaerobic digestion of microalgae using information from metatranscriptomics analysis (Joyce and Palsson 2006) and investigated the anaerobic digestion of perennial grass to produce second-generation biogas using metaproteomics in conjunction with 16S rRNA sequencing of DNA and cDNA. Similar to this, omics research can be used to create optimization paths for industrial applications with significant environmental and economic impacts.

2.4

Sequential Workflow of Omics

Studying numerous variables in every omics research is a computation-intensive procedure that necessitates significant effort to uncover important correlations and real connections. This is made even more challenging by the reality of course that micro- and macromolecular life forms frequently produce nonlinear interconnections and combined effects of several attributes, making it challenging to separate actual biological signals from background fluctuations. In order to extract useful information from and between these high-dimensional datasets, it can be difficult when biological systems are present. A growing number of research use biological datasets

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Fig. 2.2 Sequential workflow of multi-omics analysis

along with a broad range of comparatively more recent omics methods like fluxomics, ionomics, microbiomics, and glycomics to identify and predict overall status or results from interventions. Given that data originate from many technologies, data standardization is essential prior to omics scale data integration. A generalized integrated omics workflow is shown in Fig. 2.2. The first step of multi-omics workflow is collection of samples of interest. Then extraction and separation procedures are followed to collect pure form of the sample of interest. Detection and identification of the sample was further done to determine the multi-omics sample. Huge amount of data was produced while doing multiomics protocol. After the generation of data using all those abovementioned steps, different data analysis steps were followed.

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Integrative Analysis of Omics Data

Large amounts of information are produced by the analytical methods, necessitating sophisticated technology (bioinformatics and statistics) for its data analysis. The genomes and transcriptomics microarray results are frequently extensive and complex, and if they are not handled appropriately, they frequently lead to false-positive results. In order to identify and/or quantify proteins, the theoretical database for proteomics is usually compared with experimental research, but for metabolomics, raw data analysis is performed to produce relevant and comprehensible data. Therefore, the main objective of the data analysis is to display the data in a legible and intelligible style so that new hypothesis can be tested without producing falsely positive results.

2.6

Omics Data Analysis Using Programming Language

Through a variety of advanced approaches in genomics, cytomics, proteomics, transcriptomics, metagenomics, and metabolomics during the last few decades, enormous amounts of data have been collected. Numerous new technologies have been developed in the past decades that have facilitated fresh study and discoveries. Each “omics” dataset, including those from genomics, cytomics, proteomics, and transcriptomics, is typically analyzed using statistical techniques like the t-test and ANOVA. Making sense of the data deluge is the present goal because data production is useless otherwise. There are several programs available for analysis of data that provide fantastic features for these samples. These comprise the packages that are extremely effective at processing enormous amounts of data from various samples, also reduce the dimension and evaluate the data precisely, and, finally, are useful for giving improved information to the studies. When discussing the integrated approach for life science, it is necessary to analyze both datasets in order to comprehend the different levels of “omics.” To help with this, the platforms like R along with Bioconductor offer packages for the analysis of high-throughput data produced by “omics.” When it comes to sifting through massive amounts of high-throughput “omics” data, R and Bioconductor are crucial tools. A cohesive, intuitive, and easy to use language for data analysis is R console. R differs from other programming languages due to its graphical user interface (GUI), which allows for quick and easy data transfer. In silico software tools are available in Bioconductor (www.bioconductor. org) for the processing of “omics” data produced by various studies, including microarray, serial analysis of gene expression (SAGE), mass spectroscopy, etc. The packages on the Bioconductor platform can be divided into three categories: software, annotation data, and experiment data. Phenotype analysis is carried out through the cpma package. The data’s numerical values are regarded as the input for this package. Multiple linear models can be fitted together with the help of the mlm program. The rationale is made up of two

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parts: statistical data, which is the model’s variable, and an equation, which is a definition of the models. For the researcher who aims to provide a few alluring characteristics, the mixOmics package presents a several variable-based approach for the omics data interpretation (Rohart et al. 2017). An R software tool supraHex is used to preprocess, organize, normalize, and visualize omics data. This console program creates a supra-hexagonal manual to handle the data, and it offers flexible capabilities for after-dissection of the manual and, more critically, considers adding additional information for multiple omics data analyses (Fang and Gough 2014). OmicCircos is an R programming library that produces excellent circular plots for visualizing genomic variations, such as alignment patterns, shift patterns, and modification patterns (Hu et al. 2014). The aforementioned examples can be employed to develop scatterplots using this technique. Machine-learning algorithms are beneficial for combining investigations of integrated omics datasets with clinical data to facilitate clustering, dimension reduction, pathway analysis, association with diagnostic and therapeutic measurements, and other ailment prediction (Li et al. 2018). Other packages like Affy packages, Expression View, Annmap, DEGseq, etc., are employed in omics data integrity assessment and interpretation.

2.7

Applications of Omics in Industrial Microbiology

The approaches in systems biology are dependent on “omics” technologies applicable in understanding and manipulation of complex metabolic pathways which facilitate the biomanufacturing at industrial level (Baidoo and Teixeira Benites 2019). Omics technologies have numerous applications in engineering microbes for the increased production of biofuels, therapeutic metabolites, proteins, enzymes, and crop development (Amer and Baidoo 2021). Some of the applications of omics technologies have been listed in Table 2.1. The applications of omics in different industrial microbiology have been discussed below in detail:

2.7.1

Application in Food Processing

The discipline of omics dedicated to the study of food and nutrition has been named as “foodomics.” Advanced omics techniques are being used to assess food quality in terms of nutrition, screen foodborne pathogens associated with disease outbreaks, study the effect of food metabolites on essential biochemical pathways to prevent possible diseases, and add value properties to food items. Multiple molecular biology-based methods have been developed to validate the composition and quality of food by analyzing the presence or absence of certain proteins and DNA sequences. Several polymerase chain reaction (PCR)-based methods have proved beneficial in the identification of allergens in food. Both genomics and transcriptomics are used to study the mechanism of regulation of biosynthesis of pigments in fruits and

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Table 2.1 Applications of omics technologies Omics technique Genomics

Target molecules Genome

Transcriptomics

RNA

Proteomics

Proteins

Metabolomics

Metabolites such as sugars, amino acids, lipids, etc.

Applications • Diagnosis of diseases caused by unknown pathogens • Analysis of horizontal gene transfer • Microbial diversity analysis • Study of gut microbiome • Identification of unique bioactive compounds • Discovery of novel antibiotics • Identification of industrially relevant enzymes • Development of personalized medicine • Diagnostics and profiling of diseases • Forecasting antibiotic resistance based on interactions between host and pathogen • Identify genes responsible for biotic and abiotic stresses in plants • Compare expression levels of different genes • Early detection of cancer biomarkers • RNA-based therapeutics • Study alterations in protein expression in diseased heart • Study plant phenotype based on protein expression • Detection of plant pathogens • Development of therapeutics for diabetes • Study of neurodegenerative diseases like Alzheimer’s and Parkinson’s • Discovery of molecular markers • Toxicity assessment • Bioremediation • Tumor and cancer detection based on elevated levels of choline compounds • Assess the efficacy of cancer medicine • Characterize toxicity from environmental pollutants • Study plant phenotypes based on metabolic profiling • Assess nutritional value of plant products • Study of plant growth promoters

vegetables. Microorganisms are being genetically modified to produce natural pigments used in food industry (Li et al. 2017). Omics-based technologies have helped study the formation of biofilms used by microorganisms as a barrier to survive under stressful conditions (Yuan et al. 2021). The processing and quality of seafood and animal meat is monitored by combining various omics technologies (Power et al. 2022). Proteomics and immunoassays are being used to identify

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biomarkers for the presence of pathogenic bacteria and fungi present in food. Metabolomics is used to identify and quantify the toxic metabolites and determine their concentrations with respect to the status of the foodborne disease. Transcriptomics-based techniques like next-generation sequencing (NGS), RNA-Seq, microarray, etc., are used to study biofilm development and mechanism of spoilage in food. Thus, omics-based techniques are high-throughput methods that can be used to address the challenges related to the food processing industry.

2.7.2

Application in Dairy Industry

The application of combination of omics-based technologies in dairy industry has helped improve the quality and nutritional value of dairy foods. Transgenic technologies are being used for decades to introduce gene(s) of interest in livestock for the improved production of milk and flesh. In dairy industry, cheese was earlier made using rennet, a proteinase that coagulates milk and is extracted from calf stomachs. However, omics-based methods have helped to find alternative sources of proteinases from plants and microorganisms. Microbial rennet has been produced from fungi Mucor miehei, Endothia parasitica, Aspergillus niger, etc. (Chawla et al. 2018). Major proteins present in milk like casein and whey proteins have been used to study the quality of milk and its products. More than 151 proteins have been identified in milk using omics technologies (Palmer et al. 2006). Two-dimensional electrophoresis has been used in the identification of proteins present in very less concentrations in milk and colostrum. Omics technologies are being extensively used in lactation research in dairy cows. Mapping of genes (genomics) of livestock animals has helped in selective breeding based on ideal traits. Genomic studies are used to identify mastitis by determining the somatic cell count in dairy cows and have helped overcome economic losses incurred in milk production. Proteomics techniques help determine the changes occurring in the pattern of expression of proteins, their availability, and posttranslational modifications (Li et al. 2018).

2.7.3

Application in Beverage Industry

Knowledge of metabolic activities of microorganisms and their impact on the quality of beverages in terms of flavor, fragrance, and nutritional value can be obtained using omics technologies. The presence of pathogenic microorganisms in beverages may adversely impact human health. Their identification before these beverages reach consumers is very crucial. Beverage industries are using omics technologies to identify pathogenic strains. Omics approaches have been used to produce genetically modified variants of tea and coffee (most consumed beverages in the world) to meet their increasing global demand. High-throughput omics approaches have helped understand the complexity of wine microbiome and improve its taste and aroma. Techniques like high-throughput sequencing, metabarcoding, and denaturation gradient gel electrophoresis (DGGE) have helped study microbial composition at

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various stages in wine making (Sirén et al. 2019). Consumers’ concerns with dairybased beverages have popularized the plant-based drinks and substitutes of milk produced from grains and legumes. Fermentation by lactic acid bacteria has helped overcome the undesired physicochemical properties associated with flavor and nutrient availability. Ideal lactic acid bacteria isolates are selected using nextgeneration sequencing (NGS) approach for the fermentation of plant substrates not just to produce wine and beer but also for plant-based milk products (Sirén et al. 2019). A combination of different omics techniques like genomics, proteomics, and metabolomics is also used to understand the nutritional aspects of probiotic drinks on human health. Also, various microbial strains have been identified for improved production of probiotic beverages, especially lactic acid bacteria and bifidobacteria.

2.7.4

Application in Pharmaceutical Industry

Pharmaceutical research is heavily dependent on omics techniques like genomics, transcriptomics, proteomics, and metabolomics and their combinations for drug discovery, drug development, targeted drug delivery, personalized medicine, and safety assessment. Toxicogenomics, toxicoproteomics, and toxicometabolomics are commonly used to predict the potential drug toxicity by studying the mechanism of action of drug toxicity on the gene expression, protein expression, and production of endogenous metabolites, respectively, and carry out drug safety assessment (Gresham and McLeod 2009). Omics-dependent studies are conducted to understand the drug-induced hepatotoxicity in human liver. Pharmacogenomics has helped understand the effect of inherited genes on the response of human body to medications against cancer, cardiotoxicity, etc. Epigenetic changes (heritable changes) in the genome of humans have been studied using epigenomics. Transcriptomics has been used to identify the markers for Alzheimer’s disease by studying the gene expression profiles of the neurofibrillary tangles. A number of cancer-related proteins like cathepsin B, pfetin, heat shock protein 27, etc., has been identified using proteomics. Proteomics has helped identify a number of novel anticancer drug leads (Yan et al. 2015). Thus, omics approaches are an integral part of pharmaceutical research, and their extensive application may prove beneficial in targeted drug development.

2.7.5

Application in Agricultural Biotechnology

Agricultural biotechnology is utilizing “omics” technologies to improve desired phenotypic features in crops like color, flavor, drought tolerance, pesticide resistance, etc. Omics approaches are being used in the development of biofertilizers consisting of essential microbes that help increase nutrient uptake in plants from soil ram. Genomics, transcriptomics, and metabolomics have helped in identifying functional genes to develop crop varieties that can be grown with less water requirements and have better nutrient uptake properties. Omics has enabled scientists

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to identify genes and biochemical pathways through selective breeding to develop improved food crops that have added health benefits, better nutrition, high minerals, and vitamins. A variety of microorganisms are present around the crop plants, and not all of them are beneficial and cause plant diseases that cause financial loss to the farmers. Omics technologies are used understand the interactions between plants and pathogens and yield valuable data for the prevention of plant diseases. Transcriptomics has been used to study the changes in genetic expressions in food crops arising from biotic and abiotic stress. Proteomics is used to study the effect of pathogenic fungi and bacteria on the pattern of proteins in plants. The development of insecticide- and herbicide-resistant varieties of crops like cotton, soybean, brinjal, tomatoes, corn, etc., has been possible only because of the extensive use of omics technologies. Gene amplification helps establish the relationship between resistance and adaptation in host plants. Tomato and banana plants have been genetically modified to produce low-cost edible vaccines against diseases like pertussis, measles, diphtheria, and tuberculosis (Ahmad et al. 2012).

2.8

Future Prospects and Limitations

Although combined or individual omics techniques have been employed effectively, they still have several drawbacks that prevent the creation of a systems biology framework for the production of industrial strains. First, proteome profiling advances our understanding of cellular metabolic state since the majority of cellular metabolic processes are somehow mediated by proteins. Proteome analysis does identify protein spots in 2D gels that exhibit changing intensities in response to protein hyper-secretion or other important stressors, but it is labor-intensive, time-consuming, and hard to automate. Chromatography technologies may be used for highthroughput quantitative study of metabolites during the conversion of interest, and a comparison of metabolite profiles under alterations may be useful for determining the physiological status of the cells. The investigation of the metabolome is much more difficult in terms of timeframe and analytical apparatus as compared to the proteome and transcriptome (Pinu et al. 2017). At the time of sampling, all metabolic activity must be immediately quenched in order to account for the quick turnover of metabolites inside the cell. The consequent impacts on integrity of cell, which frequently involve metabolite leakage, might significantly skew the metabolome data (Bolten et al. 2007; Bolten and Wittmann 2008). In addition to the challenges mentioned above, our capacity to access and comprehend the intricate metabolic networks has been substantially enhanced by the tremendous advancements in expression systems and omics technologies during the last 10 years. However, since our understanding of metabolic networks has grown more sophisticated due to advances in genome sequencing, a variety of optimization techniques are needed to incorporate microbial activities into a wide range of industrial processes.

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Conclusion

The massive rise of sequencing data from metagenomes, genomes, metaproteomes, and metatranscriptomes and over the past 10 years has continued to reveal the immense range of metabolic potential in nature that is yet untapped. With the help of multi-omics datasets which are publicly accessible, many environmental microorganisms are moving from the unknown-unknown to the known-unknown search area. Now it is needed to examine these datasets in greater detail and utilize them to answer our ecological and exploratory questions about the microbiome members and their functions in the natural settings we are researching. Multiomics techniques, however, have considerably more promise than only been used as instruments for explanation. They provide theories that are awaiting testing utilizing cutting-edge culture techniques to cultivate a variety of existing bacteria that were previously unculturable. The possibilities for data integration are numerous, ranging from targeted isolation to multi-omics-inspired medium development to high-throughput screening of numerous colonies or enriched cultures. The additional sources of knowledge acquired from high-end molecular tools offer incredible advantages that could result in significant discoveries and shouldn’t be disregarded because microbial cultivation is laborious and time-consuming. The chances of finding new microbial isolates are increased, and the search space is reduced when multi-omics knowledge is incorporated into cultivation research. Acknowledgement This work was partly supported by the Indian Institute of Technology (Banaras Hindu University) Varanasi’s Seed Grant Ref. No. IIT (BHU)/Budget/19-(14)/2021-22/ 2268 dated July 6, 2021, Department of Science and Technology (DST), Government of India’s Project No. DST/SEED/SUTRA/2020/132(G) dated June 24, 2021, and Science and Engineering Research Board (SERB), Government of India’s Project No. SRG/2022/000147 dated October 27, 2022, all to ASD.

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Databases and Tools for Microbial Genome and Human Microbiome Studies Sibasree Hojaisa and Anupam Nath Jha

Abstract

The term microbe refers to the diverse spectrum of microscopic archaea, bacteria, protist, fungi, and algae, of which some are beneficial, whereas others might be harmful. Being omnipresent, these microbes impact the entire biosphere with the human body being no exception. All the microbiota that reside within and outside the human body constitute the human microbiome, the study of which in recent times has garnered much importance due to the advancement in the understanding of the role of microbes in normal human physiology as well as pathology. This has led to a notable surge in the available sequenced genome data generated from various microbial genomics and metagenomic studies. This big data being heterogenous in nature makes it difficult for the researchers to analyze and interpret them. As such, databases and bioinformatic tools, both existing ones and newly emerging, aim to curate this data for easy access and analysis by the people. This chapter thus summarizes various microbe-related databases, specifies the information stored and retrievable from them, and attempts to shed light on various bioinformatic tools and their application in microbial genomic and human microbiome studies.

S. Hojaisa Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India Department of Zoology, Biswanath College, Biswanath Chariali, Assam, India A. N. Jha (✉) Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_3

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Keywords

Microbial genome · Human microbiome · Database · Bioinformatic tools · Genomics

Abbreviations AIDS AMDB BV-BRC CGED DB DDBJ DNA EBI eHOMD EMBL GEO GOLD GTDB HGD HGFs HGMA HIFs HIV HPC IMG/M IMG/VR INSDC IRD JGI KEGG MADET MASI MBGB MiST MLST NCBI NGS NIH NMDC OMIM PaVE PDB

Acquired immunodeficiency syndrome Animal microbiome database Bacterial and Viral Bioinformatics Resource Center Cancer gene expression database Database DNA Data Bank of Japan Deoxyribonucleic acid European Bioinformatics Institute Expanded Human Oral Microbiome Database European Molecular Biology Laboratory Gene Expression Omnibus Genomes OnLine Database Genome Taxonomy Database Human Genome Database Host genetic factors Human Gut Microbiome Atlas Host immune factors Human immunodeficiency viruses High-performance computing Integrated Microbial Genomes/Microbiome Integrated Microbial Genomes/Viral International Nucleotide Sequence Database Influenza Research Database Joint Genome Institute Kyoto Encyclopedia of Genes and Genomes Microbiomics of Anticancer Drug Efficacy and Toxicity Microbiota-Active Substance Interactions database Microbial Genome Database Microbial Signal Transduction Database Multilocus sequence typing National Center for Biotechnology Information Next-generation sequencing National Institute of Health National Microbiome Data Collaborative Online Mendelian Inheritance in Man Papillomavirus Episteme Protein Data Bank

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Databases and Tools for Microbial Genome and Human Microbiome Studies

RNA RVDB SGD UCSC VEP VEuPathDB ViPR

3.1

43

Ribonucleic acid Reference Viral Database Saccharomyces Genome Database University of California-Santa Cruz Variant Effect Predictor Eukaryotic Pathogen, Vector and Host Informatics Process Virus Pathogen Database and Analysis Resource

Introduction

A microbe also referred to as a microorganism is minute unicellular or multicellular organisms that are not visible to the naked eye. This term encompasses a diverse form of organisms that are numerous in number and differ from each other in morphology, habitat, physiology, and many other characteristics. The microbial diversity is absolutely astounding with members occurring in all three domains of life: the Archaea, Bacteria, and Eukarya. Viruses and newly recognized prions although acellular are also regarded as microbes. Microbes thrive throughout the biosphere even in extremities and create conducive conditions for the survival and well-being of other living organisms. Although extremely diverse, microbes can be grouped as shown in Fig. 3.1.

Fig. 3.1 Simplified grouping of microbes

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3.1.1

S. Hojaisa and A. N. Jha

Prokaryotic Microbe

Bacteria are unicellular microbes that can be found in every habitat on Earth including the human body. They are devoid of a nucleus with its circular DNA suspended in the cytoplasm and the presence of extra circular DNA called plasmid all enclosed within the cell membrane composed of predominantly peptidoglycan. Bacteria in soil are an integral part of the nutrient cycle, serving as decomposers, improving soil quality and water-holding capacity, and much more. Living in harmony with the host, these microbes perform many vital functions, including digestion, detoxification, and supporting immunity (Scotti et al. 2017). In pharmaceuticals, they serve as sources for antibodies and vaccine manufacturing. Benefits are numerous, yet many are pathogens with the capacity to cause grave illness to mankind and other organisms as well. To name some organism such as, Mycobacterium tuberculosis causative agents for tuberculosis (WHO 2022a), typhoid life-threatening infection caused by Salmonella typhi (WHO 2022b), various infections caused by Pseudomonas aeruginosa (Rather et al. 2022) and several others. Archaea although morphologically similar to bacteria are much more diverse in shape and inhabit the hostile environment. They are mostly said to be mutualistic in relationships with other living organisms (Australian Academy of Science 2022).

3.1.2

Eukaryotic Microbe

Protists are unicellular eukaryotic microbes constituted chiefly by fungi-like, plantlike, and animal-like organisms, namely, slime mold, algae, and protozoa. Slime molds are decomposers and recyclers in the food web. Algae being photosynthetic contribute to oxygen production, serve as a food source, and provide products like agar for use in scientific studies. Some protozoa zooplanktons are food for higher organisms, whereas some being parasitic cause diseases like malaria (Plasmodium sp.), amoebic dysentery (Entamoeba histolytica), visceral leishmaniasis (Leishmania donovani) and several others are widely studied with experimental and computational methods (Das et al. 2021; Guillén 2023; Saha and Jha 2023; Rajkhowa et al. 2021). Fungi are both unicellular (yeast) and multicellular (mold) organisms functioning mainly as decomposers. Yeast performs fermentation yielding in food products like bread and alcohol. However, it is also responsible for the spoilage of food and fungal infection like vaginal candidiasis (CDC 2022). Mold also is a source of antibiotics like penicillin and toxin that are allergens.

3.1.3

Acellular Microbe

Viruses as they can survive only short term outside the host body are considered nonliving organisms. They reproduce by hijacking the cellular machinery of the host. They are packages of nucleic acid (either DNA or RNA) confined within a

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protein capsule and sometimes lipid. These microbes are the cause of numerous detrimental diseases like HIV-AIDS, Ebola, and so forth. Prions are newly recognized proteinaceous infectious agents which are abnormally folded. On contact, they induce other proteins to fold causing a cascade of havoc.

3.2

Microbial Genome

From the above, we can establish how significant the microbes are for sustaining life on Earth. The above characteristics of different microbes are attributes of their genome, thus prompting researchers to dive into the microbial genome (complete set of an organism’s genetic information) studies for pure and applied research purposes. The branch dealing with the identification and characterization of genetic constituents of the microbe is called microbial genomics.

3.3

History of Microbial Genome Sequencing

The history of microbial genome sequencing can be traced back to the early 1970s when the first viral genome of single-stranded RNA bacteriophage MS2 was sequenced at the Laboratory of Molecular Biology, Belgium, by Walter Fiers and his team (1976) (Fiers 1995; Fiers et al. 1976; Koonin and Galperin 2003). The viral genome sequenced was only 3569 base pairs long. Around this period with the advent of the chain termination method by Frederick Sanger (Koonin and Galperin 2003; Sanger et al. 1977), the first viral DNA genome sequencing was also carried out for bacteriophage ϕX174 of 5386 base pairs, at the Medical Research Council Laboratory of Molecular Biology, Cambridge, UK, by F Sanger and his team (Fiers et al. 1976). Viral and organelle genome sequencing has flourished through this period. However, with an aim to hasten the process of sequencing, Messing in the year 1981 developed the Sanger-based shotgun method of DNA sequencing, a computational approach (Messing et al. 1981). This was employed by R.D. Fleischmann and his team to sequence the first cellular genome of bacteria Haemophilus influenzae Rd. with 18,30,137 base pairs in the year 1995 at the Institute of Genomic Research, Maryland, USA (Cooper 2000; Fleischmann et al. 1995; Land et al. 2015; Loman and Pallen 2015). It has been 27 years since the first cellular microbial genome was sequenced. From then on, microbial genomics has grown to an extent from sequencing of a single organism to metagenomic studies. All this evolution at the grace of advancing technology from early sequencing method to next-generation sequencing (NGS) technology allows massive parallel and high-throughput sequencing. It was reported by the year 2000 that a total of 30 microbial genomes were sequenced (Fraser et al. 2000), which gradually increased to 73 unicellular species in 2002 (Koonin and Galperin 2003), 261 microbial genomes in 2005 (Rasko and Mongodin 2005), and henceforth. Today, these numbers have reached millions as is

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evident and supported by enormous microbial genome data publicly available in biological repositories, in particular the National Center for Biotechnology Information (NCBI) (Sayers et al. 2022), Ensembl Bacteria (Yates et al. 2022), Genomes OnLine Database (GOLD) (Mukherjee et al. 2022), and Genome Taxonomy Database (GTDB) (Parks et al. 2022). As of December 2022, the NCBI Genome houses 4,76,069 prokaryotic (bacterial and archaeal) and 54,184 viral genome (Sayers et al. 2022). The Ensembl Bacteria, a nonredundant bacterial genome database, in its latest update, has published a total of 31,332 bacterial and archaeal genomes (Yates et al. 2022). Genomes OnLine Database (GOLD) is a metadata repository that maintains a catalog of genome and metadata projects and has so far reported 55,518 studies (4022 metagenomic and 51,496 non-metagenomic sequencing projects) which amounts to 4,76,190 organisms with a sequenced genome, out of which 406,333 were bacterial, 4978 were archaeal, and 18,327 were viral genomes, respectively (Mukherjee et al. 2022). The Genome Taxonomy Database (GTDB) also according to its latest release comprises 311,480 bacterial and 6062 archaeal genomes organized into 62,291 bacterial species clusters belonging to 148 phyla and 3412 archaeal species clusters belonging to 18 phyla, respectively (Parks et al. 2022).

3.4

Introduction to Databases

This explosive increase in data volume in genomics and all branches of omics has been massive, from just a few megabytes in the 1970s to petabyte and exabyte in nearly two decades, such that biologists today are said to have joined the big data club. Big data in biology can be seen as an endless exploration opportunity, but the heterogeneity and complexity of this data stemming from different experiments have brought forth challenges of their own. Data of such size demands special storage and also tools for easy access, retrieval, sharing, analysis, and interpretation. It is here where the integration of biology with informatics gained popularity and a new branch of life sciences, bioinformatics, emerged. Bioinformatics is a term that has been coined by Paulien Hogeweg and Ben Hesper in the 1970s where they defined it as “the study of informatic processes in biotic systems” (Hogeweg 2011). Today, as defined by NCBI, “Bioinformatics, is the field of science in which biology, computer science, and information technology merge into a single discipline” (NCBI 2022). And this discipline harbors three principal aims: 1. Creation of databases for storage and retrieval of different categories of biological information 2. Development of efficient bioinformatic tools, algorithms, and statistics for biological data analysis 3. Exploitation of the above tools for analysis and meaningful interpretation from the data available Looking back at the first database Atlas of Protein Sequence and Structure by Margaret Oakley Dayhoff and colleagues (1965) (National Biomedical Research

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Fig. 3.2 Simplified classification of biological database

Foundation 1973), databases and bioinformatic tools in the twenty-first century have evolved at a great pace in number and complexity. The 2021 Nucleic Acids Research (NAR) database issue has reported a total of 1641 databases (Rigden and Fernandez 2021). For a better understanding of how diverse the nature of data stored in these databases is, we can categorize them as shown in Fig. 3.2. 1. Based on the data source and level of curation: (a) Primary database: These are databases that archive the experimental data directly submitted to it by researchers, for example, GenBank (Benson et al. 2017) and Protein Data Bank (PDB) (Berman 2008). They are redundant in nature with minimal annotation. (b) Secondary database: These databases comprise highly curated and annotated data derived after analysis of the primary data, for example, Ensembl and UniProt (UniProt 2022). (c) Composite database: These databases amalgamate data from different primary sources providing a unified platform while eliminating redundancy and trivial entries, for example, OWL – composite protein sequence database with entries from six primary databases (Bleasby et al. 1994; Bleasby and Wootton 1990).

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Table 3.1 Different categories of databases based on data types Databases Disease database Enzyme database Gene expression database Genomic database Metabolic pathway Nucleotide sequence database Organelle database

Organismspecific database Protein database

Information stored Publish information on various organism-related diseases Contains comprehensive information on enzyme Gene expression information

Explore the genome of an organism Molecules involved in different cellular pathways and understanding protein-protein interaction Comprises information DNA and RNA sequence Information about different organelles

Contain information about a specific organism Protein sequence Protein structure

Example Online Mendelian Inheritance in Man (OMIM 2022) BRENDA (Chang et al. 2021) and LIGAND (Goto et al. 2002) ArrayExpress (Athar et al. 2019) and Gene Expression Omnibus (GEO) (Barrett et al. 2013) Ensembl (Cunningham et al. 2022) and IMG (Chen et al. 2022) BioCyc (Karp et al. 2019) and KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al. 2022) GenBank and DNA Data Bank of Japan (DDBJ) (Fukuda et al. 2021) NCBI Organelle Genome Resources (Sayers et al. 2022) and MITOMAP – human mitochondrial genome database (Lott et al. 2013) EcoCyc (Keseler et al. 2021) and Saccharomyces Genome Database (SGD) (Cherry et al. 2012) UniProt Protein Data Bank (PDB)

2. Based on data types: Bioinformaticians have attempted to present the diversified information available in biology by creating different comprehensive databases which roughly can be categorized as given in Table 3.1. Thus, with an increasing number and variety of databases comprising different information, biologists are facing a new challenge of decision-making regarding which database to resort to. Thus, to aid microbiologists, we have attempted to curate and list out a database dealing with microbial genomics.

3.5

Microbial Genome and Human Microbiome Databases

The publicly available databases were scrutinized and compiled on the basis of the rich data content and tools, relatively high degree of usage, active maintenance, and continued development with new releases or updates after 2018. After thoroughly inspecting the vast array of databases, we have put together a list of 36 microbial genome and microbiome-related databases which have been categorized as given below:

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Table 3.2 Global genomic databases along with its data type for use in microbial research Sl. no 1

Name BioCyc

1.1.

BsubCyc

1.2.

YeastCyc

1.3.

EcoCyc

2

DNA Data Bank of Japan (DDBJ)

3.

Ensembl Genomes

3.1.

Ensembl Bacteria

3.2.

Ensembl Protist

3.3.

Ensembl Fungi

4.

GenomeNet

4.1.

VarDB

4.2.

Virus-Host DB

4.3.

Kyoto Encyclopedia of Genes and Genomes (KEGG) KEGG GENOME

4.3.1.

4.3.2.

URL https://biocyc. org/?sid= biocyc13-388111 8535 https://bsubcyc. org/?sid= biocyc16-388111 8484 https://yeast. biocyc.org/?sid= biocyc16-388111 8517 https://ecocyc. org/?sid= biocyc16-388111 8471 https://www. ddbj.nig.ac.jp/ index-e.html http:// ensemblgenomes. org/ https://bacteria. ensembl.org/ index.html http://protists. ensembl.org/ index.html http://fungi. ensembl.org/ index.html https://www. genome.jp/en/ http://www. vardb.org/vardb/ https://www. genome.jp/ virushostdb/ https://www. genome.jp/kegg/

https://www. genome.jp/kegg/ genome/

Data type Pathways and genomes

References Karp et al. (2019)

Bacillus subtilis subtilis 168 reference genome

Saccharomyces cerevisiae S288c model genome

Escherichia coli K-12 substr. MG1655 reference genome

Keseler et al. (2021)

Nucleotide sequence, genome, bioprojects, biosample, metabolomics, and genomic variation Non-vertebrate genome

Fukuda et al. (2021)

Yates et al. (2022)

Bacterial and archaeal genome Protist genome

Fungal genome

Unified genomic database web portal Antigenic variation Virus-host relationship

Genomic, chemical, systems, drug, and disease information

GenomeNet al. (2022) Hayes et al. (2008) Mihara et al. (2016) Kanehisa et al. (2022)

Complete organism and viral genome

KEGG GENES (continued)

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Table 3.2 (continued) Sl. no

Name

4.3.3.

KEGG Metagenomes

4.3.4.

KEGG Orthology (KO)

5.

5.3.

Joint Genome Institute Genomes OnLine Database (GOLD) Integrated Microbial Genomes/ Microbiomes (IMG/M) Integrated Microbial Genomes/Viral (IMG/VR) MycoCosm

6.

NCBI

7.

PhylomeDB

8.

UCSC genome browser

5.1.

5.2.1.

5.2.2.

3.5.1

URL https://www. genome.jp/kegg/ genes.html https://www. genome.jp/kegg/ catalog/org_list3. html https://www. genome.jp/kegg/ ko.html https://jgi.doe. gov/ https://gold.jgi. doe.gov/index

Data type Catalog of gene and protein in a complete genome

References

Metagenome

Functional orthologs and genome annotation Unified genomic database web portal Genomic and metagenomic sequencing project and related metadata

Nordberg et al. (2014) Mukherjee et al. (2022)

https://img.jgi. doe.gov/cgi-bin/ m/main.cgi

Microbial genome and microbiome dataset

Chen et al. (2022)

https://img.jgi. doe.gov/cgi-bin/ vr/main.cgi

Viral genome

Camargo et al. (2022)

https://genome. jgi.doe.gov/ programs/fungi/ index.jsf https://www. ncbi.nlm.nih.gov/ guide/genomesmaps/ http://phylomedb. org/ http://genome. ucsc.edu/index. html

Fungal genome

Grigoriev et al. (2014)

Genome sequence and maps, bioprojects, pathogenic microbe genome sequence

Sayers et al. (2022)

Gene phylogenies’ catalog

Fuentes et al. (2022) Nassar et al. (2022)

Vertebrate and non-vertebrate genome sequence

Global Genome Databases

This category of databases encompasses those resources serving as repositories for genomic information (given in Table 3.2) of all domains of life and tools needed for its visualization, retrieval, assembly, annotation, variant analysis, comparative genomics, and henceforth. Most of them function as integrated platforms for numerous

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databases like NCBI, DDBJ, Ensembl, GenomeNet (GenomeNet 2022), and Joint Genome Institute (JGI) (Nordberg et al. 2014) web portals. However, while compiling such composite databases, we have included only those that are relevant to microbes. BioCyc is a massive collection of databases with genomic information on 20,026 organisms along with tools for genome visualization, alignment, comparative genome, and pathway analysis (Karp et al. 2019). Among many databases of BioCyc here are mentioned a few popular microbe-related resources. DDBJ, NCBI, and EMBL-EBI (European Molecular Biology Laboratory – European Bioinformatics Institute) as contributors to International Nucleotide Sequence Database (INSDC) (Karsch-Mizrachi, Takagi, Cochrane,, and International Nucleotide Sequence Database 2018) primarily serve as a nucleotide sequence database. However, with time they have grown to include sources for genome, transcript sequences, bioprojects, biosample, and much more. Ensembl Genomes is one such extension of EMBL-EBI, a genome browser for non-vertebrate species, namely, for the plant, metazoan, protist, fungi, and bacteria, and tools for assembly, annotation, alignment, and comparative genomic studies. Here Ensembl Bacteria with 31,332, Ensembl Fungi with 1506, and Ensembl Protist with 189 genomes have been included of the above five mentioned (Yates et al. 2022). NCBI Genomes and Maps is a collection of databases offering multitudinous genomic information such as assembly, bioprojects, complete genome, and disease-associated microbial genome sequences as well. NCBI provides its users myriad of tools for genomic similarity search, viewing, remapping, variation viewing, and much more. GenomeNet is also a suite of databases with services for genomic research. KEGG being one of its most cited databases is dedicated to services for deciphering and understanding functional relationships from genomic information. KEGG has integrated a total of 16 databases that put together genomic and chemical information to generate high-level system information for in-depth knowledge of biological systems and their practical application. KEGG at present holds information for 835 eukaryotic, 7409 bacterial, 405 archaeal, and 359 viral genomes (Kanehisa et al. 2022). JGI maintains a total of six genomic databases along with tools of which IMG, GOLD, and MycoCosm (Grigoriev et al. 2014) are relevant to our interest. GOLD is an enormous comprehensive collection of genomic and metagenomic sequencing projects and related metadata. Integrated Microbial Genomes (IMG) content prioritizes the distribution of microbial genome and microbiome dataset along with annotation and other analysis tools. Integrated Microbial Genomes/Viral (IMG/VR) (Camargo et al. 2022) is viral-specific IMG resource, whereas MycoCosm is specific for fungal genome access, visualization, and analysis. UCSC Genome Browser (Nassar et al. 2022) is also a hub for visualization, annotation, and assembly for both vertebrate and non-vertebrate organisms.

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Microbial Genome Database

Here we have grouped those databases having information and tools only for microbial genomic visualization and analysis. Thus, by nature, it is focused on microbes; however, they do serve as repertoire for the additional host or vector information that is associated with microbes. Ensembl Bacteria, Ensembl Protist, Ensembl Fungi, KEGG Virus, IMG/M maintained by JGI, and NCBI microbial genome could very well fit in this grouping. However, they have been placed in global genomic resources as they are not a standalone web portal but are maintained and developed by a parent institute besides other nonmicrobial resources. Microbial Genome Database (MBGB) (Uchiyama et al. 2019) assists in the comparative analysis of microbial sequence by creating an ortholog gene cluster, whereas Microbial Signal Transduction Database (MiST) (Gumerov et al. 2020) is a signal transduction profiling database for bacterial and archaeal genome. The Eukaryotic Pathogen, Vector and Host Informatics Process (VEuPathDB) (Amos et al. 2022) is a unified platform for diverse genomics and datasets associated with eukaryotic pathogens, invertebrate vectors, and hosts. They function as genome browsers and have tools like Apollo for annotation and curation and Galaxy for RNA-seq, ChIpSeq, variant analysis, and appreciably more. At present, genomic information of 689 organisms is stored in this database (Amos et al. 2022). VEuPathDB site has several databases for specific organism integrated which has been listed in Table 3.3.

3.5.3

Bacterial, Archaeal, and Viral Genomic Database

Making the classification more specialized, we have grouped together those microbial resources which purely provide information about bacteria, archaea, and viruses only (provided in Table 3.4). BacMap is an interactive database for 2989 highly annotated chromosomal maps of the bacterial genome (Cruz et al. 2012). The Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (Olson et al. 2022) is a newly transitioned database with tools for bacterial and viral genome visualization and analysis, built by integrating previously existing PATRIC (bacterial and archaea databases), Influenza Research Database (IRD), and Virus Pathogen Database and Analysis Resource (ViPR). This database however focuses more on disease-related microbes and thus provides host information and additional tools for protein, metagenomic, and also phylogenetic analysis. BacWGSTdb (Feng et al. 2021) aims to assist microbiologist in the accurate identification of pathogenic bacteria by providing genomic data and associated metadata. DoriC (Dong et al. 2022) has replication origin information for the prokaryotic genome, whereas GTDB as the name suggests uses genome phylogeny to construct a microbial taxonomy. Reference Viral Database (RVDB) (Goodacre et al. 2018) is an attempt to help the easy detection of viruses by providing a repertoire of the annotated viral nucleotide sequence. ViralZone (Hulo et al. 2011) is a resource that provides information about viral diversity along with proteomic and genomic details.

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Table 3.3 Microbial genomic databases along with their data type for use in microbial research Sl. no 1.

3.1

Name Microbial Genome Database (MBGD) Microbial Signal Transduction Database (MiST) VEuPathDB Eukaryotic Pathogen, Vector and Host Informatics Process 3.1AmoebaDB

3.2.

CryptoDB

3.3.

FungiDB

3.4.

GiardiaDB

3.5.

HostDB

3.6.

MicrosporidiaDB

3.7.

PlasmoDB

3.8.

PiroplasmaDB

3.9.

TrichDB

3.10.

ToxoDB

3.11.

TritrypDB

3.12.

Vector DB

2.

3.

3.5.4

URL https://mbgd. nibb.ac.jp/ https://mistdb. com/ https:// veupathdb.org/ veupathdb/app https:// amoebadb.org/ amoeba/app https://cryptodb. org/cryptodb/ app https://fungidb. org/fungidb/app https://giardiadb. org/giardiadb/ app https://hostdb. org/hostdb/app https:// microsporidiadb. org/micro/app https:// plasmodb.org/ plasmo/app https:// piroplasmadb. org/piro/app https://trichdb. org/trichdb/app https://toxodb. org/toxo/app https://tritrypdb. org/tritrypdb/ app https://trichdb. org/trichdb/app

Data type Microbial genome Bacterial and viral genome single transduction profile Pathogenic microbial, invertebrate vector, and host genome

References Uchiyama et al. (2019) Gumerov et al. (2020) Amos et al. (2022)

Amoeba genome

Cryptosporidium genome

Fungal and oomycete genome Giardia genome

Pathogenic host genome Microsporidia genome

Plasmodium genome

Piroplasma genome

Trichomonas genome Toxoplasma genome Kinetoplastid genome

Invertebrate vector genome

Species-Specific Genomic Database

Although we have encountered many species-specific databases in the previous genomic and microbe resources, here we have mentioned only those independent databases dedicated to one model organism. Papillomavirus Episteme (PaVE) (Van

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Table 3.4 Genomic databases specialized for bacteria, archaea, and viruses Sl. no 1.

2.

Name BACMAP genome atlas

3.

Bacterial and Viral Bioinformatics Resource Center (BV-BRC) BacWGSTdb

4.

DoriC

5.

Genome Taxonomy Database (GTDB)

6

Reference Viral Database (RVDB)

7.

ViralZone

8.

viruSITE

URL http:// bacmap. wishartlab. com/ https://www. bv-brc.org/

Data type Bacterial genome

References Cruz et al. (2012)

Pathogenic bacterial and viral genome

http://bacdb. cn/ BacWGSTdb/ http://origin. tubic.org/ doric/home https://gtdb. ecogenomic. org/ https://rvdb. dbi.udel.edu/

Pathogenic bacterial genome

Olson et al. (2022) Feng et al. (2021)

Replication origin for prokaryotic genome

Dong et al. (2022)

Microbial taxonomy derived from microbial genome phylogeny Viral annotated nucleotide sequence

Parks et al. (2022)

https:// viralzone. expasy.org/ http://www. virusite.org/ index.php

Viral diversity, proteomic, and genomic data Viral genome

Goodacre et al. (2018) Hulo et al. (2011) Stano et al. (2016)

Doorslaer et al. 2017) is for papillomavirus, PomBase (Harris et al. 2022) for fission yeast Schizosaccharomyces pombe, Pseudomonas Genome DB (Winsor et al. 2016) for bacteria Pseudomonas aeruginosa PAO1, and SGD for yeast Saccharomyces cerevisiae (listed in Table 3.5).

3.5.5

Human Microbiome Databases

In this section, we have emphasized only human microbiome resources. To avoid repetition, IMG/M and KEGG Metagenomes databases have not been mentioned here, and, for reference, you can find them in the global genomic resource group. The Animal Microbiome Database (AMDB) (Yang et al. 2022) aims to assist in studying the relationship between gut microbiota and animal hosts with the help of a 16 rRNA gene profile. The Expanded Human Oral Microbiome Database (eHOMD) (Escapa et al. 2018) is specialized for human oral and aerodigestive tract bacterial microbiota. They have accumulated information on their taxonomy, genome, and proteome and tools for respective analysis. eHOMD at present is home to 774 oral bacterial species (Escapa et al. 2018). GMrepo (Dai et al. 2022) and gutMEGA (Zhang et al. 2021) is

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Table 3.5 Species-specific genomic databases Sl. no 1.

Name Papillomavirus Episteme (PaVE)

2.

PomBase

3.

Pseudomonas genome DB

4.

Saccharomyces Genome Database (SGD)

URL https://pave. niaid.nih.gov/ index https://www. pombase.org/ https://www. pseudomonas. com/ https://www. yeastgenome. org/

Data type Papillomavirus genome

References Van Doorslaer et al. (2017)

Schizosaccharomyces pombe knowledgebase Pseudomonas aeruginosa PAO1 genome Saccharomyces cerevisiae genome

Harris et al. (2022) Winsor et al. (2016) Cherry et al. (2012)

a gut metagenome repository. GMrepo so far has information for a total of 2747 species of 895 genera (Dai et al. 2022). GIMICA (Tang et al. 2021) is a specialized database as it provides information on host factors that impacts the inhabiting microbiota. The host factors emphasized here are the host genetic factors (HGFs) and the host immune factors (HIFs). MGnify (Mitchell et al. 2020) maintained by EML-EBI is a site offering microbial genome submission, browsing, and analysis tool. Microbiota-Active Substance Interactions database (MASI) (Zeng et al. 2021) is just as specialized as GIMICA; the only difference is here they have information on the interaction of human microbiota with active substances, particularly those with those substances having therapeutical significance. mBodyMap (Jin et al. 2022) has curated and accumulated extensive information on human microbiota. MicrobiomeDB (Oliveira et al. 2018) is one such standalone web portal for all microbiome-related experiments/studies and associated metadata. The National Microbiome Data Collaborative (NMDC) (Wood-Charlson et al. 2020) is a platform for microbiome data exploration presently dissipating microbial metagenomic, metatranscriptomic, proteomic, metabolomic, and environmental information (Table 3.6).

3.6

Bioinformatic Tools for Genomic Analysis

Genomic sequence of an organism can be used for a variety of studies from assembling, predicting genes, and annotating to comparative studies. Figure 3.3 is showing a comprehensive and simplified workflow for microbial genomic analysis. Depending on the interest of the research, the workflow will deviate accordingly. For every section of the workflow, there is a plethora of bioinformatic tools available. To provide an idea of the myriad of tools, a few are listed in Table 3.7.

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Table 3.6 Human microbiome databases Sl. no 1.

Name Animal Microbiome Database (AMDB)

URL http://leb.snu.ac.kr/ amdb

2.

AMADIS

3.

4.

Expanded Human Oral Microbiome Database (eHOMD) GIMICA

http://gift2disease. net/GIFTED/ https://www.homd. org/

5.

GMrepo

7.

GutMega

8.

Magnify

https://www.ebi.ac. uk/metagenomics/

Microbiome

9.

https://www. madet.info/

11.

Microbiomics of Anticancer Drug Efficacy and Toxicity (MADET) Microbiota-Active Substance Interactions database (MASI) mBodyMap

Microbiota and cancer therapeutic drug association Interaction of human microbiota with active substances Human microbiota

12.

MicrobiomeDB

13.

National Microbiome Data Collaborative (NMDC)

10.

http://gimica. idrblab.net/ttd/ https://gmrepo. humangut.info/ home http://gutmega. omicsbio.info/

http://www. aiddlab.com/ MASI/ https://mbodymap. microbiome.cloud/ #/mbodymap https:// microbiomedb.org/ mbio/app https://data. microbiomedata. org/

Data type Bacterial 16 rRNA gene profile of animal gut microbiota Human microbiota and associated disease Human oral and aerodigestive tract bacterial microbiota HGFs’ and HIFs’ impact on inhabiting microbiota Human gut microbiota

References Yang et al. (2022)

Gut metagenome

Zhang et al. (2021) Mitchell et al. (2020) Zhang et al. (2022) Zeng et al. (2021)

Microbiome studies and associated metadata Microbiome knowledgebase

Li et al. (2021) Escapa et al. (2018) Tang et al. (2021) Dai et al. (2022)

Jin et al. (2022) Oliveira et al. (2018) WoodCharlson et al. (2020)

Apart from the above, even most of the databases mentioned in the previous segment have tools of their own. To name some Ensembl Variant Effect Predictor (VEP), NCBI BLAST, and several others. BIOWULF, a web portal maintained by NIH high-performance computing staff, has a catalog of sequence analysis scientific tools, algorithms, pipelines, and software packages with description for easy

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Retrieval of sequenced genomic data from genomic repositories

Raw Reads Quality Control Data Preprocessing Trimming Genome Assembly

Assembly Validation Structural Annotation Functional Annotation

Genome Annotation Genome Visualization Comparative Genomic analysis

Fig. 3.3 Simplified genomic workflow

understanding and access (HPC NIH 2022). KBase web portal also has a dedicated app catalog to read processing, assembly, annotation, metabolic modeling to expression, and numerous more (Arkin et al. 2018). According to the necessity of the research, the source and size of the reads different tools have to be utilized respectively. A pipeline is a series of connected analyzing tools where the output of one becomes the input for the other to produce a desirable result. Among the myriad of pipelines developed here, we have listed a few commonly used command-based as well as automated pipelines for different genomic analyses along with their references (Table 3.8).

3.7

Conclusion

Bioinformatics is still evolving at a great pace and so are the associated databases and tools. Nonetheless, in this chapter, we have attempted to compile and assemble to the full extent the databases and bioinformatic tools available and utilized for

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Table 3.7 Bioinformatic tools for microbial genome analysis Function Read quality and preprocessing

Tool FASTQC

Alignment

Trimmomatic MultiQC Burrows-wheeler aligner (BWA) Bowtie2

Genome assembler Assembly quality validator Codding gene annotation tRNA, rRNA, and noncoding RNA Plasmid profiling Antimicrobial resistance profiling Virulence gene detection MLST (multilocus sequence typing)

MUMmer SPAdes Velvet QUAST Glimmer GeneMark tRNAscan RNAmmer Infernal Staramr ResFinder AMRFinder Abricate stringMLST SRST

References Babraham Bioinformatics (2022) Bolger et al. (2014) Ewels et al. (2016) Li and Durbin (2009) Langmead and Salzberg (2012) Marcais et al. (2018) Prjibelski et al. (2020) Zerbino and Birney (2008) Gurevich et al. (2013) Kelley et al. (2012) Besemer et al. (2001) Chan and Lowe (2019) Lagesen et al. (2007) EddyRivasLab (2022) Bharat et al. (2022) Bortolaia et al. (2020) Feldgarden et al. (2019) Tseemann (2022) Gupta et al. (2017) Inouye et al. (2012)

microbial and human microbiome studies. A total of 36 databases categorized into five groups have been tabulated along with URL, brief description, and references to assist the reader in understanding which database suits their work the best. Most database among the above mentioned are genome browser equipped with tools for data submission and analysis. Equivalently multifarious analytical tools have developed parallelly. A few popularly used microbial genome analysis tools have been mentioned here. These tools can be used in various combinations to serve the purpose of different researches. As such emphasis was given to already developed and cited pipeline used for different genomic analyses. We have listed few pipelines along with reference that shall help in understanding the workflow and integrated tools serving different functions. This chapter shall aid as an introduction to microbial databases and analytic strategies.

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Table 3.8 A list of different pipelines for multitudinous microbial genomic analysis Sl. no 1.

Description Microbial genome assembly pipeline

References Coil et al. (2015)

3.

Pipeline A5-Miseq (Andrew and Aaron’s awesome assembly pipeline) Genome assembly + annotation pipeline (GAAP) Unicycler

Microbial genome assembly and annotation pipeline Bacterial genome assembly pipeline

4.

MOCAT2

Metagenome analysis pipeline

5.

Bacterial genome annotation pipeline

6. 7.

DIYA (do-it-yourself annotator) Funannotate (fungi) IMG annotation pipeline

Kong et al. (2019) Wick et al. (2017) (Kultima et al. 2016) Stewart et al. (2009)

8.

MAKER

9.

Microbe annotator

10.

NCBI prokaryotic genome annotation pipeline (PGAP) Prokka (rapid prokaryotic genome annotation) RAST (rapid annotation using subsystem technology) JCVI viral genome ORF reader pipeline (VIGOR) GATK variant calling pipeline

2.

11. 12.

13. 14.

Fungal genome annotation pipeline DOE JGI’S IMG microbial genome annotation pipeline Genome annotation pipeline Metabolic annotation of microbial genome Prokaryote genome annotation pipeline Prokaryote genome annotation pipeline Prokaryote genome annotation pipeline

Viral genome annotation pipeline Variant calling

15.

DeepVariant

Variant calling

16.

rMAP (rapid microbial analysis pipeline)

17.

Bactopia

Microbial genome analysis for ESKAPE pathogens (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter species Complete analysis of bacterial genome

18.

BAGEP

19.

ASA3P

Whole bacterial genome analysis pipeline Bacterial genome analysis

20.

MicroPIPE

Bacterial genome assembly

Chen et al. (2019) Campbell et al. (2014) Ruiz-Perez et al. (2021) Tatusova et al. (2016) Seemann (2014) Brettin et al. (2015) Wang et al. (2010) Broad Institute (2022) Poplin et al. (2018) Sserwadda and Mboowa (2021)

Petit 3rd and Read (2020) Olawoye et al. (2020) Schwengers et al. (2020) Murigneux et al. (2021) (continued)

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Table 3.8 (continued) Sl. no 21.

Pipeline TORMES

22.

BacPipe

23.

Multiphate2

24.

FunGene

25.

SNVPhyl

26.

Snippy

Single nucleotide variant phylogenomics pipeline for microbe SNP pipeline

27.

NASP

SNP pipeline

28.

antiSMASH

29.

De novo genome analysis pipeline (DeNoGAP)

Bacterial and fungal secondary metabolite genome mining pipeline Comparative prokaryotic genomic studies

30.

MetaSanity

Description Whole bacterial genome analysis pipeline Whole bacterial genome analysis pipeline Functional annotation and phage genome comparison Functional gene pipeline

Microbial genome annotation and evaluation pipeline

References Quijada et al. (2019) Xavier et al. (2020) Ecale Zhou et al. (2021) Fish et al. (2013) Petkau et al. (2017) Seemann (2022) Sahl et al. (2016) Blin et al. (2021) Thakur and Guttman (2016) Neely et al. (2020)

Acknowledgment We would like to acknowledge the Department of MBBT, Tezpur Central University, for providing computational facilities.

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CRISPR/Cas9 System: An Advanced Approach for the Improvement of Industrially Important Microorganisms Sharmilee Sarkar, Mohit Yadav, and Aditya Kumar

Abstract

Genetic engineering is a complex process that refines genes at the molecular level. The field of industrial biotechnology relies much on genetic engineering techniques which have played a significant role in the improvement of this area during the past few years. The discovery and characterization of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has brought a breakthrough in genome-editing techniques owing to its high versatility and efficiency. CRISPR/Cas systems are bacterial and archaeal adaptive immune defense systems repurposed as versatile genetic editing tools in a broad range of microorganisms. The technique has potential for numerous microbial engineering applications, including microbial strain typing, autoimmunity, culture immunization, and engineering metabolic pathways for improved synthesis of biochemicals. At present, the RNA-guided endonuclease CRISPR/Cas9 (CRISPR-associated protein 9) approach is also used to engineer various microbes for industrial applications. This chapter focuses on the recent trends of CRISPR/Cas9 approach in editing the genome of industrially important microorganisms for producing various value-added chemicals and compounds. Keywords

CRISPR/Cas9 system · Genetic engineering · Microorganisms · Industrial biotechnology · Genome editing

S. Sarkar · M. Yadav · A. Kumar (✉) Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_4

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S. Sarkar et al.

An Introduction to Industrial Microbiology

The utilization of microbial strains to obtain a product of economic value constitutes the idea of industrial microbiology (Shi et al. 2022). The most common microorganisms used in industrial microbiology include prokaryotes such as various species of bacteria as well as fungi (yeasts and molds). Industrial microbes are metabolic specialists which are capable of synthesizing one/more industrially relevant products in high yield. Microbiologists often use classical genetic strategies for the selection of high-yielding microbial strains. Novel genetic methods which include metabolic engineering and combinatorial biosynthesis along with their modifications contribute vastly to the development of enhanced industrial processes. The goal is to obtain industrially important products in high yields so that an economically feasible process is possible. The nature of the actual production strain following alteration is very different from the original wild-type version. The microbial product may be microbial metabolites, intracellular/extracellular enzymes, and chemicals produced on utilization of the medium components or the added substrate. Processes such as molecular breeding, combinatorial biosynthesis, and metabolic engineering play a significant role in the improvement of industrial processes. Genome-based strain reconstruction strategy is used for the creation of a better strain with mutated genes which are essential for hyperproduction of desired end products but not accumulating random mutations from brute-force mutagenesis and screening (Adrio and Demain 2010). The directional increment in product formulation by modifying specific biochemical reactions or introducing new pathways through the RDT process is termed as metabolic engineering. Utilizing multiple analytical techniques, fluxes are quantified as well as controlled with molecular biology techniques. Reverse metabolic engineering is an alternate approach which (1) selects a strain with an advantageous cellular phenotype; (2) assesses and identifies the genetic, environmental, and/or other factors that have an impact on the phenotype; and (3) changes the genetic and environmental elements directly to transmit that phenotype to another strain. Molecular breeding techniques mimic enabling in vitro homologous recombination to promote natural recombination. DNA recombination and point mutations are introduced at a controlled rate by the process of DNA shuffling. This process of gathering and recombining pieces of comparable genes from many animals has produced significant advancements within a short amount of time. By combining the benefits of multi-parental crossing and whole genome recombination, shuffling of the entire genome is a novel method for strain enhancement.

4.2

CRISPR/Cas System: An Introductory Overview

The idea of CRISPR was first brought to notice in 1987, when the team from Japan led by Ishino was focusing on the iap gene, which codes for the alkaline phosphatase gene in Escherichia coli. The group found out certain repeats in the DNAs of the

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bacterial genome which were not similar to other regular sequences in the genome (Barrangou 2013). These recurring DNA sequences possibly could be the parts of reoccurring DNA sequences today popularly named as “clustered regularly interspaced short palindromic repeats” (CRISPR). In CRISPR systems, small DNA repeats are followed by segments of short length spacers that are acquired through the typical bacterial route via a bacteriophage or plasmid (Hille and Charpentier 2016). The repetitions found in the genome are related to nucleases and helicases where specific DNA sequences are unwinded (Adiego-Pérez et al. 2019; Barrangou 2013). The American scientist Jennifer A. Doudna and the French scientist Emmanuelle Charpentier shared the 2020 Nobel Prize in Chemistry for developing the gene scissor: CRISPR/Cas (CRISPR-associated systems). This system can specifically recognize and excise DNA, a method now widely used for the precise editing of microbial, plant, and animal DNA. CRISPR/Cas (henceforth abbreviated here in this text as CRCa) system in prokaryotic genomes is the adaptive immune system which exists in prokaryotes, mostly archaea and bacteria and to prevent their infection by nonhost genetic elements such as phages, viruses, etc. The efficient expression of guide RNA constitutes an integral part of the CRISPR system since the guide RNA consists of spacer sequence which is indispensable for the target DNA binding. This helps in editing the loci and is associated with both on-target and off-target effectiveness. A CRCa system normally expresses one or more single guide RNAs (sgRNAs). In some instances, CRISPR repeat-spacers are translated later to yield CRISPR-RNA (crRNA), trans-activating CRISPR-RNA (tracrRNA), and a group of CRISPR-associated (Cas) genes that code for the Cas protein, which together make up the CRCa system (Haurwitz et al. 2010; Jinek et al. 2012; Charpentier and Dounda 2013). The CRISPR system requires careful gRNA design, because a well-designed gRNA minimizes the possibility of off-target effects (DSBs induced by CRISPR at desired regions of the genome) and maximizes editing efficiency at the targeted location (on-target activity) (Ryan et al. 2018; Yin et al. 2018). Different types of web tools and algorithms are available for the designing of gRNA in various species. Guidelines for gRNA scoring include self-complementarity, probable binding sites with mismatches in the spacer sequence, GC content, and the presence of polyT. Designing gRNAs for both gene suppression and activation is made easier with the aid of CRISPR-ERA and CHOPCHOP (Liu et al. 2015b; Labun et al. 2019). The following table (Table 4.1) enlists a handful of websites for gRNA design in different species. Apart from Cas proteins, protospacer adjacent motifs (PAMs) are essential elements of the CRCa system. PAMs are brief (two to six base pairs, bp) sequences that are found around the Cas protein-targeted sequences in the microbial genome (Gleditzsch et al. 2019). On invasion of the prokaryotic system with exogenous DNA, these foreign genetic elements can be excised into small fragments by the Cas protein. The Cas enzymes recognize PAMs and are not able to cleave DNA unless when a PAM sequence is present. PAMs ensure that only external DNA is cleaved and not CRISPR arrays. The Cas proteins cut foreign DNA into pieces that

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Table 4.1 Websites used for the design of gRNA Name CHOPCHOP v3 E-CRISPR

Organism More than 200 genomes More than 50 genomes ATUM H. sapiens sapiens M. musculus S. cerevisiae E. coli A. thaliana CRISPRdirect More than 200 species CRISPRH. sapiens sapiens ERA M. musculus R. rattus D. rerio C. elegans E. coli D. melanogaster B. subtilis S. cerevisiae CC TOP 102 species

Function References Nanopore enrichment, activation, Labun et al. (2019) repression, knockout, and knockin Pair of designs, a single design Heigwer et al. (2014)

Naito et al. (2015) Gene repression, activation, and editing

Liu et al. (2015a)

Off-target prediction using gRNA

Stemmer et al. (2015)

are subsequently incorporated as new spacers into the CRISPR array. If this same foreign DNA infects the host system again, it is recognized by the crRNA which in turn pairs with the freshly invading DNA, guiding the Cas nuclease to the target sequences, which thus protects the host.

4.3

Classification of the CRISPR/Cas Systems

CRCa systems showcase high diversity, which include core genes rendered by different CRCa variations, structure of the chromosomal locus, and the gene architecture along with the original sequences (Makarova et al. 2020). Two categories (class 1 and class 2), six kinds (types I through VI), and a number of subtypes can be used to categorize the CRCa systems. Under the class, multi-Cas nuclease effector complexes are categorized, and a single effector protein is classified under class 2 system. Transcripts for Cas1, Cas2, Cas5, Cas7, and Cas6 types are present in type I Cas system. Big and small subunits make up the cascade complex, which is encoded for by Cas1 and Cas2. The processing of the pre-crRNA transcripts is aided by the remaining Cas proteins, Cas5, Cas6, and Cas7 (Liu et al. 2020) (Fig. 4.1). Type I Cas system has six subgenotypes, I-A through I-F, each of which has a distinct gene and functional organism. Type II CRCa system comprises the Cas9 gene, encoding for and controlling the functions of the cascade complex via a multidomain protein. The Cas9 protein has

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Fig. 4.1 Diagram illustrating the various types of CRISPR system. (a) When the PAM sequence (NGG) is present in type II Cas9 system, the targeting effect of sgRNA is used to drive the Cas9 protein to cleave both the complementary and noncomplementary strands, resulting in the development of a blunt-ended nick. (b) The operating system for type V is the Cas12a protein. When the PAM sequence (NTTT) is present, the targeting action of crRNA is used to signal the Cas12a protein to cleave both the complementary and noncomplementary strands, creating a sticky-ended nick. (c) When the PAM sequence is present in type I Cas system, the Cas3 protein is directed by the targeting activity of crRNA to cleave the noncomplementary strand, leaving a large gap. (d) Without the aid of a PAM sequence, the targeting function of sgRNA is used in type III Cas system to instruct the Csm protein to cleave the noncomplementary strand, resulting in the creation of short nucleic acid fragments. The scissors symbolize the cleavage site of nucleases, the green transverse U represents sgRNA or crRNA, and the red nucleotide sequences stand for the PAM sequence

six domains, including the REC I and II, Bridge Helix, PAM-associating, HNH, and RuvC. The primary subunit of this protein complex is Rec-I which is responsible for binding to the RNA. The REC II domain’s function is not yet known. When the target DNA is bound, the cleavage process is started in the part of the linked helix that is rich in arginine. Target DNA binding requires the PAM-associating region. The ssDNA cleavage process is efficiently catalyzed by the nucleases found in the HNH and RuvC domains (Qi et al. 2013). There are three subtypes of type II CRCa system: IIA, IIB, and IIC. Type III CRCa system has Cas10 as the main gene which encodes for a protein similar to the multidomain protein used by PolBcyclases and polymerases. While encoding for Cas1 and Cas2 proteins, type III CRISPR/Cas system utilizes crRNAs which are type I or type II CRCa system linked. Type IV CRCa system lacks both Cas1 and Cas2 proteins but exists with the plasmid genome of numerous bacteria which is not commonly linked to CRISPR arrays. Type V CRISPR/Cas12 system was created especially for genome editing uses including gene activation, gene repression, and epigenomic editing, large-scale genome screening, and others. Lastly, handling RNA requires type VI CRISPR/Cas13 system in terms of RNA interference (RNAi), RNA visualization in vivo, and detection of nucleic acid (Fig. 4.2).

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Fig. 4.2 List of several CRISPR/Cas system configurations

Table 4.2 Classification of the CRISPR/Cas systems Class 1 1 1 2 2 2 2 2 2 2 2 2 2 2

Type I III IV II II II II V V V VI VI VI VI

Subtype A, B, C, D, E, F, U A, B, C, D A, B A A B C A B C A B C D

Effector Cascade Cascade Cascade SpCas9 SaCas9 FnCas9 NmCas9 Cas12a Cas12b Cas12c Cas13a Cas13b Cas13c Cas13d

Target dsDNA ssRNA dsDNA dsDNA dsDNA dsDNA/ssRNA dsDNA dsDNA dsDNA dsDNA ssRNA ssRNA ssRNA ssRNA

Requirement of tracrRNA – – – Yes Yes Yes Yes – Yes Yes – – – –

The classification of the CRCa systems is summarized in Table 4.2. The distribution of CRCa systems is nonuniform among bacterial and archaeal genomes. CRCa loci are found majorly in archaea (85.2%), including almost all hyperthermophiles; however, only 40% of bacterial genomes contain CRCa systems (statistics based on the study of 13,116 genomes containing bacteria and archaea).

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The manufacture of microbe-based metabolic products offers a feasible path to a continuous future. Presently, various microbial strains are frequently utilized to create a range of metabolic products which are essential for various industries like food, pharma, and others. Genome editing is widely used for the increment in the yield of metabolic products. Genomic modification is a genetic alteration type where single DNA bases are influenced by the addition, removal, or alteration of the microbial genome. However, most microbial strains showcase The main barrier is difficulties in their genetic regulation in the metabolic engineering process. Conventional techniques such as the transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have been widely used for the genetic moderation of microbial genomes. Both these genetic alterations are based on the fundamental understanding of DNA-protein interaction. ZFNs are engineered proteins which have two domains: a DNA cleaving domain and a synthetic zinc finger DNA binding domain which bind to the desired DNA. ZFNs were withdrawn owing to limitations like the technique being time-consuming and high cost of production of the target enzymes and low specificity. TALEN is a second type of gene-editing technology which replaced ZFNs. TALEN consists of repeats of DNA sequences which facilitate in vivo homologous recombination (Bikard et al. 2013; Gaj et al. 2013). Howsoever, these two methods are hindered by the need to create a unique pair of nucleases for every target genome. Also, neither can successfully target several genes simultaneously. Hence, CRCa system for genomic editing is a new method that enables targeting multiple genes simultaneously to create better, industrially significant microbial strains.

4.4

CRISPR/Cas9 System

The CRISPR/Cas9 (CRCa9) type II system is constructed from the Gram-positive bacterium Streptococcus pyogenes (SpCas9) and is one of the most used categories among the CRISPR/Cas systems (Ran et al. 2013). The CRCa9 system consists of a single guide RNA and RNA-guided Cas9 endonuclease. A guide RNA is a combination of crRNA and tracrRNA. Cas9 is a 160 kDa protein whose HNH and RuvC, two nuclease domains, break the target DNA single strand (Lei et al. 2017). This Cas9 protein and a guide RNA make up the Cas9 complex of ribonucleoproteins which attaches to the target DNA before cleaving it. Also, the PAM sequence is essential for the binding of Cas9 protein to the target DNA (Fig. 4.3). A double-strand break (DSB), which can be repaired via either the HDR (homology-directed repair) pathway or the NHEJ (nonhomologous end joining) pathway, is created during the genome-editing process by recruiting the Cas9 endonuclease to a genome-specific location (Fu et al. 2014) (Fig. 4.4).

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Fig. 4.3 Schematic of the gRNA-Cas9 complex. An illustration of the interaction between the Cas9 protein and the CRISPR gRNA to target endonuclease activity close to the PAM sequence

Fig. 4.4 The mechanism of genome editing in prokaryotes. Both the nonhomologous end joining (NHEJ) route and the homology-directed repair (HDR) pathway can repair double-strand breaks caused by nucleases

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Role of CRISPR/Cas9 in Improvement of Industrially Important Microorganisms

In industrial microbiology, the majorly used microorganisms include fungi, yeasts, and prokaryotes which include species of the bacteria belonging to the genus Streptomyces. Industrial microorganisms are also known as metabolic specialists which are capable of synthesizing product(s) in high yield. Industrial biotechnology relies on the techniques of introduction of the foreign gene for the engineering of new pathways for the biosynthesis of engineered products. CRCa9 offers the advantage of introduction of DSBs in the genome over other conventional genome-editing techniques which increases the recombination rate when used with appropriate donor DNA molecule. The CRCa9 technique has evolved as a novel tool for engineering microbial cells to enhance biofuel and inhibitor tolerance and also enhance the production of modifying enzymes such as cellulases and hemicelluloses. Several microorganisms are well known for their ability of fermenting sugars to alcohols on an industrial scale. Such microbes include yeasts such as Saccharomyces cerevisiae and bacteria such as T. brockii, C. thermosaccharolyticum, C. thermohydrosulfuricum, and additional species. Site-specific editing techniques include REM (RNA-guided endonuclease-mediated) and MEM (modified endonuclease-mediated) for the genome which are presently being applied for microbial strain editing and improvement. The CRCa9 system is a well-known example of a REM-based method for genome editing that directs Cas9 to a specific genomic sequence using a guide RNA. This RNA-guided genetic engineering technique is regarded as one of the most recent developments in biology and has numerous creative applications in the production of biofuels (Javed et al. 2019).

4.6

CRISPR/Cas9 Applications in Bacteria

Escherichia coli is frequently employed in industrial settings to produce a range of significant compounds and pharmaceuticals. Traditional methods of knocking out genes in E. coli were inefficient and unsuitable for recombination at multiple sites. For improving the efficiency of genomic engineering, a tri-plasmid system was constructed which led to improvement in the genetic modification efficiency and contributed to development in the field of industrial biology. In earlier research, two distinct plasmids were used to produce the Cas9 enzyme and gRNA as opposed to both being expressed simultaneously as it would burden the metabolism of the organism and lead to cell death. Thus, the Cas9/gRNA repression is necessary before the editing of the genome. It is possible to combine Cas9 and gRNA into a single plasmid that has the pBAD promoter. Glucose acts as a repressor for this cassette, and a temperature-sensitive replicon called repA101ts and arabinose act as inducers. Thus, transformed E. coli is able to grow on plates with varying glucose levels and can be induced by arabinose. Because only a single generated plasmid and transformation process were required for many loci, this genome-editing approach

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can be used continually (Zhang et al. 2020). For further improving the efficiency of editing at multiple loci, a CRCa9-facilitated genome-editing method was developed. This approach was gradually implemented to improve the central carbon metabolism and boost the MEP pathway. In fed-batch fermentation, the strain with the highest production yielded 2.0 g/L of β-carotene (Li et al. 2015). Apart from E. coli, CRISPR/Cas-based genetic engineering has also yielded satisfactory performances in other bacteria. Genetic engineering using CRCa9 for Clostridium saccharoperbutylacetonicum N1–4 was developed which improved the genetic engineering efficacy from 20% to 75% (Wang et al. 2017). This was achieved by selection of the PJ23119 promoter for the gRNA expression, from E. coli. The greatest reported quantities of butanol from batch fermentations were produced in Clostridium saccharoperbutylacetonicum N1–4 after deletion of two key genes for phosphotransacetylase (which produces acetate) and butyrate kinase (which produces butyrate). Additionally, editing tools based on CRCa9 have been used to produce bulk compounds like succinate from Synechococcus elongatus, γ-amino-butyric acid (GABA) from Corynebacterium glutamicum, and others (Liu et al. 2017; Ferreira et al. 2018; Liu et al. 2019). This frequent and extensive use of the CRCa9 system in numerous bacterial species highlights how crucial it was for the emergence of the bioindustries.

4.7

CRISPR/Cas9 Applications in Yeasts

Yeasts are essential to industrial biology because they can produce a wide range of materials, including biopharmaceuticals, biocatalysts, biofuels, and other goods. They are strong candidates for cultivation in difficult growth conditions, like low pH and high temperatures, owing to their robust physiology. Additionally, yeasts enable the introduction of posttranslational modifications, which are necessary for the generation of biochemicals but are absent in bacterial host systems. To date, various genetic modification tools based on CRCa9 have been utilized in yeast to improve the efficacy of genomic moderation. Since long Saccharomyces cerevisiae has been extensively studied model organism for research and application. For testing the efficacy of the CRCa9 system in S. cerevisiae, CAN1 which encodes for the arginine permease was chosen as a target gene. Designing ds oligonucleotides with an internal stop codon and sequences that are similar to the target region improved the efficacy of gene knockout. The frequency of recombinants selected from canavanine (an arginine analogue lethal to cells)-supplemented medium carrying a functional CAN1 gene was nearly 100%, which led to the basis for powerful and simple genome-editing tools in yeasts. With the aid of this approach, numerous genes in the yeast S. cerevisiae ATCC 4124, including LEU2, TRP1, URA3, and HIS3, were knocked out with an efficiency of up to 60%. Additionally, employing this genome-editing method, a strain with mutations in the LEU2, TRP1, URA3, and HIS3 genes was successfully created (Zhang et al. 2020). The USER cloning technology has been employed for assembling of multiple sgRNAs in a single plasmid for smooth disruption of the

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target gene and thus improves the efficiency of CRCa system. Using this genome engineering approach, efficiencies ranging from 50% to 100% were achieved. The CRCa9 method is additionally utilized to facilitate gene insertion using donor DNA that is homologous to the target gene. To protect the 5′ end of sgRNA from the activity of the 5′ exonucleases, the HDV ribozyme was introduced at the 5′ end of the sgRNA, allowing for precise cellular level regulation of appropriately folded sgRNA. Yeasts had the genes for endogenous glucosidase (gh1–1) and heterologous cellodextrin transporter (cdt-1) introduced using this HDV-gRNA hybrid expression system, which dramatically improved the efficiency of multiplex genome editing. Due to SDM of the cdt-1 and gh1–1 genes using the CRCa9 system, the cellobiose utilization efficiency increased by a factor of ten. It is possible to introduce the genes for xylose reductase, xylitol dehydrogenase, and xylulokinase (XYL1, XYL2, and XYL3) into the PHO13 and ALD6 loci in the yeast S. cerevisiae through the CRCa9 system. The reengineered strains gained the ability to utilize xylose and could serve for large-scale fermentations. Another genetic engineering method, Cas9-facilitated multiple loci integration of in vivo assembled DNA parts (CasEMBLR), was created with a 31% engineering efficiency to assemble the ADE2, HIS3, and URA3 loci with CrtYB, CrtI, and CrtE gene expression cassettes (Jakočiunas et al. 2015). Other industrial yeasts, including Schizosaccharomyces pombe, have employed the CRCa9 approach to establish an expression cassette between the sgRNA sequence and the promoter by including the leader RNA from the rrk1 gene. To carry out 3′ sgRNA processing, the HH ribozymes were fused with the mature sgRNA’s 3′ end. When a donor template was co-transformed, this system in turn produced a high efficiency (98%) result.

4.8

CRISPR/Cas9 Applications in Fungi

Considering the enormous economic value of their products, filamentous fungus is used to create several industrially significant compounds and metabolites, including PUFA, organic acids, and antibiotics (Liu et al. 2015b; Nødvig et al. 2015). The extension of genome engineering techniques in filamentous fungi was a challenging task due to the scarcity of plasmids and promoters which could deliver them through the fungal cell wall. Additionally, ineffective editing hindered the continued use of filamentous fungus in the sector and the fact that it was a labor-intensive procedure. In the instance of filamentous fungus, the new CRCa9 resulted in a breakthrough in the realm of genetic manipulation. Deletion of KU70 or KU80 heterodimer is the widely used traditional genetic engineering strategy to improve the specificity of homologous recombination (Weld et al. 2006; Koh et al. 2014). However, interruption of KU70 or KU80 results in the sensitivity of filamentous fungi to growth circumstances with precisely specified chemical needs, such as phleomycin and bleomycin (Liu et al. 2015b). To overcome such hindrance, the CRCa9 system was applied in T. reesei RNA transcription in vitro and a specific, optimized codon (Zou et al. 2021). A microhomologymediated joining of ends method in Aspergillus fumigatus was also developed

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based on CRCa9. Through this, target-gene-editing accuracy was achieved with a 95–100% efficiency rate (Zhang et al. 2016). This system is also applicable in different fungal species, thus widening the CRCa9 system application. Thus, the CRCa9-mediated genome-editing technology can perfectly edit individual target gene as well as perform multigene editing for genetic manipulations in filamentous fungi.

4.9

Applications of CRISPR/Cas9 in Microbes

Utilizing a variety of editing techniques, including gene knockdown, integration, CRISPR interference, editing, etc., the CRCa9 system is capable of altering microbial strains and hence improves the output of industrially significant products (Shi et al. 2022) (Fig. 4.5).

Fig. 4.5 Construction of microbial cell factories for chemical synthesis with the aid of the CRISPR-Cas toolset. (a) Assembling the biosynthetic processes necessary to produce a desired chemical. When several biosynthetic enzymes are combined to form useful metabolic structures, the integrated copy number can be modified simultaneously by picking the appropriate intergenic loci. (b) Metabolic flux optimization for desired chemical synthesis. Genetic manipulation is established using multiplexed and combinatorial methods to balance or improve metabolic flow to the target molecules. (c) Identifying substitute enzymes for a biocatalyst. Direct protein integration and continually targeted in vivo mutagenesis are two methods for producing enzymes with altered or increased activity. Protein variations could be produced via (semi-) rational design or error-prone PCR. In vivo mutagenesis could be achieved by directing modified DNA polymerases (nCas-Pol) to the target loci using CRISPR-guided nickases. (d) There have been significant efforts made to effectively substitute substrates with a higher theoretical yield or utilize renewable substrates. (e) Through the use of functional gene screening, metabolic resilience or tolerance to diverse cell stresses has been enhanced

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Genome Editing

Due to significant development in the genome sequencing domain, researchers have gathered a huge amount of genomic information, which sheds light on the scope for the development of science and technology. Traditional genome-editing technique through the process of homologous recombination restrains their use in various organisms. Present evidences suggest that dsDNA breaks can activate the error-prone NHEJ or HDR at particular gene positions which lays down the basis for the necessity of a technology for genome editing. CRCa9 gene editing has advantages over conventional methods, features with great specificity and efficiency, and a simple design. CRISPR-modulated insertions and deletions are made possible by tracrRNA: crRNA targeting of gene sequences and the addition of donor templates (Xu and Li 2020). The procedure is made simpler when the tracrRNA:crRNA combination is replaced by sgRNA. The CRCa9 D10A approach, which facilitates quick and meticulous in-frame chromosomal deletions and site-specific changes, was created for the genomic engineering of Lactobacillus casei. Cyanobacteria being oligoploid in nature and due to the instability of the introduced gene are limited in their application for their use as cell factories for the production of biofuels and various biochemical products. Li et al. developed CRCa9-mediated simultaneous glgc knockout and gltA/ppc knockin strains and hence modified cyanobacteria to accelerate the production of succinic acid titer to an amount that is 11-fold more when compared to the wild-type counterpart (Li et al. 2016). Feng et al. (2018) created CRCa9-mediated modular assembly-based E. coli multiple genome-editing technology (CMGE) to create microbial cell factories. The modification efficiencies of the second, third, and fourth sites were 100%, 88.3%, and 30%, respectively. A technique for multiple genome editing was created by Zhang et al. of S. cerevisiae, GTR-CRISPR, which has the ability to use effective gRNA which results in the interruption of eight genes simultaneously with efficiency ranging up to 87%. Additionally, lightning GTR-CRISPR system has the ability to knock out six genes in just 3 days (Zhao et al. 2020). The CRCa9 system’s high efficiency and degree of specificity have sped up the development of precise gene-editing methods. NHEJ or HDR can be used to repair DSBs brought on by Cas9. The DSB ends are first processed via the error-prone NHEJ pathway before being recombined by internal DNA repair processes, which leads to alterations at the junctional locations. When plasmid or ssODN, an exogenously supplied repair template, is present, HDR properly delivers alterations at the target location. HDR can be activated by single-stranded nicks in the DNA. When Cas9 transforms into a DNA nickase, the HDR pathway closes the single-strand gap. To improve the CRCa9 system, two Cas9 nickases that target opposite strands of a target site are guided by a pair of sgRNAs in order. The citrate synthase gene’s 5′-UTR sequence has also been precisely edited using the CRCa9 approach in order to lower the level of expression and reroute the carbon from acetoacetyl-CoA to the citric acid cycle (Zhao et al. 2020).

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Transcriptional Control

Besides genome editing, CRCa9 technology also finds its application in the gene expression regulation at both transcriptional and translational levels. A deactivated nuclease inactive Cas9, known as dCas9, binds to specific target sequence of DNA under the control of sgRNA, thus modulating the levels of expression of particular genes. CRISPR interference is the nomenclature of the dCas9 technique used to limit gene expression when sgRNA is the guide (CRISPRi) (Donohoue et al. 2018). In order to stop the transcription process from starting, dCas9 restricts the RNA polymerases’ ability to attach to promoters. It also prevents the RNA polymerases from sliding along the dsDNA, which stops the transcriptional extension phase from occurring. Constitutive expression of endogenous/exogenous genes offers the risk of feedback inhibition which in turn hinders cell growth and leads to cytotoxic effects. Permanent genetic changes, such as gene knockouts, are never the only means to impose metabolic control over a microbe to increase yields of the desired metabolite or end product. Flux can be controlled through transcriptional regulation, which also avoids the drawbacks of gene overexpression. In this regard, transcriptional regulation based on CRISPR interference (CRISPRi) aids in the adjustment of any gene’s expression level (La Russa and Qi 2015). Mutating key residues in the Cas9 protein (H840A in HNH and D10A in RuvC domains, respectively) gives rise to a catalytically deactivated dCas9 protein which lacks the endonuclease activity; however, dCas9 retains the ability of binding to the nucleic acid target. Thus, CRISPR activation (CRISPRa) is achieved by fusing dCas9 to a transcriptional activation domain. Thus, simultaneous repression and activation of specific genes can be achieved by a combination of CRISPRi/a system (Ryu et al. 2018; Yao et al. 2018; Meaker et al. 2020). For example, CRISPRi is used to target essential microbial genes, which limits the growth of bacteria to repress a polyketide antibiotic expression in S. coelicolor. Additionally, it is employed to discover that gene suppression in S. cerevisiae results in resistance to antifungal drugs due to chemo-genetic interactions. These studies demonstrate how CRISPRi can be used to block metabolic pathways or as an in vivo system for identifying genes involved in biochemical synthesis. When dCas9 is fused to an RNA polymerase (RNAP) complex subunit in E. coli, the remaining RNAP subunit components are effectively recruited, increasing the synthesis of the desired protein product by about 2.5-fold. Bidirectional regulation, a method by which various genes can be simultaneously activated and repressed, has also been achieved with CRISPRi/a systems. The RNA polymerase subunit’s surface and SOX interact which has high conservation among gamma-proteobacteria, alpha-proteobacteria, bacteroides, and Grampositive bacteria (Zhao et al. 2020). The E. coli CRISPRa system can be transferred to other bacteria with a variety of crucial biological activities due to its conservatory nature. It is used in the creation of artificial bacterial cell-based medicinal and commercial biosynthetic systems (Ding et al. 2020).

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Additionally, CRISPRi/a systems have demonstrated to be useful for creating logic gates, which may act as the fundamental components of synthetic biological circuits. Gene circuits control metabolic pathways where the dynamics of genomic components increase the yield of desired products, making them intriguing. Before upregulating a gene, gene circuits can be made to have several inputs, or they can be constructed so that the expression of a single gene downregulates the expression of another gene. Circuit layering creates complex regulatory networks where a single signal can trigger a series of actions. The restricted availability of high specificity transcription factors that regulate the expression of circuit components is one of the main challenges in using gene circuits. This issue can be solved with CRISPRi/a systems because of their customizable targeting, high specificity, and quick binding kinetics. Implementing the CRISPRi/a system in synthesis pathways enables the control of the microbial metabolic flux at a level that has never before been possible. Thus, the CRISPRi/a method for manipulating microbial strains for bioproduction is made easier to apply by the capability to modify endogenous gene expression without promoter modification (Lv et al. 2022). Biofuels, chemicals, and other products can be produced in microbial cell factories. To achieve the highest production, metabolic engineering of these microbes is therefore required (Wu et al. 2020). As a result, altering numerous metabolic engineering target genes, such as those that encode rate-limiting enzymes, critical genes, and pathways that compete, is frequently necessary. However, the development of a conjugative metabolic engineering technique to modify the host genome in a high-throughput manner is the key to producing effective microbial cell factories. A three-function orthogonal CRISPR system serves as the foundation for the combinatorial method known as CRISPR-AID. It combines gene deletion, transcriptional interference, and activation of transcription. Using the CRISPRAID method, beta-carotene production was tripled in a single step. The endoglucanase display on the surface of yeast was boosted by around 2.5 times through the integration and optimization of numerous metabolic engineering target genes (Lian et al. 2017). For the expression of proteins and the creation of pathways, gene expression refinement is a crucial step, but it is difficult due to hierarchical gene regulation at several levels. While co-expressing dCas9 with transcriptional regulators, positionspecific gRNA was developed to activate/repress the expression of a wide variety of different genes. The efficient regulation of target gene expression is made possible by the timing of dCas9 expression. Hierarchical enzyme expression levels were adjusted under dCas9 control in order to research the ideal level of gene expression in the yeast S. cerevisiae. To span an over 40-fold expression range, the glycolytic promoter was changed utilizing the repressor and activator coupled with dCas9. Finally, for the glycerol biosynthesis pathway, this strategy was applied, and the titer increased significantly (Jensen et al. 2017). Due to the rapid reduction in the price of DNA synthesis and the development of parallel sequencing technologies, this approach may also be used in the future to supplement genome-scale metabolic models.

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The demand for genome editing in microorganisms is to develop improved tolerance toward end products of metabolic processes since many biofuel products have antimicrobial properties. Due to a frameshift mutation in the NADH dehydrogenase enzyme gene, random or targeted alterations result in either an increase or a decrease in ethanol level tolerance as well as ethanol production. The fluidity of the bacterial membrane can also be altered by changes to the enzymes that catalyze the conversion of trans-fatty acids to cis-fatty acids, which in turn affects membrane accessibility and tolerance. The CRCa9 genome-editing approach is effective for carrying out these types of genome alterations. CRCa9 gene introduction and deletion has found its place in the genome editing of several industrial microbes since 2013. Cellular repair of CRCa9-modulated DSBs via the NHEJ pathway might lead to frameshift mutations which ultimately results in gene knockouts (KOs). By removing genes that promote undesired catabolism of carbon or its precursor metabolites, these KOs can be exploited to reroute the metabolic flux. For the effective deletion of many genes in bacteria, yeast, and filamentous fungi, multiple sgRNA transformation is used. Some examples of engineered industrial microbes using CRISPR/Cas system are enlisted in Table 4.3: Microorganisms can produce a variety of compounds, some of which are unnatural to the host; therefore, the creation of heterologous pathways is the solution. For the consistent levels of expression of different pathway genes, the chromosome’s heterologous pathways are knocked in with the help of the CRCa system. To produce 5-methylpyrazine-2-carboxylic acid (MPCA), large synthetic pathway genes are integrated into the chromosome of E. coli. By choosing different integration sites, the copy number of integrated xylM and xylA can be changed, which improves MPCA production and results in a higher yield (Gu et al. 2020). Similarly, pathway integration in Pichia kudriavzevii and Zymomonas mobilis leads to the production of itaconic acid and lactate, respectively (Sun et al. 2020; Shen et al. 2019). Integration of two or more genes simultaneously in the microbial chromosome to build multiplex integration platform can lead to the production of betalain or kauniolide. For increasing the copy number of a gene, DSBs can be generated at delta sites through CRCa modification system, and integration up to 25 copies might be achieved for the efficient production of industrial chemicals. Multiplex pathway editing is required to adjust cell metabolism in microorganisms due to the complex regulatory metabolic network in microbes. CRCa9-mediated multiple gene integration system in Kluyveromyces marxianus helped in the optimization of three shikimate pathway genes, namely, ARO4, ARO7, and PHA2 via the creation of a 33 combinatorial library (Li et al. 2021). The creation of effective chemical manufacturing routes relies heavily on enzymes. Naturally occurring enzymes have the drawback of feedback inhibition. CRCa systems possess the ability to integrate proteins of interest into microbial genomes with an efficiency of 98% or greater. The creation of a genome-wide library with sequence deletions and substitutions was made possible by the CRCa9 technology. Increased resistance of yeast mutants to environmental changes, such as higher temperatures and the presence of

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Table 4.3 CRISPR/Cas system-mediated engineered industrial microbes Species A. aculeatus A. carbonarius A. niger A. oryzae B. subtilis

C. beijerinckii

Industrial importance Source as well as producer of enzymes Source as well as producer of enzymes Source as well as producer of enzymes Industrial fermentation Produces low molecular weight substances Catabolizes phenol and formaldehyde Ferments of CO, CO2, and H2 into biofuel ethanol and 2,3-butanediol Produces biofuels

C. glutamicum E. coli

Produces amino acids Common strain for production

K. lactis L. reuteri

P. chrysogenum P. pastoris

Common strain for production Probiotics Biotherapeutic strain Produces industrially relevant lignocellulosic enzymes Produces β-lactam group of antibiotics Common strain for production

S. cerevisiae

Common strain for production

S. thermophilus

Probiotics and industrial fermentation strain Produces industrially relevant secondary metabolites

C. albicans C. autoethanogenum

N. crassa

S. coelicolor

Modifications Mutagenesis Mutagenesis Mutagenesis Mutagenesis Recombination Recombination Recombination Recombination CRISPRi CRISPRi Recombination CRISPRi Multiplexed CRISPRi Multiplexed recombination Recombination Recombination Recombination Multiplex mutagenesis recombination Donor-mediated gene disruption Multiplexed recombination CRISPRa/CRISPRi Engineered immunity Recombination CRISPRi

substances like hydroxyurea and others, has been discovered. Sixty-eight tiny open reading frames (ORFs), which were responsible for the cell’s toughness, were the cause of this resistance. Since a few years ago, highly effective CRCa9-based genomic editing systems have been created. To enable genomic editing, the methods of mutant production introduce one or two dsDNA breaks (DSB) at the target region, which are then repaired by several DSB repair mechanisms. In bacteria, homologous recombination using specially created recombination templates is necessary for precise deletions. Cytosine to thymine (CRISPR-cBEST) and adenosine to guanine (CRISPR-aBEST) base editors, which are very effective genome-editing methods with precision up to single base pair, are included in the highly efficient CRISPR/deaminase-mediated base editors (CRISPR-BEST) for Streptomyces sp. In the CRISPR-BEST system,

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Cas9n serves as the deaminase delivery vehicle. CRISPR-cBEST prefers TC as a substrate over other NC combinations. Similarly, CRISPR-aBEST system prefers TA over any other NC combinations as the substrate for editing (Tong et al. 2019).

4.12

CRISPR/Cas9 Optimization: Improvement of Editing Efficiency

4.12.1 Reduction of Off-Target Effects The selection of sgRNA at a locus with low specificity results in off-target effects, and the spatial control of Cas9 levels is poorly understood. This occurs on the simultaneous expression of Cas9 protein and gRNA on a single plasmid. In the present scenario, multiple CRCa9 optimization schemes have been developed to accelerate the CRCa9 specificity (Zhao et al. 2020).

4.12.1.1 Reduction of Off-Target Effects: sgRNA Design Approach Higher specificity can be attained by adding deoxynucleotides to the CRISPR system to produce RNA/DNA hybrids (Yin et al. 2018). The specificity is increased by chemical changes, such as the introduction of 2′-O-methyl-3′-phosphonoacetate at target locations on the recognition of the DNA sequence of gRNA ribose phosphate (Ryan et al. 2018). Reduction of the CRISPR system’s tolerance for base pair mismatches and elimination or insertion of nucleotides can lower the off-target share. Various algorithm-based tools, such as CRISPR Design and ChopChop, are also based on variables including sequence similarity, the number of mismatches, and the locations of the mismatches. They were created to minimize off-target effects (Hsu et al. 2013; Chapman et al. 2017). The sgRNA lowers the off-target efficiency by selecting a target position where there are no or few homologous genes. Therefore, the gRNA alteration can improve the CRCa9 system’s selectivity. 4.12.1.2 Reduction of Off-Target Effects: Modification in the Cas9 Protein Cas9 transformation can minimize off-target effects. In order to place two copies of Cas9n at two adjacent target sites where the active sites of HNH/Ruv-C are altered, two sgRNAs can be employed. The selectivity of gene targeting may be increased by the concurrently produced ds breaks. When compared to WT Cas9, a single Cas9n is substantially less effective at introducing mutations and can leave gaps in the target DNA. At specific genomic locations, highly frequent insertions and/or deletions could take place; as a result, using two copies of Cas9n:sgRNA to prevent these effects could be beneficial. To get around the problem, Cas9n was switched out for Cas9d, and the active sites of HNH and Ruv-C were altered. The gRNA was coupled with the FokI nuclease to form the RNA-guided FokI-Cas9d nuclease (RFN). To exert its nuclease activity, FokI works as a dimer. Two gRNAs can drive two copies of RFN to nearby locations, improving the specificity and cutting efficiency. This

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will trigger the dimerization of FokI and its nuclease activity (Guilinger et al. 2014; Bolukbasi et al. 2015). The Cas9 nuclease’s non-catalytic REC2 domain plays a critical function in off-target recognition. Single-molecule fluorescence studies between nontarget DNA and the Cas9 protein have helped to understand the conformational kinetics of their interaction and revealed that the REC2 regulates the nontarget DNA strands reshuffling via the positively charged surface residues to perform cleavage reactions. As a result, highly specific Cas9 variations were logically created for the goal of genome editing (Sung et al. 2018).

4.12.2 Reduction of Cas9 Toxicity Effects The CRCa9 system exhibits toxicity in a significant number of microorganisms, which may result in lethal chromosomal breakage, which ultimately may lead to decreased transformation efficiency and gene-editing failure. In the process of editing the genome of Clostridium sp., a severe vector integration event was seen where the inducible production of Cas9 was paired with plasmid-borne editing templates for homologous recombination (Wang et al. 2016). Cas9 toxicity levels can be decreased in two ways: first, by controlling cas9 gene expression and, second, by utilizing prokaryotes’ innate immune systems to combat Cas9 toxicity and poor transformation efficiency.

4.12.2.1 Reduction of Cas9 Toxicity: Regulation of the Cas9 Protein Expression Numerous bacterial species die from Cas9-induced DSBs because the NHEJ pathway is not present. Therefore, controlling Cas9 expression is an important step in the gene-editing process. The Cas9 and gRNA expressing cassettes were redesigned to tolerate Cas9 toxicity and make it easier to quickly and effectively stop gRNA transcription. A CRCa9 genome-editing toolbox was created for S. glutamine. The relatively frequent mutation of Cas9 may be avoided by co-transforming Cas9 and gRNA expression plasmids, enabling the efficient gene deletion and insertion procedure with efficiencies between 60.0% and 62.5% (Liu et al. 2017). 4.12.2.2 Reduction of Cas9 Toxicity: Exploitation of Endogenous CRCa System Numerous bacteria are particularly hazardous to the production of heterologous Cas9; hence, researchers have suggested using the endogenous CRCa system to lessen the toxicity of the CRCa9 system (Charpentier and Dounda 2013; Wang et al. 2013). The endogenous CRCa system is presently being exploited in several bacteria and archaea for genome editing and transcriptional regulation. The endogenous CRCa system’s ability to be transformed with great efficiency was demonstrated. The IB-type CRCa system was successfully reused with 100% efficiency for repeated genome editing in butyric acid-producing bacteria (Zhang et al. 2018). A lactose-inducible promoter was used in place of the leader sequence to control the

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endogenous CRCa system’s expression levels. The 1.7 cfu/mL donor transformation efficiency as a result was achieved. The failure of any plasmid carrying these Cas9 or nCas9 proteins driven by the same inducible promoter to successfully transform any CRISPR/Cas9/nCas9 system is evidence that the implementation of the intrinsic CRCa system could be made possible to avert toxicity caused by the heterologous CRISPR/Cas9/nCas9 system. One of the highest amounts of butanol synthesis ever observed was achieved by the butanol production mutant created by substituting cat1 with alcohol dehydrogenase adhE1/adhE2 gene using the endogenous CRCa system (Zhao et al. 2020).

4.12.3 Optimization of crRNA 4.12.3.1 SOMACA For the creation of crRNA arrays, a synthetic oligonucleotide-mediated assembly approach (SOMACA) was developed, achieving a full efficiency of 100% in the in-frame knockout of double genes, numerous point mutations, and single gene insertion (Wu et al. 2020). The breakdown of N-acetylglucosamine was facilitated by the genes NAGA, NAGB, NAGP, and GAMA as well as by-products of acetic acid and lactic acid synthesizing pathways. SOMACA was utilized to carry out nonsense mutations in these genes, which increased the N-acetylglucosamine titer by 50.9%. 4.12.3.2 Optimization of crRNA Length The genes in C. glutamicum were simultaneously edited and controlled using RE-CRISPR, a dual-purpose clustered regularly spaced short palindromic repeat system. The efficiency of transcriptional repression was examined in relation to crRNA length using the reporter genes GFP, rfp, and lacZ. When compared to crRNAs of other lengths, those between 15 and 16 nucleotides long displayed better repression efficacy. In C. glutamicum, the RE-CRISPR system was used to generate a high cysteine and serine metabolism (Liu et al. 2019). Additionally, the simultaneous application of the RE-CRISPR system resulted in the deletion of AECD and suppression of mcbR, which in turn generated a further rise in cysteine titer of roughly 3.7-fold (Zhao et al. 2020).

4.12.4 Optimization of sgRNA The Cas9 endonuclease is directed to the target location in the genome by the sgRNA’s base pair complementarity in the CRCa9 system. The sgRNA possesses a 20-base-pair target sequence at its 5’end. Following sgRNA genome targeting, Cas9 mediates DSBs. Thus, for the Cas9sgRNA complex to assemble and function properly, careful design of the sgRNA expression cassette is required (Zhao et al. 2020).

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4.12.4.1 Optimization of the sgRNA Promoter The promoter and the architecture of sgRNA are two important considerations to make when creating the sgRNA expression cassette since the efficiency of gene editing is tied to sgRNA promoters. Continuous production of sgRNA is not necessary in either gene knockout or knockdown situations. A suitable sgRNA promoter must be chosen when the system is set up in an organism. For instance, the CRCa9 system in fungi widely uses the promoter for RNA Pol III of the spliceosome U6 snRNA, whereas the U6 promoter is absent in A. niger. Depending on the microbial species, different sgRNA promoters have varying levels of geneediting effectiveness. As a result, A. niger developed a unique CRCa9 system that expresses the sgRNA and is based on one endogenous U6 promoter and two heterologous U6 promoters. The polyketide synthase alba gene in A. niger can be successfully interrupted by all three U6 promoters, and the gene can then be successfully inserted at the target locus using donor DNA of 40 bp. The U6 promoter requires GTPs to begin transcription, which reduces the availability of the CRCa target site. Due to its high conservation and expression in eukaryotes, the 5S rRNA gene is widely employed as a gRNA promoter in the eukaryotic CRCa9 system (Mali et al. 2013; Ranganathan et al. 2014; Liu et al. 2015b; Zheng et al. 2017, 2018a, b). 4.12.4.2 Optimization of the sgRNA Structure For Cas9 endonuclease to function, partly sgRNA must first create a secondary structure. The sgRNA gene contains two variable regions. A 20-bp protospacer makes up one region, and an inverted 6-bp repeating region makes up the other. The first 20 nucleotides of the sgRNA serve as a guide for the target DNA. By adding more nucleotides or altering the 5′ end, the sgRNA’s ability to instruct Cas9 to cleave the nucleotide sequence may be impaired (Haurwitz et al. 2010). Designing an artificial gene that, following transcription, generates a ribozyme-gRNA-ribozyme (RGR) cassette is a standard technique for effective gRNA generation in vitro and in vivo (Gao and Zhao 2014). The two ribozymes on either side of the gRNA aid in its maturation, which can then successfully direct the target gene’s precise cleavage. Additionally, this method has been utilized to increase the effectiveness of gene editing in filamentous fungi (Nødvig et al. 2015). To successfully express genes unique to cells and tissues, a proper promoter must be chosen.

4.12.5 Increase in Recombination Rates Since endogenous recombination system has the drawback of poor efficiency, heterologous recombination system is essential for the application of CRCa system for editing of the genome in microorganisms. Bassalo et al. proposed a recombination technique based on the association of CRISPR and λ-red systems. When the Cas9 gene and the λ-red recombinase were inserted into E. coli at the same time, it was discovered that the Cas9 nuclease can effectively operate in many genomic contexts with the λ-red recombinase. This technique allows for the efficient labeling of a 10 kb gene that encodes for the whole isobutanol synthesis pathway in a single

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day (Bassalo et al. 2016). The metabolic transformation of microorganisms is a quick process, thanks to their exceptional capacity for recombination. Multiple genes in S. glutamine might be successfully knocked off using CRISPR and λ-red (Cho et al. 2017). Thus, in industrially significant microbial strains, this type of genomic engineering speeds up the metabolic engineering process. Donor DNA was co-transformed into E. coli cells along with sgRNAs found in pTargetF using the CRCa9 method to perform genomic alteration (Jiang et al. 2015). With 100% maximum efficiency, this led to genomic modifications such gene deletions and insertions. To increase the recombination efficiency, many genes might be edited simultaneously. The amount of recombination that occurred when homologous sequences were cut down in length or the number of target genes raised was significantly decreased. It is believed that dsDNA transformation in E. coli is not as effective as it may be. The recombination efficiency can be increased by utilizing ssDNA as a donor or by lengthening the homologous sequence since the λ-red recombinase encourages the recombination of more compact ssDNA pieces. The use of CRCa in medical field has become extremely relevant nowadays. CRISPR is now used for diagnostic purposes as well as simple typing of the pathogenic bacterium M. tuberculosis. This has helped researchers to unravel the strategies employed by pathogenic microbes for their transmission by keeping a track of the discrepancies between related strains’ spacer composition. This method has been termed as spacer oligotyping and has been adapted for microbes such as Salmonella enterica, Yersinia pestis, and C. diphtheriae (Hille and Charpentier 2016). CRCa also finds its use in antimicrobial resistance. Targeting the genes responsible for virulence and antibiotic resistance allows artificial CRISPR arrays to eradicate disease-causing bacteria (Gholizadeh et al. 2020). However, this approach exclusively targets pathogenic strains of bacteria, not nonpathogenic types. Genetic engineering’s foundation is the exact insertion of changes into an organism’s genes. The CRCa9 system is typically preferred over ZFNs and TALENs due to its simplicity, efficacy, and ability to address numerous specific locations in the genome.

4.13

Applications of CRISPR/Cas Systems in Gene Therapy

Foreign genes are inserted into target cells during gene therapy in order to treat particular disorders. Somatic cells as well as germ line cells are the two basic categories into which target cells fall. Somatic cell gene therapy is the only type of gene therapy now available because germ line gene therapy raises ethical and security concerns. Either homologous recombination or lentiviral delivery is used in conventional gene therapy. Due to homologous recombination’s ineffectiveness and the random insertions made by lentiviral vectors into the recipient genome, clinical diagnosis may be at risk. CRCa systems are presently used extensively in gene therapy to treat a wide range of infectious and human disorders. Clinical trials for some CRISPR-based genome-editing treatments are now underway. Table 4.4

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Table 4.4 A summary of genome-editing technology-based gene therapy clinical trials S. no. 1. 2. 3. 4. 5. 6. 7. 8.. 9 10. 11. 12. 13. 14.. 15 16. 17. 18. 19. 20. 21. 22. 14.. 15 16. 17. 18.

Diseases HIV HPV-related malignant neoplasm Hemophilia B Mucopolysaccharidosis I Metastatic non-small cell lung cancer Mucopolysaccharidosis II Acute myeloid leukemia HPV-related malignant neoplasm HIV-1 B cell lymphoma Esophageal cancer Transfusion-dependent beta-thalassemia T cell malignancy Sickle cell disease Thalassemia Solid tumor Liposarcoma Multiple myeloma B cell acute lymphoblastic leukemia Acute myeloid leukemia Thalassemia major B cell malignancy CD19+ leukemia Gastrointestinal cancer Multiple myeloma Renal cell carcinoma Advanced hepatocellular carcinoma

Nucleases ZFN ZFN ZFN ZFN CRISPR/Cas9 ZFN TALEN TALEN CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 ZFN CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 CRISR/Cas9 CRISPR/Cas9 TALEN TALEN TALEN CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9

Year 2009, 2015 2016 2016 2016 2016 2017 2017 2017 2017 2017 2017 2018 2018 2018 2018 2018 2018 2019 2019 2019 2019 2019 2019 2020 2020 2020 2020

provides a comprehensive summary of the present genome-editing technologybased gene therapy clinical trials (Mollanoori et al. 2018; Uddin et al. 2020).

4.14

Delivery Methods

A significant limitation of gene therapy is the inability to effectively distribute the CRCa components to the target cells, tissues, and organs for precise genome editing. The best delivery vectors should be biodegradable, highly effective, well targeted, nontoxic, and efficient. Presently, physical, viral, and nonviral approaches are the three main techniques used to administer CRCa components. Electroporation and microinjection are the most straightforward methods of delivering CRCa components. These are quick and effective techniques that can boost gene expression and are frequently used in in vitro research. In addition, lentiviral vectors,

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adenoviruses, and adeno-associated viruses (AAV) are often used for in vitro, ex vivo, and in vivo distribution due to their excellent delivery performance (Xu et al. 2019; Mengstie et al. 2022). A significant problem that needs to be resolved is the safety concern related to viral vectors. Consequently, more study is being done on the utilization of liposomes and nanoparticles at the moment. They are presently emerging as the most effective tools for the delivery of CRCa components due to their benefits of safety, simplicity, and affordability. Since each delivery method has advantages and disadvantages, it is essential to develop a complex of viral and nonviral vectors that maximizes the advantages of both kinds of vectors. Different carriers can be altered using various techniques to improve delivery effectiveness and lessen toxicity. For the distribution of CRCa components, new vectors like graphene and carbon nanomaterials can also be used.

4.15

Conclusion

Despite being discovered roughly 20 years ago, research on CRCa systems is still in its inception, and the area needs to be investigated. Different modes of activity could result from the existence of numerous CRISPR families. Determining the biological roles of CRCa systems is therefore crucial, in addition to choosing the right microorganism model. Genomic characteristics of CRISPR organization have been used for typing. Large-scale industrial milk fermentations exploit the S. thermophilus phage resistance phenotype developed utilizing CRCa technology. The next few years will bring about fresh and intriguing discoveries as well as its applications due to the enormous diversity of CRCa systems, their prevalence in the microbial world, and the knowledge gaps in their mode of action. The ever-expanding knowledge of information related to the genome necessitates the development of tools to construct and then grasp the biological meaning of this genomic data. A revolutionary strategy for enhancing the commercial and industrial production of desired microbes is offered by CRCa. A comprehensive collection of tools is now available for the alteration of the genomes of the most widely used E. coli and S. cerevisiae strains; using CRCa systems in atypical organisms, massive engineering efforts requiring several concurrent insertional events have been completed. Together with other methods already in use, the use of CRCa systems as tools for gene introduction and gene deletion has advanced the production of a wide range of chemicals in a variety of microorganisms. CRCa systems offer a variety of unique methods for RNA-guided interferencebased molecular biology applications. Applications in the industry include strengthening phage resistance to improve microbial culture. The “vaccination” of bacteria against the acquisition of undesirable genetic material, such as antibiotic resistance as well as virulence traits, may also use interference with DNA acquisition. The potential of these systems is highlighted by recent discoveries on the mechanism of CRCa action, which also offer a molecular foundation for the use of the Cas proteins. As a result, in addition to prokaryotic cell lines, the capacity to

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create designed RNA-guided nucleases offers prospects for next-generation editing of the genome.

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Biomedical Application of Industrial Microbiology Komal Bana and Sachin S. Tiwari

Abstract

Recombinant DNA (rDNA) technology advancements over the past few decades have completely changed the field of microbial biotechnology by altering microbial genomes to create cells for the mass manufacture of goods like nutraceuticals and biomedicines. The development of antibiotics and the large-scale manufacture of vaccinations have both been aided by biomedical industrial applications using microbial platforms. Nutraceuticals is a catch-all phrase for pharmaceuticals with bioactive ingredients. Probiotics are made up of bacterial species like Lactobacillus, Bifidobacterium, and others that not only combat harmful bacteria but also generate the body’s vital vitamins (B1, B2, B5, and B6). Novel microbial culture techniques have made it possible to produce in large quantities the bacteria, algae, and yeast that serve as a suitable source of protein in dietary supplements. The manufacture of vaccines today makes use of diseasecausing microorganisms that have been attenuated. This chapter will highlight the positive effects of microbial engineering in healthcare and outline the main areas of pharmaceutical biotechnology development. Keywords

Microbes · Industrial biotechnology · Vaccine · Nutraceuticals · Biopharmaceuticals

K. Bana · S. S. Tiwari (✉) Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_5

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Introduction

The field of microbiology is divided into two broad areas: • Basic microbiology: the study of fundamental properties and nature of microorganisms • Applied microbiology: employing the info learned from basic microbiology to use microorganisms in beneficial ways

5.1.1

Basic Microbiology

Basic microbiology encompasses fundamental knowledge about microbial cells and populations. The subjects of basic research are summarized as follows: • Morphological characteristics: microbes’ size and shape, their internal structures’ function, and chemical composition • Physiological characteristics: specific temperature, pH, osmolarity, and nutrient conditions required for microbes’ growth and reproduction • Genetic characteristics: variability and inheritance of characters during reproduction • Biochemical activities: how microbes obtain energy by breaking down nutrients and utilizing their power to do cellular activities • Ecological characteristics: understanding the relationship between microbes and the environment Microorganisms are easy to manipulate in labs compared with plants and animals. So, they have become the experimental organism of choice in molecular biology. Much of the present-day understanding of mammalian genetics has been learned from the research with microbes because many fundamental processes like replication and translation are the same in all life forms. Combining various laboratory procedures and experiments with cell cultures and animal models, microorganisms have helped explain the nature of multiple diseases such as cancer. Bacteria, such as Escherichia coli, provide clues to life’s metabolic and general characteristics and are considered essential in biological research. Scientists have applied microbes to their important purposes by going beyond the basic microbiology principles.

5.1.2

Applied Microbiology

Microbiology’s practical applications are unlimited in their variety and scope. The marked fields of microbiology include those focusing on the environment or medicine, food, and dairy products. Whether it is a less expensive method to make

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vaccines or a more effective method of producing recombinant proteins, microbiology provides the best solution to the problem. Numerous chemical substances, ranging from vitamins to more sophisticated antibiotics and catalytic enzymes, are produced by microorganisms. Industrial microbiology is the study of large-scale microbial chemical production. Large bioreactors are used to cultivate microorganisms that are collected for use in humans or as animal feed. Some microbes are harvested for human food supplements called single-cell protein (SCP). Pharmaceutical and food industries widely use carbohydrates produced from algae. These inexpensive sources of food and nutrients are very attractive in a world with inadequate food resources. The most dramatic microbiology applications in recent years have been seen in medicine. These discoveries helped health professionals understand, diagnose, and treat previously misunderstood diseases. They came to know that conditions like some types of ulcers and tooth decay are related to microorganisms. Detailed knowledge about microbes leads to better treatments and diagnosis, perhaps even a vaccine against ulcers. Better treatments for diseases like AIDS, Legionnaires’ disease, and Lyme disease have been found through microbiology. Medical microbiology and genetic engineering clubbed together to bacteria releasing enzymes that can dissolve blood clots. Human vaccines are also produced using viruses. Microbiology is also improving the preexisting vaccines and drugs for health betterment. Making transgenic animals with the help of retroviral vectors allows human genes to express in animals. For example, transgenic goats make human antithrombin proteins. Viral vectors are coming as the front edge for gene transfer, and bacterial use in gene cloning is heading the importance of microbiology in the biomedical future. You must know that basic microbiology builds the foundation for microorganisms, and applied microbiology uses the knowledge for humankind’s betterment. Both approaches of microbiology help in understanding the complex world of life. Even if you ignore the presence of microorganisms, they are always with us. Louis Pasteur once said, “The bacteria will have the last word.”

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Products and Processes for Industrial Microbiology

Manipulated microorganisms have been used for making thousands of commercially used essential products ranging from basic food items to vaccines and anticancer therapeutic agents (Von Schwerin et al. 2015). These commercial products are produced by various biological and industrial processes and can be divided into several groups (Table 5.1): (a) Production of pharmaceutical chemicals: This category includes antibiotics and steroid drugs. Some other products are also made using RDT by inserting genes of interest into engineered bacteria, such as insulin, interferons, growth hormones, etc. (b) Production of nutraceuticals: Includes dietary fibers, PUFA, proteins, amino acids, etc. The most frequently used probiotic microorganisms include Lactobacillus, Bifidobacterium, and Enterococcus spp. They aid in proper digestion and improve intestinal health. (c) Production of commercially valuable chemicals: Includes various types of solvents and enzymes that can be extracted from modified microbes in large quantities. (d) Production of vaccines: Microbial cells, their parts, or their metabolic products are used as immunizing agents to prevent disease. Table 5.1 Microbial products used in health sciences Substances Medical products Antibiotics Transformations in steroids Alkaloids Insulin, interferons, human growth hormone, somatostatin Industrial products Ethanol (from glucose) Acetone and butanol 2,3-Butanediol Enzymes Food additives Amino acids (e.g., lysine) Organic acids (citric acid) Nucleotides Vitamins Polysaccharides

Microorganisms Penicillium, Bacillus, Streptomyces Rhizopus, Arthrobacter Claviceps purpurea Escherichia coli, Saccharomyces cerevisiae Saccharomyces cerevisiae Clostridium acetobutylicum Serratia, Enterobacter Aspergillus, Mucor, Trichoderma Corynebacterium glutamicum Aspergillus niger Corynebacterium glutamicum Eremothecium, Blakeslea, Ashbya Xanthomonas

Source: Demain AL. Antibiotics: natural products essential to human health. Med Res Rev. 2009 Nov;29(6):821–42. doi: 10.1002/med.20154. PMID: 19291695 Celedón RS, Díaz LB. Natural Pigments of Bacterial Origin and Their Possible Biomedical Applications. Microorganisms. 2021 Apr 1;9(4):739. doi: 10.3390/ microorganisms9040739. PMID: 33916299; PMCID: PMC8066239

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(e) Production of bio-food supplements: Microbial cells such as bacteria, algae, and yeast may be used as food supplements since they can be quickly grown. (f) Production of metabolites: Microbes produce primary metabolites such as vitamins and metal ions essential for cell growth and secondary metabolic products such as antibiotics.

5.3

Microbiology in Antibiotic Production

Treating disease with chemical compounds is known as chemotherapy. With time due to random discoveries, researchers discovered that microorganisms also release chemotherapeutic agents termed antibiotics. The beginnings of chemotherapy’s modern era were led by the findings of Alexander Fleming, Paul Ehrlich, and Gerhard Domagk.

5.3.1

Fleming and the Discovery of the Antibiotic Penicillin

Natural chemotherapeutic agents are termed antibiotics, a substance produced in small quantities by a microorganism that kills or inhibits the growth of nearby microbes. These substances were known long before their actual discovery. For example, to treat boils, Chinese used moldy soybean curd and molds for treating foot infections. In 1881, John Tyndall described the antimicrobial properties of molds. While experimenting with culture media, he observed that the bacterial population died with mold grew in the culture. Pasteur and Joubert also concluded that

Penicillium colony

antibiotics do exist

Staphylococci undergoing lysis

.

Normal staphylococcal colony

The modern era of antibiotics began in 1927. Alexander Fleming, a microbiologist, surprisingly discovered penicillin. He was working searching for new antibacterial agents against wound infections. So, he grew different bacterial colonies and found that the plate culture of Staphylococcus aureus was contaminated with mold. The bacterial population near the mold was killed, and clear zones appeared around them. Then he started the research on the mold and found that the mold belongs to the genus Penicillium, species Penicillium chrysogenum, which

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produces toxins against S. aureus, a very harmful pathogen. Fleming named the substance penicillin, and it became a new research topic during the discovery of potential chemotherapeutic agents

.

On February 12, 1941, the first clinical trial was done on an oxford policeman dying from staphylococcus infection. The administration of penicillin improved his health, but after 5 days, the penicillin stock was exhausted, and he died.

5.3.2

Commercial Production of Antibiotics

The American team of N.G. Heatley and his colleagues explored the means of largescale penicillin production. In 1941, the US Office of Scientific Research and Development worked on the plan of Heatley. It started producing penicillin in large bioreactors with an average yield of 900 units/mL, which was originally 2 units/mL during Fleming’s culture plates (presently 50,000 units/mL). In 1945, Fleming, Howard W. Florey, and Ernest Chain were awarded a Nobel Prize for their work with penicillin, and, later on, many antibiotics came into the pharma market. Factors responsible for the achievement of successful manufacture of many other antibiotics were the following: 1. Discovery of better penicillin-producing species, P. chrysogenum. 2. Improvements in the culture media and their composition. 3. Development of submerged culture technology. Large-scale production using bioreactors containing large volumes of the culture media (thousands of gallons)

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Fig. 5.1 Drug development process. (a) Microbe collection and making pure cultures (b)Streak plating (c) Growth of bacteria in fermentation vessels followed by concentrating the antibiotic material (d) Determination of antimicrobial spectrum and then toxicity testing (e) Regrowing the selected bacteria in large ferments and then creating vial of purified antibiotics (f) Vials then go for chemical characterisation and clinical trails

and sterile air were forced through the medium; this created the perfect environment for abundant growth (Fig. 5.1) (Decker and Reski 2008). Different kinds of antibiotics based on the chemical structure. • The beta-lactam antibiotics This category comprises penicillins, monobactams, carbapenems, and cephalosporins. The characteristic feature is a four-membered ring called a beta-lactam ring. • The macrolides This class of antibiotics contains the most well-known antibiotic, erythromycin from Streptomyces erythreus, which features a large lactone ring connected with amino sugars. This category also works against some Gram-negative and Grampositive bacteria. • The aminoglycosides Amino sugars and a ring structure known as aminocyclitol make up this group. Major antibiotics in this class include neomycin and streptomycin (Streptomyces griseus). • The tetracyclines Different tetracyclines are a result of varied chemical groups connected to the primary ring in this family of antibiotics’ conventional chemical structure, known as a naphthalene ring. Doxycycline, minocycline, tetracycline, chlortetracycline,

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and oxytetracycline are among the medications in this family. The species Streptomyces produces all tetracyclines, which are helpful against a variety of illnesses brought on by Gram-negative bacteria.

5.4

Recombinant DNA Technology (RDT)

The capacity of the host to absorb the recombinant DNA is necessary for gene cloning and the production of foreign gene products within host cells. As a host for DNA cloning, microorganisms like Escherichia coli, Bacillus subtilis, and budding yeast Saccharomyces cerevisiae are frequently selected because they are simple to culture and can be kept in high quantities on Petri plates. Fortunately, humans have learned to grow microbes cheaply and efficiently on an industrial scale. Bakers and brewers learned how to use yeast cells in producing bread and beers, and for human health, the essential product ever made by microorganisms is antibiotics. DNA cloning involves cutting and pasting the required gene of interest (GOI) into a DNA vector, and then the vector is introduced in a suitable host cell (microorganism). As the host cell replicates, GOI also replicates, resulting in the formation of clones. For the expression of genes, expression vectors are required. They are designed to carry a cloned gene and produce the protein of interest. They have an origin of replication, promoter sequence, GOI, terminator sequence, reporter gene, and selectable gene to detect the transformants. The uptake of DNA by bacterial cells is known as transformation. After DNA cloning, the GOI is packed inside the virus head, and the virus is allowed to infect the zygote transferring the gene inside the cell and integrating it within the genome (transfection). Zygote cells will express the foreign proteins. Types of vectors obtained from microorganisms: (a) (b) (c) (d) (e)

Plasmid vector Viral vector Cosmid vector Bacterial artificial chromosome Yeast artificial chromosome

Expression vectors within microorganisms produce many proteins required for human health. Once a recombinant gene is constructed and introduced in host microorganisms like bacteria and yeast, expressed protein provides more excellent quality and purity than the original extracted protein. Large fermenters are for the production of recombinant proteins that can be used to produce biopharmaceuticals on a large scale. Added benefits of switching from natural protein to recombinant protein reduce the variation of biological activity and less inadvertent contamination.

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Biopharmaceuticals

The introduction of biotechnology and microbiology to health sciences introduced many new proteins and peptides (Gouilleux et al. 1995). The agents include: (a) Erythropoietin, granulocyte colony-stimulating factor (GCSF), granulocytemacrophage colony-stimulating factor (GM-CSF), clotting factors VIIa, VIII, and IX, and GPIIb/IIIa are examples of hematopoietic growth and blood coagulation factors. (b) Cytokines and interferons for anti-infective and cancer therapy. (c) Hormones and their derivatives. (d) Enzymes and their derivatives. (e) Recombinant proteins for vaccines. Pharmaceutical companies produce transgenic animals (pharm animals) using viral-mediated gene transfer (retrovirus). Virus containing the gene of interest infects the zygote and transfers the gene into the zygote’s genome, and protein expression takes place. Transgenic goats produce human antithrombin protein. Transgenic rabbits produce human fibrinogen protein. Transgenic cows produce human myelin basic proteins.

5.5.1

Enzymes

Various bacteria and fungi secrete pharmaceutically valuable enzymes. Molds such as Penicillium, Aspergillus, Rhizopus, and Mucor secrete invertase, amylase, pectinase, and protease. Amylase hydrolyzes the starch to dextrin and sugars. Invertase hydrolyzes sucrose to glucose and fructose and produces syrups that do not crystallize (Table 5.2). Table 5.2 A number of the microorganisms that produce enzymes Microorganisms Actinomycetes Bacteria

Fungi

Actinoplanes sp. Klebsiella pneumoniae Bacillus cereus Escherichia coli Bacillus coagulans Aspergillus flavus Candida lipolytica Penicillium funiculosum Saccharomyces cerevisiae Trichoderma reesei Penicillium notatum Aspergillus niger

Enzymes Glucose isomerase Pullulanase Penicillinase Penicillin acylase, b-galactosidase a-Amylase Urate oxidase Lipase Dextranase Invertase Cellulase Glucose oxidase Amylases, protease, pectinase, glucose oxidase

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Vitamins and Amino Acids

Through fermentation technology and metabolic engineering, microorganisms are effectively employed for the commercial manufacture of vitamins, such as vitamin B complex, vitamin C (ascorbic acid), provitamin A (P-carotene), and provitamin D (ergo sterol) (Wang and Luo 2022). The food industry uses amino acids produced by microbes as nutritional supplements. For a large amount of amino acid production, regulatory mutant strains of microbes are taken that cannot limit the synthesis of end product and give large production. A mutant of Corynebacterium glutamicum is mainly used to produce glutamic acid and lysine amino acid. Mutated strains cannot process α-ketoglutarate to succinyl-CoA in the TCA cycle. A low level of biotin in culture further increases the excretion of glutamic acid. To meet their needs, mutated bacteria shift to the glyoxylate pathway during the growth phase. After some time, the stationary phase approaches due to changed nutrient availability, and 81% molar conversion of isocitrate to glutamate occurs (Tables 5.3 and 5.4).

5.5.3

Organic Acids

In the study of human health, organic acids generated by microbes throughout their metabolic pathways—such as citric acid, lactic acid, fumaric acid, and gluconic acids—play a significant role. Earlier, citric acid was extracted from citrus fruits, but things changed after discovering organic acid-producing microbes. Today, most of the commercial citric acid production comes from Aspergillus niger. Twenty percent of the acid is used by pharmaceutical companies, 70% by food industries as a preservative, and the rest by other companies. Table 5.3 Fat-soluble vitamins from microorganisms Vitamins Arachidonic acid Dihomo-y-linolenic acid Eicosapentaenoic acid Vitamin K2 Vitamins E and K, side chains

Microorganism Mortierella alpina Mortierella alpina Mortierella alpina Flavobacterium sp. Geotrichum candidum, Geotrichum sp., Candida sp., etc.

Method Fermentative production from glucose Fermentative production from glucose by a Δ5desaturase-defective mutant Δ17-desaturation of arachidonic acid or conversion from α-linolenic acid Conversion of quinone and side-chain precursors to the vitamin Enzymatic conversion from (E)-3-(1.3′-dioxolane-2′-vl)-2-butene-1-ol

Source: Yuan P, Cui S, Liu Y, Li J, Du G, Liu L. Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives. Appl Microbiol Biotechnol. 2020 Feb;104(3): 935–951. doi: 10.1007/s00253-019-10,157-x. Epub 2019 Dec 14. PMID: 31838543

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Table 5.4 Microbes’ water-soluble vitamins and coenzymes Vitamins Biotin

Coenzyme A

Nicotinamide Nicotinic acid Pantothenic acid Vitamin C

Microorganism Serratia marcescens Bacillus sphaericus Brevibacterium ammoniagenes

Rhodococcus rhodochrous Rhodococcus rhodochrous Fusarium oxysporum Corynebacterium sp.

Method Using a mutation of B. sphaericus’ biotin biosynthesis enzyme system, a genetically modified bacterium produces glucose by fermentation from diaminopimelic acid Conversion by enzymatic coupling of ATP-generating system and coenzyme A biosynthesis system of B. ammoniagenes with D-pantothenic acid, L-cysteine, and AMP as substrates Hydration of 3-cyanopyridine 3-Cyanopyridine is hydrolyzed to produce nicotinic acid and ammonia By stereospecific hydrolysis, D,L-pantolactone is converted to D-pantoic acid and L-pantolactone 2,5-Diketo-D-gluconate produced by fermentation is converted chemically to L-ascorbic acid after being converted enzymatically to 2-keto-L-gluonic

Source: Kanmani P, Satish Kumar R, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Probiotics and its functionally valuable products-a review. Crit Rev. Food Sci Nutr. 2013;53(6):641–58. doi: 10.1080/10408398.2011.553752. PMID: 23627505

Aspergillus niger is also used in gluconic acid production and contributes to 95% of the total yield. Under ideal circumstances, the microbe’s glucose oxidase enzyme catalyzes the single-step oxidation of glucose to gluconic acid. When there is a deficit, gluconic acid is employed as a calcium and salt transporter (Kaur and Chaudhary 2021).

5.5.4

Biopolymers

Microbially produced polymers, especially polysaccharides, are employed in various areas of pharmaceutical industries (Table 5.5). Polymers like dextran are used as blood expanders and absorbents. A strain of Acetobacter produces cellulose microfibrils that are used as food thickeners. Essential polymers used by pharmaceutical industries are cyclodextrins produced by Thermoanaerobacter. The sugars in cyclic oligosaccharides that make up cyclodextrins are connected by α-1,4 links (Fig. 5.2). They can easily bind with specific substances, alter their physical properties, and have various purposes. The bitterness and chemical odors of medications are covered up by cyclodextrin’s binding to them and boosting their solubility in blood. It may also be used by the food industry as a selective absorbent to take cholesterol out of eggs and shield species from oxidative damage.

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Table 5.5 Biomedical compounds produced by microorganism and their effects Compound type Antitumor agents

Enzyme inhibitors

Source S. peucetius subsp. Caesius S. caespitosus S. verticillus Actinoplanes sp.

Immunosuppressants

Tolypocladium inflatum S. tsukubaensis S. hygroscopicus

Polyethers

Streptomyces cinnamonensis Aspergillus terreus Penicillium citrinum + actinomycete Claviceps purpurea

Statins

Uterocontractants

Specific product Doxorubicin Mitomycin Bleomycin Acarbose

Cyclosporin A FK- 506 Rapamycin Monensin Lovastatin Pravastatin Ergot alkaloids

Process/organism affected Treatment of cancer Treatment of cancer Treatment of cancer Intestinal glucosidase inhibitor (decreases hyperglycemia and triglyceride synthesis) Organ transplants Organ transplants Organ transplants Coccidiostat and ruminal growth promoter Cholesterol-lowering agent Cholesterol-lowering agent Induction of labor

Source: Otten SL, Stutzman-Engwall KJ, Hutchinson CR. Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius. J Bacteriol. 1990 Jun;172(6):3427–34. doi: 10.1128/jb.172.6.3427-3434.1990. PMID: 2345153; PMCID: PMC209154 Yang X, Feng P, Yin Y, Bushley K, Spatafora JW, Wang C. Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment. mBio. 2018 Oct 2;9(5): e01211–18. doi: 10.1128/mBio.01211-18. PMID: 30279281; PMCID: PMC6168864 Huang X, Men P, Tang S, Lu X. Aspergillus terreus as an industrial filamentous fungus for pharmaceutical biotechnology. Curr Opin Biotechnol. 2021 Jun;69:273–280. doi: 10.1016/j. copbio.2021.02.004. Epub 2021 Mar 10. PMID: 33713917

Fig. 5.2 Chemical structure of α-CD. CD cyclodextrin

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Prebiotics and Probiotics

Besides microorganisms’ role in fermentation technology, they are used as food sources for humans and animals. Microorganisms like bacteria, yeast, and especially fungi are a good source of nutrients. Agaricus bisporus is the most important fungi for nutrients (Shanahan et al. 2009). Microorganisms are also used as supplements and are then known as single-cell proteins. Cyanobacterium Spirulina is one of the most popular microbial food supplements commonly used in powdered or dried cakes in Africa and the United States. The latest emerging age of microbial utilization as food is probiotics. They are living bacteria that give the host health advantages; prebiotics are the probiotics’ food. In 1965, Lilly and Stilwell accredited the word “probiotic” as a microorganism that affects other organisms, i.e., substance secreted by microorganisms supports the growth of other beneficial microbes. Lactobacillus and Bifidobacterium are the microorganisms used to develop probiotics; adding these to diet improves health beyond basic nutritional value (Khalesi et al. 2014). The health benefits which may be possible are anticancer effects, healthier blood pressure, control of diarrhea, and possible modulation of severity of Crohn’s disease, depression, obsessivecompulsive disorder (OCD) and autism (Table 5.6). A few applications of Lactobacillus acidophilus are to reduce antibiotic therapy’s side effects and decrease E. coli presence in beef cattle. The cattle showed a markedly lower carriage of E. coli O157:H7 after spraying the desired bacteria on the feed. Table 5.6 List of various probiotics and their beneficial effects on the human body Strain Lactobacillus plantarum Saccharomyces boulardii Bifidobacterium animalis subspecies lactis Bifidobacterium longum subspecies infantis Bacillus coagulans Lactobacillus reuteri

Lactobacillus reuteri Prodentis for oral health Lactobacillus reuteri Protectis

Lactobacillus paracasei,Lactobacillus johnsonii, Lactobacillus johnsonii

Claimed potential effect on body Affects IBS symptoms Treatment and prevention of antibiotic-associated diarrhea Impact on the digestive system Relieves constipation, bloating, and stomach discomfort Reduce bloating and stomach discomfort in IBS sufferers Aid in shielding kids against diarrhea, H. pylori infection, antibiotic-related adverse effects, fever, and diarrhea Effect on gingivitis and periodontitis, decrease in bad breath, and decrease in cancer risk factors Decreases a child’s chance of developing diarrhea and has an impact on constipation and stomach discomfort in youngsters Reduce the frequency of gastritis and inflammation brought on by H. pylori

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A synbiotic system is a combination of prebiotics and probiotic microorganisms, which can increase propionic and butyric acid levels and increase Bifidobacterium in the human intestine (Salminen et al. 2021). Probiotics are successfully used with poultry, which was recently proved by USDA. When given to chicken, a probiotic strain of Bacillus subtilis called Calsporin that was deemed GRAS resulted in enhanced body weight and feed conversion. This probiotic could aid in lowering pathogen populations in agriculture and the need for antibiotics while growing poultry. Spraying a proprietary combination of 29 bacteria on chicks can reduce the risk of Salmonella contamination. Following ingestion of the bacterial combination, a useful microbial community develops in the cecum, preventing Salmonella from colonizing the stomach. Competitive exclusion is the term given to this process. A product called PREEMPT was given US Food and Drug Administration approval for usage in 1998).

5.7

Vaccines and Immunizations

To develop lifelong immunity, a chemical is administered as a vaccine to humans or animals. It might be made up of a combination of weak, inactive, or dead microorganisms or one of their component elements. Vaccination, sometimes referred to as active immunization, guards against contagious illnesses. The biggest accomplishment of contemporary biopharmaceuticals may be vaccines (Medzhitov 2007). The smallpox vaccine created by Edward Jenner in 1789 from the cowpox virus (Latin vacca, cow) and the rabies and anthrax vaccines created by Louis Pasteur in 1789 marked the beginning of the era of vaccination. However, it was not until the nineteenth century when vaccinations for additional illnesses were created. Eventually, in the twentieth century, vaccinations against the majority of the epidemic illnesses, including diphtheria, measles, polio, and mumps, were created via trial and error. The most important aspect in improving world health at the time was vaccination, and it is still the most effective way to avoid infectious illnesses today. Mass manufacture of vaccinations against numerous infectious illnesses began with improvements in cell culture technology. Vaccination in children begins after 2 months of birth. Till then, they are protected by passive immunity by antibodies (IgA) from breastfeeding. Veterinary professionals and agricultural workers need to be routinely immunized against anthrax, rabies, and plaques because they often come into contact with diseased animals and products. Passive immunization is the process of injecting premade antibodies created by people, animals, or in vitro. It is referred to as passive since the recipient’s immune system is not needed. For illnesses including botulism, rabies, tetanus, hepatitis, and diphtheria, passive immunization is required. Unfortunately, the defense only lasts as long as the recipient’s body still has the antibody. Antibodies from other humans can persist for several months, but antibodies from animals and in vitro only last a few weeks.

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Types of Vaccines

The immune response triggered by the original infection is reproduced by vaccines that resemble the pathogens. With the aid of vaccinations, several severe illnesses including rotavirus illness, tetanus, and hepatitis A and B will soon be exterminated. Smallpox has been totally eradicated, and polio is also on the approach of extinction. However, vaccinations for a number of severe illnesses, including AIDS, hepatitis C, and malaria, have not yet been created. These illnesses are brought on by recurrent infections in which the pathogen weakens or disables the human immune system. Medical sciences have yet to find a solution to the problem of creating a vaccination that mimics a persistent infection. Present vaccines are being developed using a variety of techniques in order to stimulate immune responses and the generation of memory cells. Vaccinomics is a subfield of medical immunology that employs genomes and bioinformatics to create vaccinations for diverse bacteria and parasites as well as provide novel vaccine design concepts.

5.7.1.1 Whole-Organism Vaccines The most present vaccines in the market are whole-organism vaccines, and they utilize the complete microbes either attenuated (live but nonvirulent) or killed. So, this category is divided into two types, inactivated vaccines and attenuated vaccines. In attenuated vaccines, microorganisms can multiply within the host and require only a single booster. These vaccines are very effective in developing humoral and cell-mediated immunity, but the problem with these vaccines is that they can revert to a virulent form, e.g., Varivax is an attenuated vaccine against chickenpox. Vaccines for rubella, measles, and mumps are also attenuated. Inactive vaccines are also effective, but since the microorganisms cannot replicate, they require multiple boosters. They can induce humoral immunity but not cellmediated immunity. The best thing about these vaccines is that they cannot revert back to a virulent form and do not require refrigeration because they are stable at room temperature. Although whole-organism vaccinations are regarded as the “gold standard,” they are often ineffective for some illnesses. They can trigger allergic responses and be problematic for some people since they are made up of entire infections and maintain all the antigenic chemicals. For individuals with AIDS and cancer treatment who have impaired immune systems, attenuated vaccinations might cause issues. Reversion to the attenuated vaccine’s virulent form is unbearable when it comes to fatal infections. 5.7.1.2 Subunit Macromolecules as Vaccines These vaccines were developed to overcome the side effects of the whole-organism vaccines. Subunit vaccines only utilize specific, purified macromolecules derived from pathogenic microbes. Presently, three different macromolecules are used for vaccines:

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(a) Surface antigens: hepatitis B vaccine (b) Polysaccharides of the capsule: Haemophilus influenzae type b vaccine (c) Toxoids, inactivated exotoxins secreted by pathogens: diphtheria and tetanus vaccine

5.7.1.3 DNA Vaccines DNA vaccines utilize pathogens’ genetic materials, elicit strong immune responses (humoral and cell-mediated), and produce long-lasting memory cells. Pathogen’s antigen-producing genes are inserted into plasmids and then injected into muscles. Muscle cells take up the plasmid, integrate the foreign genome in its nucleus, express antigen genes, and produce antigenic proteins. Antigens released by the muscle cells are ingested by the antigen-presenting cells (APCs), and they break the antigen into pieces and display them to T-helper cells (TH cells). Upon binding with APCs, TH cells get activated and secrete cytokines for the activation of B-cells and T-cytotoxic cells (TC cells). Activated B-cells start differentiating into antibodysecreting plasma and memory cells. TC cells engulf the infected muscle cells and retain the memory of the antigen using memory T-cells. Presently, many DNA vaccines are under trial for human use against AIDS, malaria, herpesvirus, and influenza. Cancer-specific vaccines are also in trials. 5.7.1.4 Recombinant Vector Vaccines Recombinant vector vaccines are designed differently from other types of vaccines. They require live vectors such as nonvirulent viruses and bacteria. Antigenproducing genes of the pathogens are inserted into these nonvirulent vectors. Recombinant vector vaccines are usually delivered with the help of a needle or a gene gun. Attenuated vectors inside the host body replicate and produce the pathogen’s antigens. Antigens, once released, initiate an immune response activating B and T cells. Vaccines for various viral and bacterial diseases have been developed till now (Tables 5.7 and 5.8). Microorganisms presently used to produce recombinant vector vaccines include vaccinia, canarypox, adenovirus, and attenuated strains of Mycobacterium and Salmonella.

5.8

Clinical Use of Microbiology in the Detection and Therapy of Disease

Apart from biomedical applications, microbiology also has many laboratory applications to detect the presence of antigens in blood, harmful molecules in drugs, and carcinogenic compounds and to study protein-protein interactions (McFarland and Elmer 1995). A few clinical uses of microorganisms are listed below.

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Table 5.7 List of viral diseases and types of vaccines used against them Viral diseases Chickenpox Rabies Brain infection Influenza A Measles, mumps, rubella Hepatitis A Hepatitis B Smallpox Respiratory disease Yellow fever Poliomyelitis

Vaccines Attenuated Oka strain (Varivax) Inactivated virus Inactivated Japanese encephalitis virus Quadrivalent flu vaccine-inactivated virus The MMR vaccine—a combination of attenuated viruses Inactivated virus (Havrix) Recombivax HB—hepatitis B viral antigen Live attenuated vaccinia virus Adenovirus—live but attenuated Attenuated virus Oral poliomyelitis vaccine—attenuated or inactivated

Table 5.8 List of bacterial diseases and types of vaccines used against them Bacterial diseases Tuberculosis Plague Typhoid fever Typhus fever Q fever Cholera Anthrax Diphtheria, pertussis, tetanus Haemophilus influenzae type b

5.8.1

Vaccines Attenuated Mycobacterium bovis (BCG vaccine) Fraction of Yersinia pestis Ty21a (live attenuated, polysaccharide) Killed Rickettsia prowazekii Inactivated Coxiella burnetii Fraction of Vibrio cholerae Unencapsulated B. anthracis’s extracellular components DPT vaccines—toxoid of diphtheria, inactivated Bordetella pertussis, and tetanus toxoid Bacterial polysaccharide or polysaccharide conjugated protein

Carcinogenicity Testing

Bruce Ames created the Ames test in the 1970s to determine if a substance is carcinogenic using specialized strains of Salmonella typhimurium. It operates according to the mutational reversion theory. In the Ames test, the chemical to be examined is introduced after plating S. typhimurium histidine auxotrophic strains on the dish. S. typhimurium, the test material, and potential mutagens are combined with a little quantity of histidine in molten agar. The molten mixture is put on top of thin agar plates, and an incubation time of 2–3 days is allowed at a physiological temperature of 37°C. The bacteria will become prototroph if the chemical causes a DNA mutation, and colonies will develop on a sparse medium.

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5.8.2

Phage Therapy

Phages are viruses that infect bacteria. Phage therapy uses genetically engineered viruses to treat bacterial infections by controlling the growth of harmful bacteria in the human body. In the 1920s and 1930s, phage therapy was used in the United States and Europe, but success rates were not adequately established because of fewer clinical trials. Interest in phage therapy was developed because of antibacterial-resistant bacteria. Companies like Novolytics, Gangagen, and Intralytix, foundations, and universities worldwide started focusing on phage therapy.

5.8.3

Medical Devices

In many medical gadgets, microorganisms serve an essential purpose. Fluorescent fusion technique allows for the quick and accurate identification of pathogens in tissue samples, providing information about the illness. By focusing on the producers of the necessary cell, immunofluorescence investigations are used, which may even assist in locating individual cells in the complicated cell mixture.

5.8.3.1 Biosensors In the field of bioelectronics, microorganisms are also playing a crucial role. Biosensors are developed using living microorganisms or their organelles or enzymes attached to the electrodes, and these biosensors convert their biological reactions into electrical currents. They detect glucose, organic acids, ethanol, and BOD levels. They are also developed to measure medicines’ specific components and air pollutants and detect flavor compounds and biofilm concentration gradients. Measures the concentration of vitamin B complex, nicotinic acid, as well as cephalosporin levels. New biosensors with more capabilities using detection systems based on immunochemistry have been developed. They detect toxins, proteins, DNA, antigens, etc. Rapid advances have been made in biosensor technology. They are even using the streptavidin-biotin recognition system (from Streptomyces avidinii). Microorganisms and metabolites can be measured, contributing to modern medicine development (Fig. 5.3). Potential biomedical applications are: (a) (b) (c) (d) (e)

Clinical diagnosis and biomedical monitoring Detection of microbial contamination during drug development Biological measurement of pheromones Veterinary analysis Concentration measurement of compounds in medicines

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Function- molecule discrimination

Receptacle substances (Antibiotic, antigen or enzymes) Physical and chemical changes

Substance to be measured

Signal conversion

Electric signal

Transducer Receptor

Fig. 5.3 Figures showing the basic functioning principle of biosensors

AD X DBD UAS

Promoter

Reporter gene

AD X RNA Polymerase II

DBD UAS

Promoter

Reporter gene

Fig. 5.4 Figure showing binding of activator domain (AD) with DNA binding domain (DBD) linked with prey and bait proteins and activation of reporter gene

5.8.4

Yeast Two-Hybrid System (Y2H System)

Song and Fields used a microbiological environment to find protein-protein interactions in the yeast Saccharomyces cerevisiae (a type of fungus) in 1989. This finding transformed the study of protein interactions and gave researchers a fresh tool for studying eukaryotic transcription factors in a healthy cellular setting (in vivo). This method looks at the protein machineries engaged at the atomic level, the signaling pathways involved in cellular activities, studies of the neuromuscular junction, age-related illness, cancer, and cardiovascular disease to identify disease in eukaryotes. The interaction of two proteins known as bait and prey, linked DNA binding domain and activator domain, respectively, is the fundamental working principle of the Y2H system. Reporter genes are activated when bait and prey interact, producing a certain color response or kind of growth on a particular medium. By using this method, it is possible to pinpoint the roots of several important synthetic human interactions and analyze the pathophysiology of human disease (Fig. 5.4).

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Summary

(a) Microbiology has two branches: one is basic microbiology and second one is applied microbiology. Basic microbiology provides knowledge about microorganisms’ community, while applied microbiology uses the knowledge provided by the basic microbiology. Applied microbiology has achieved great heights in industrial sectors, especially the pharmaceutical and food industry. (b) Health sciences apply microorganisms to produce compounds important to human health. The discovery of antibiotics was done by Alexander Flaming and made penicillin (from Penicillium chrysogenum) against Staphylococcus aureus. (c) Different antibiotics have been discovered and are broadly classified into four groups: beta-lactam, macrolides, aminoglycosides, and tetracyclines. (d) The major antibiotic-producing genus is Streptomycin. With the development of cell culture technologies, large-scale production of antibiotics started. (e) The advancement of recombinant DNA technology led to the development of biopharmaceuticals. This category includes various types of proteins and peptides produced by microorganisms. Agents are cytokines, interferons, hormones, enzymes, and recombinant proteins. (f) Transgenic animals produced by viral-mediated gene transfer methods produce human antithrombin, fibrinogen, and myelin basic proteins. Actinoplanes sp. makes glucose isomerase for patients. Aspergillus niger produces amylases, protease, and pectinase. (g) Microorganisms can cure vitamin deficiency. Corynebacterium glutamicum produces glutamic acid and lysine amino acids, and Geotrichum candidum produces vitamins E and K. Organic acids are the third category created by the microorganisms. Aspergillus niger is used by the pharmaceutical industry because of its capability to form various organic acids. Cyclodextrin is a polymer produced by Thermoanaerobacter; it binds to pharmaceuticals and increases their solubility. Food industry also uses it as a selective absorbent. (h) Probiotics are good bacteria which provide nutritional value. Lactobacillus and Bifidobacterium are the microorganisms used to develop probiotics. Food of probiotics is known as prebiotics, e.g., oligosaccharides. Probiotics, along with prebiotics, form synbiotic. (i) A vaccine is a preparation of killed, live, weakened microorganism or their component administered to animals or humans to initiate protective immunity. Vaccinomics is a branch of medical immunology that uses genomics and bioinformatics to develop vaccines against various microorganisms and parasites and bring up fresh ideas for designing vaccines. (j) Four broad categories of vaccines are whole-organism vaccines, subunit macromolecular vaccines, DNA vaccines, and recombinant vector vaccines. (k) Microbiology has various laboratory uses in addition to biological ones. Microbes are utilized in protein-protein interaction investigations, phage treatment, biosensors, and carcinogenicity tests using the yeast two-hybrid method.

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References Decker EL, Reski R (2008) Current achievements in the production of complex biopharmaceuticals with moss bioreactors. Bioprocess Biosyst Eng 31(1):3–9 Gouilleux F, Pallard C, DusanterFourt I, Wakao H, Haldosen LA, Norstedt G, Groner B (1995) Prolactin, growth hormone, erythropoietin and granulocyte macrophage colony stimulating factor induce MGF Stat5 DNA binding activity. EMBO J 14(9):2005–2013 Kaur N, Chaudhary V (2021) Biotherapeutics and its applications in microbiology. Environ Conserv J 22(SE):63–78. https://doi.org/10.36953/ECJ.2021.SE.2207 Khalesi S, Sun J, Buys N, Jayasinghe R (2014) Effect of probiotics on blood pressure: a systematic review and meta-analysis of randomized, controlled trials. Hypertension 64:897–903 McFarland LV, Elmer GW (1995) Biotherapeutic agents: past, present and future. Microecol Ther 23:46–73 Salminen S, Collado MC, Endo A, Hill C, Lebeer S, EMM Q, Sanders ME, Shamir R, Swann JR, Szajewska H, Vinderola G (2021) The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat Rev Gastroenterol Hepatol 18(9):649–667. https://doi.org/10.1038/s41575-021-00440-6. Epub 2021 May 4. Erratum in: Nat Rev Gastroenterol Hepatol. 2021 Jun 15;: Erratum in: Nat Rev Gastroenterol Hepatol. 2022;19(8):551. PMID: 33948025; PMCID: PMC8387231 Wang X, Luo X (2022) Precursor quantitation methods for next generation food production. Front Bioeng Biotechnol (10):849177. https://doi.org/10.3389/fbioe.2022.849177. PMID: 35360389; PMCID: PMC8960114 Medzhitov R (2007) Recognition of microorganisms and activation of the immune response. Nature 449(7164):819–826 Shanahan F, Stanton C, Ross P, Hill C (2009) Pharmabiotics: bioactive from mining host-microbe dietary interactions. Funct Food Rev 1:20–25 Von Schwerin A, Stoff H, Wahrig B (2015) Biologics, a history of agents made from living organisms in the twentieth century. Routledge, New York

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The Role of Whole-Genome Methods in the Industrial Production of Value-Added Compounds Kaushika Olymon, Upalabdha Dey, Eshan Abbas, and Aditya Kumar

Abstract

The rate of microbial genome sequencing has accelerated with the introduction of high-throughput sequencing technologies and pertinent analytical methods, which has led to the emergence of new scientific disciplines that focus on characteristics of whole genomes, often known as whole-genome methods. The development of bio-based products with economic interest employing wholegenome methods has become a hot topic in the present scientific era. However, it is commonly known that just a few of the biosphere’s microbial species have so far been successfully cultivated in a lab, leaving a wealth of microbial knowledge unexplored. This is where the whole-genome approach comes into play which has drawn significant interest recently and provides potential solutions for several critical issues, such as new drugs and antibiotic discovery, toxic chemical degradation, understanding pathogen virulence and disease mechanisms, and revealing the human microbiome. So, let us explore state-of-the-art tools and methods of the whole-genome domains of genomics, transcriptomics, and proteomics to identify future research priorities and opportunities. Along with that, we’ll also make an effort to comprehend their importance and relevance for biotechnology research, particularly in the area of industrial microbiology. Keywords

Whole-genome methods · Value-added compounds · Human health · Therapeutics

K. Olymon · U. Dey · E. Abbas · A. Kumar (✉) Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_6

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Introduction

An integral part of the biotechnology industry is biomanufacturing, or the industrial production of biological products. Most bioproducts are proteins and metabolites that can be procured from different parts of an organism, be it the cell or from the tissue and sometimes from the organ itself (Amer and Baidoo 2021). Biotechnological or naturally occurring processes can generate these bioproducts. These bioproduct-generating biological systems can be modified through genetic, metabolic, protein engineering, and synthetic biology approaches (Zhang et al. 2017). Understanding the physiology and biochemistry of different organisms has been made possible by pioneering advancements in the field of omics. Economically viable organisms that add value to a variety of industrial processes are not an exception in this regard (Babar et al. 2018). Additionally, developments in metabolic and protein engineering as well as synthetic biology have been applied to research on renewable energy sources to develop advanced biofuels and production of hydrogen by engineered microbes (Jagadevan et al. 2018). The food industry has also used these approaches, ideas, and tools to manufacture synthetic consumables making use of biocatalytic processes in an eco-friendly manner (Akoh et al. 2008). The extensive knowledge gained from systems biology investigations prompted by omics data can be applied to the improvement of metabolic engineering as well as the creation of synthetic biology tools (Amer and Baidoo 2021). This makes it easier to alter intricate biological systems and build reliable platforms for industrial biomanufacturing (Baidoo and Teixeira Benites 2019). The thorough knowledge of how different gene products are annotated has been facilitated by the efficacious whole-genome sequencing methods. New scientific disciplines have emerged as a result of developments in these fields. These include disciplines that focus on aspects of whole genomes and are known as whole-genome methods, and they are all extremely effective and employ a high-throughput mechanism (Manzoni et al. 2018). The industrial output has increased lately as a consequence of latest developments in the subject area of genomics, metagenomics, transcriptomics, proteomics, and metabolomics, which has enhanced the findings in the biomedical field (Amer and Baidoo 2021). These technological developments will enhance pharmaceutical research, expand our knowledge of how genes influence phenotypes and traits, reveal the factors that lead to disease, investigate the relationship between variations in the genome and therapeutic targets, and boost the development of new and novel biologics (Sindelar 2013). Some of the biotechnology applications and methods discussed in this chapter are well known and frequently used, resulting in potential pharmaceutical drugs that are presently undergoing progress, including clinical trials (Mohs and Greig 2017). A greater proportion of drug development is anticipated to be in the field of biologics as the products produced by biotechnologies continue to expand exponentially (KesikBrodacka 2018). According to the rationale, there is no high omics method that can provide a comprehensive overview (Wu et al. 2019). Hence, to accomplish the industrial objectives, various strategies must be employed and modified. These

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Fig. 6.1 Overview of whole-genome methods

whole-genome approaches are now regarded as the foundation of any procedure that is commercially viable (Pareek et al. 2011). Procedural steps in industrial production rely on the presence and production of efficacious strains of microorganisms to generate the highest possible yield of bioproducts (Adrio and Demain 2010). In addition, combining “omics” techniques is essential to tackle open queries and concerns (i.e., data-based study) that increase the prevalence with which systems metabolic engineering methods are put into practice and broaden our understanding of the system in general in commercial environments (Amer and Baidoo 2021). To enhance the general effectiveness of omics-based strategies in the industry, it is necessary to optimize the investigational protocols in accordance with the statistical designs along with computational ones (Misra et al. 2018). The following sections of the chapter would provide a concise overview of the relevance of omics in the processes of industrial microbiology. We have also given a brief overview of the experimental platforms used in omics technology (Fig. 6.1). Several challenges that limit the resilience of many commercial operations using these tools have also been addressed.

6.2

The Rise of Omics: Its Role in Industrial Biotechnology

The research community has gained a comprehensive understanding of the DNA-encoded information of an organism, since the general structure of DNA was discovered in 1953 (Watson and Crick 1953), and as a matter of fact, we are now starting to “customize” this knowledge. One of the greatest scientific

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breakthroughs in history opening a door of enormous possibilities was completing the sequencing of the entire human genome (Venter et al. 2001). A great deal of new information was available to the scientific community with the accomplishment of the Human Genome Project (Lander et al. 2001). The sequencing of the human genome has been highly beneficial in understanding the primary protein structure of humans (Nurk et al. 2022). Along with that it has been easier to decipher the role of all the regulatory elements in the genome during different stages of growth and development of an organism (Oudelaar and Higgs 2021). The significance of these involves understanding the genetic causes of diseases both frequent and uncommon, providing a method for diagnosing them, implying novel and intriguing molecular targets for treatment, and creating novel biotechnologies to eradicate them (Price et al. 2015). Further, efforts have been made to devise new and advanced methods to tailor and reconfigure the genetic makeup of different microorganisms to derive biomedical benefits directly or indirectly (Curtiss 3rd. 1976). Numerous genetic elements, which include coding and regulatory regions, have been analyzed and modified to ensure the maximum economic advantages (Te Boekhorst et al. 2008). For a thorough understanding of the fundamentals of life, an approach that considers multiple omics is hence required to decode the biological frameworks’ chemical makeup, operations, growth, and modifications (Doran et al. 2021). Precise and reliable phenotype prediction from genotype is necessary for biological engineering which demands both the study of the transcriptome (Sahraeian et al. 2017) along with the validation and verification of the altered and engineered genomes (Carroll 2017; Cheng et al. 2021). Although genomics and transcriptomics provide a dependable and trustworthy foundation for biotechnology application, the knowledge remains insufficient (Khodadadian et al. 2020). Unlike genomic analysis, phenotypic features cannot be described by genes or gene interrelationships (Baidoo 2019). To aid in the comprehension of biological systems at a deeper level, the emphasis is presently on the implementation of other “omics” tools, such as proteomics, metabolomics, and metagenomics (Table 6.1), which can constrict the disparity between genetics and final phenotypic traits. Information on expression of genes is offered by both transcriptomics (transcription) and proteomics (translation) (Buccitelli and Selbach 2020), but the latter provides a direct connection between genotype and phenotype (Diz et al. 2012). A snippet of a cell’s precise physiological and metabolic condition is provided by the metabolome, which responds quickly to genetic and/or environmental fluctuation (Wen et al. 2014). On the other hand, metagenomics, for instance, has the potential benefit of being a less biased pathogen identification technology given that it relies on directly sequencing the DNA after it has been retrieved from the specimen (Eckburg et al. 2005). This method may eliminate the need for pure culture while still capturing a representative sample of the microbial community. This has been used to investigate niche-specific microbiomes by sequencing all of the nucleic acids present in a given sample (Human Microbiome Project 2012). As such, molecular-level insight into a lifeform drives the need to integrate “omics” data with the goal of discovering novel molecules and mechanisms (Joyce

– Evaluate the roles of genes – Protein synthesis evaluation – Figure out what PTM is – Determine indicative biomarkers – Gives a readout of phenotype

– Analyze the mRNA transcripts to determine how a gene is functioning – Reconstructing phylogenies by sequencing 16S rRNA

(continued)

– Analysis of diversity patterns in microorganisms – Examine genes/ operons for desirable enzyme candidates – Learn about the secretory, regulatory, and signal transduction processes that are connected to the samples or genes of interest

Assess the benefits and drawbacks of genome editing in both engineered and naturally evolved systems

Strengths

Metagenomics DNA of all microbes – Genetic material of microbes retrieved in bulk from environmental samples (metagenome) – Diverse microorganisms – Architecture of the population – Evolutionary linkage – Functional activity

Genomics DNA – Genes in a biological system in their entirety (genome) – Genome sequence – Understanding the roles and interactions of genes – Diversity of genes

Methods Object Analysis

Metabolomics Metabolite – The sum total of an organism’s metabolites (metabolome) – Metabolites (i.e., molecules which are used in the metabolic process of cells) – Fluctuations in the pathways (i.e., analysis of metabolic concentration and/or flux) – Evaluate the roles of genes – Locate biological roadblocks in metabolic pathways – Determine indicative biomarkers (for instance, biomarkers for productivity) – Analyze the role of proteins

Table 6.1 Summary of whole-genome methods Proteomics Protein – Total set of proteins in a given organism (proteome) – Translation of proteins (i.e., gene expression) – Protein posttranslational modifications – Metaproteomics

The Role of Whole-Genome Methods in the Industrial Production. . .

Transcriptomics RNA – Total RNA transcripts in a given organism (transcriptome) – Synthesis of RNA from DNA (i.e., gene expression) – Sequence of the transcriptome – Metatranscriptomics using 16S rRNA

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❌

❌

Qualitative

Analysis of pathway

Type of data

Qualitative + quantitative

Transcriptomics RNA – Cannot suffice for a good overview of intricate biological systems (i.e., unable to characterize phenotypes) – Due to PTMs, there is not enough information to draw any conclusions – Cross hybridization and contamination

Genomics DNA Cannot suffice for a good overview of intricate biological systems (i.e., unable to characterize phenotypes)

Methods Object Weakness

Table 6.1 (continued)

✔ (e.g., by means of protein content/amount and posttranslational modification) Qualitative + quantitative

Proteomics Protein – Expense of instruments is quite high – Hard-to-measure proteins and peptides – Poor assessment of unstable PTMs – Examining proteins that do not occur frequently can be challenging – The risk of contamination during proteolytic enzyme reactions (similar peptides can originate from various proteins) – Large number of proteins makes it challenging to cover the entire proteome

Metabolomics Metabolite – Expense of instruments is quite high – In terms of chemical composition, the metabolome is extremely varied – Short half-lives in metabolites may result from their instability or the biotransformations that occur to them – Low-abundance metabolites are extremely challenging to analyze – Complicated sample collection and analysis – Sources of metabolite production and consumption in microbial communities are infamously difficult to pin down ✔ (e.g., by evaluating how proteins work and how metabolites are made) Qualitative + quantitative

✔ (e.g., by first locating the genes, then allying them with a role, and construct pathways Qualitative

Metagenomics DNA of all microbes – High amount of data is required – The vast majority of genes remain untraceable – Problems with contamination and clone sequences that are chimeric – Challenges in extracting – Standardized approach to genome annotation is required – Instrumentation with a high throughput is required – Dependent on the development of library tools, particularly sequencing technology

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Recent advances

Insights obtained Technologies and methods

Phenotype – NMR – GC-MS – LC-MS

When two LC or MS machines are used in tandem, the resolution of individual classes can be improved, as in the case of LC-MS/MS; high temperature-ultrahighperformance LC and other new technologies are allowing scientists to detect metabolites that were previously challenging to detect;

Phenotype – 2DGE – 2D-DIGE – MDLC – CE – Mass spectrometry (MALDI; SELDI; ESI; SILAC; ICAT; iTRAQ)

Highly complex proteins can be easily ionized with the help of an Orbitrap mass spectrometer; liquid chromatography coupled with multiple MS’s provides a clear representation of subsets of proteins; more reliable predictions can be made from untargeted proteomics owing to the

Genotype – Nonspecific approaches (MPSS; EST; RNA-seq; SAGE) – Specific approaches (cDNA-AFLP individual RNA sequences) – In situ hybridization; Northern blotting

Larger amounts of task accomplished utilizing next-generation sequencers (NovaSeq 6000); metatranscriptomics of extensive systems is now a reality Accurate characterization of cell lineage and cell state genetic profiles has been made possible by the

Genotype

– DNA sequence (GWAS; exome sequencing; MeDIP-seq; ChIP) – Bioarray technology (SNP; array-based comparative genomic hybridization chip, aCGH; MeDIP-Chip) – RT-PCR; TaqMan probe

Third-generation sequencing, also known as de novo sequencing technology, is a major advancement in the sequencing industry because it combines single-molecule sequencing technology (SMS) and large-scale parallel sequencing; the simultaneous determination of

The Role of Whole-Genome Methods in the Industrial Production. . . (continued)

– Cloning and sequencing the 16S ribosomal RNA gene – PCR-denaturing gradient gel electrophoresis (PCR-DGGE) – PCR-temperature gradient gel electrophoresis (PCR-TGGE) – Terminal restriction fragment length polymorphism (T-RFLP) – Next-generation sequencing technologies Lab-on-a-chip refers to microfluidic devices that perform automated evaluation of samples; Pacific Biosciences’ SMRT (single-molecule real-time sequencing) and American Helicos’ True Single-Molecule Sequencing (SMS) (also known as TSMS) (Zhang et al. 2021)

Genotype

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Methods Object

Transcriptomics RNA latest application of RNAseq approach to single-cell transcriptional profiling

Genomics DNA

epigenetic modifications to a genome is another advancement (O’Donnell et al. 2019)

Table 6.1 (continued)

availability of powerful analytical tools such as PECAN

Proteomics Protein recent developments in single-cell sorting are opening the door to powerful single-cell metabolomics

Metabolomics Metabolite

Metagenomics DNA of all microbes

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and Palsson 2006). This helps in establishing a link between genotype and phenotype by making it feasible to evaluate the communication between successive omics levels (Guo et al. 2021). In the upcoming section, we will try to decipher the basic knowledge regarding genomics, transcriptomics, proteomics, metabolomics, and metagenomics among the whole-genome methods and investigate their potential applications in the field of industrial microbiology.

6.3

Genomics

The professor of botany at the University of Hamburg, Hans Winkler, came up with the concept of “genome” in 1920 to refer to “the haploid number of chromosomes” observed in the nucleus (Noguera-Solano et al. 2013). With the advancement of sequencing technology and the identification of DNA complexity in the age of modern microbiological investigation, this idea has been expanded to include the entire collection of DNA sequences in a cell or organism. The first generation of sequencing methods was a low-efficient strategy which was soon replaced by Heather and Chain (2016) intense parallel processing of sequencing reactions making a massive difference (Margulies et al. 2005). The second generation produced large numbers of short reads in a short period of time (Bentley et al. 2008). Presently, the third generation has made great strides toward single-molecule sequence analysis and incredibly long sequencing reads (Haque et al. 2013). Since the advent of high-throughput sequence analysis, a large number of genomes across multiple kingdoms have been sequenced in full, including more than 15,000 eukaryotic genomes (Caudai et al. 2021). As such, the investigation of DNA’s configuration, activity, transformation, and editing is now also included under the overarching term “genomics” (Collins et al. 2003). The production of novel antimicrobials, diagnostics, vaccinations, medicinal interventions, and environmental cleanup techniques has been made possible by the investigation of microbial genomes (Margarit and Rappuoli 2014). Furthermore, present developments in genome editing technology and other breakthroughs in genomics hold out state-of-the-art capabilities for both designing new systems and reengineering naturally evolved ones (Castle et al. 2021). Besides this DNA sequencing using advanced mechanisms and robust bimolecular modeling of metabolic as well as signaling networks across both wild and lab-bred isolates are now possible as a consequence of genomic information (Reuter et al. 2015). Genomic research has steadily become a key component and a driving force in both basic and applied sciences, beginning with the Human Genome Project’s inception three decades ago and continuing after its completion in 2003 (Moraes and Goes 2016). Additionally, novel drug discovery and innovation are being greatly influenced by the field of genomics (Emilien et al. 2000). The integration and analysis of the petabytes of genomic data generated annually have undergone significant changes as a result of latest innovations, including the onset of cloud-based and federated strategies (Yang 2019). Advancements in gene

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regulation interpretation, the multiple functional aspects of RNA, the complex and multifaceted nucleome, single-cell genomic approaches, and planned modern technological and information processing skills for evaluating genomic datasets and variants offer a previously inaccessible opportunity to comprehend the unique and integrated functions of each gene and regulatory component (Lowe Jr. and Reddy 2015). The first step in accomplishing this is figuring out how each human gene works, including the phenotypic impact of human gene knockouts. It is essential to develop reliable experimental and computational models that infer correlation and precisely anticipate cellular and organismal phenotypes utilizing pathway and network models because genes and regulatory components do not operate independently (Molitoris et al. 2016). The nearly infinite experimental space and complexity, which include cell states and fates, temporal associations, environmental elements, and genotypic contexts, must be taken into consideration in analytical techniques alongside functional redundancy (Amer and Baidoo 2021).

6.3.1

Genomics for Industrial Application and Production

It took over 13 months to sequence the first bacterial genome, which was completed in 1995 (Fleischmann et al. 1995). Our understanding of the microbial world around us has been substantially altered by these genomic technologies and the observations made as a result (Shu and Huang 2022). The massive complexity and ambiguity of the microscopic organisms surrounding, in addition to their countless interconnections with their biotic and abiotic extreme habitats, have been revealed by the ability to interpret the nucleic acid sequence of microbial genomes (Fraser et al. 2000). These observations have enabled us to comprehend this long buried, unculturable world. From renewable resources like sugar, microorganisms are used to synthesize valuable compounds and substances (Ramamurthy et al. 2021). With the application of genomics, it may be possible to manipulate these microbes so they can use numerous different forms of renewable sources such as cellulose and wood (Johnston et al. 2016) that mostly are much more cost-effective than sugar and do not essentially clash for food usages. To cut down the period necessary to develop industrial strains and therefore enhance the proportion of compounds sufficient for mass production, it is crucial to learn about physiology, metabolic activity, response to stress, and the phenotypegenotype relationship in the microbes (Lee and Kim 2015). Genome comparison across a wide range of species and strains of microorganisms (Prentice 2004) is an emerging approach that seeks to quickly find design guidelines and comprehend the effects of engineering. It encompasses a variety of methods, including genomic sequence comparisons of strains with that of species (Snyder et al. 2005), genomewide cellular responses to industrially relevant environments (Pereira et al. 2014), and strain-crossing to identify genetic bases for favorable phenotypic traits (Sauer 2001). The effectiveness of these methods is now being used to develop industrial strains, such as selecting organisms that are suitable for particular industrial uses,

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examining genetic variation in nature to understand how industrial factors impact cells, and maximizing genetic resources to engineer strains that are more effective at producing a wide variety of products (Favaro et al. 2019), (Verstrepen et al. 2006).

6.3.2

Development of Microbial Strains

Red yeasts that manufacture carotene to generate terpenes (Melillo et al. 2013) and lipogenic fungi found in nature for the production of high-energy oil (Rutter et al. 2015) are two examples of organisms that are typically first selected because they possess features relevant to the target chemical. The growing interest in the study of novel microbes is expected to contribute in the production of newer, more genetically tractable species, despite the fact that not every species has mapped out its molecular and genetic pathways that facilitate host alterations (Alper and Stephanopoulos 2009). Finding the best strain possible by taking a good look at its attributes up front can shorten the amount of time needed to eventually modulate such qualities into the preferred microbial factory (Sardi and Gasch 2017). Due to their small genome sizes, significant evolutionary divergence durations, and high phenotypic variety, fungi are at the heart of comparative genomics (Dujon 2005). These characteristics, taken as a whole, greatly enhance the use of genomics, a field that maps attributes of interest onto an evolutionary tree to locate genes and genetic sequences that connect with phenotypes. Genome comparisons between species have been employed in several contexts to identify genes with commercial utility (Sardi and Gasch 2017). In order to uncover enzymes for plant biomass deconstruction, various researches using genomic data have taken advantage of the innate abilities of filamentous fungi that feed on decaying plant material (Solomon et al. 2016). The genomes of eight other fungi were also analyzed to find out why Aspergillus niger is the go-to fungus for manufacturing citric acid by isolating its key enzymes and studying its metabolic properties (Sun et al. 2007). In another study, 14 different Hemiascomycete fungi’s genomes were analyzed to pinpoint the genes that were specific to yeasts typically found on beetles and increased xylose fermentation when added to Saccharomyces cerevisiae (Wohlbach et al. 2011). Understanding the key mechanisms of regulation (Martinez et al. 2008) and transcriptional responses (Andersen et al. 2008) that enable xylose metabolism was further facilitated by the analysis of multiple species. Furthermore, scientists have sequenced and characterized 16 previously unknown species of Ascomycete yeast. Novel genes and mechanisms associated with the utilization of methanol, glycerol, and xylose in addition to the synthesis of esters, organic acids, and lipids were uncovered after further comparing 29 yeast genomes (Riley et al. 2016). As more species are sequenced and meticulously phenotyped, the potential of these methods will only enhance. It is feasible to identify gene expression variations that are specifically related to tolerance by contrasting and comparing expression responses in strains with various levels of tolerance to a given stimulus (Sardi and Gasch 2017). Using this strategy, it became feasible to identify genes whose activation is associated to and accountable

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for variation in nitrogen consumption (Barbosa et al. 2015), enhanced tolerance to lignocellulosic hydrolysate (Sardi et al. 2016), and ethanol (Lewis et al. 2010). stress experienced during synthesis of bioethanol (Zheng et al. 2013), and genes responsible for certain fermentation traits in yeast (Rossouw et al. 2010).

6.3.3

Fermentation and Post-fermentation Handling

When it comes to maintaining genetic consistency and purity throughout the fermentation process, microbial genomics provides invaluable insight (Parekh et al. 2000). Depending on the level of information required, various degrees of genomic studies can be performed on fermentation samples. Genome-wide (Fang et al. 2012) and loci-specific analysis support the retention of inserted genes, plasmid copy quantity (Salazar et al. 2017), and unanticipated chromosomal rearrangements or deletions (Gerashchenko et al. 2012). The analysis can explain why production output drops throughout fermentation, which can promote strain improvement (Costessi et al. 2018). Genomic analysis makes it simple to examine community viability in fermentation techniques that use mixed cultures (Parekh et al. 2000). It is possible to set up genomic analysis to enable quicker signaling and quick detection of pollutants. Genomic techniques (Bolger et al. 2014) can be used to troubleshoot recurrent contamination incidents more effectively, simplifying underlying investigations (Gallone et al. 2016).

6.3.4

Viability of Strains and Their Compliance with Regulations

Genomic analysis is a step in the regulatory approval process and can assist to assure the viability of bacteria used in the food chain (Gallone et al. 2016). The genetic characterization of bacteria employed in the food chain is necessary for regulatory authorization. Data demonstrating the genetic stability of genetically modified strains in the planned process is required. Due to the ease with which mobile elements, such plasmids, can be horizontally transferred to the environment, production strains containing these elements call for genomic investigations (Salazar et al. 2017).

6.3.5

Safeguarding Inventions and Analyzing Products

These days, patenting industrial fermentation processes is nearly impossible to do without first conducting a genomic evaluation. Using microbes in patented processes allows corporations to assert “flexibility to manage” in the relevant industrial processes. In order to apply for a patent on all these microorganisms, whole-genome sequencing must be performed, as the genome contains the innovative component of the patent.

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It is relatively easy to collect a specimen of several commercial items and evaluate for beneficial genes in cases when the source is a microbe, such as probiotics and yoghurt starter cultures. Then the genome of the strains could be scanned in silico to identify genes that are comparable and impart the desired traits. This strategy was used in a recent study of strains of commercial brewer’s yeast. A total of 157 industrial strains of Saccharomyces cerevisiae used to manufacture beer were subjected to extensive phenotyping and genotyping (Gallone et al. 2016). The findings may indicate that genes for sugar consumption, flavor synthesis, and stress tolerance were very prevalent in industrial strains and may be used as criteria for strain selection (Costessi et al. 2018). The implementation of genomics will increase dramatically owing to the upcoming technological developments, biological understanding, and commercial applications, among other things. To assist experimental researchers focus on the most enticing areas of biology and to improve the reliability of correlations in both transcriptional state and disease-relevant cell behavior, computational methods that use artificial intelligence or deep learning can serve as input filters by refining the biological space to the specific genomic or transcriptomic subgroups (Przybyla and Gilbert 2022).

6.4

Transcriptomics

In reference to a specific genome, a catalogue of all RNA molecules, including those that are translated into proteins (mRNAs) and the noncoding transcripts such as microRNAs (miRNAs), is incorporated in the transcriptome (Milward et al. 2016). The study of the “transcriptome” is known as transcriptomics. To define a comprehensive collection of transcripts, this term was initially coined by Charles Auffray. Transcriptomics is an omics technique that analyzes the dynamic gene expression profiles reflected in an organism’s RNA transcriptome under varying external and internal circumstances (Subramanian et al. 2005). This comprises their levels of transcription and expression, as well as their roles, locations, mobility, and degradation. Apart from the structures of the transcripts with reference to 5′ and 3′ end sequences, start sites, posttranscriptional modifications, and splicing patterns, it also encompasses the structures of the genes from which they were transcribed (Wang et al. 2009). With the advent of more advanced tools in the late 1990s, transcriptomics gained widespread attention and acceptance as a legitimate scientific discipline (Lowe et al. 2017). Our awareness of the correlation around transcriptome and phenotype in a diverse range of organisms is constantly developing owing to the implementation of high-throughput methods in advanced transcriptomics (Wang et al. 2009) to analyze multi-transcript expression in a number of physiological as well as pathological state. Gene predictions (Levin et al. 2010) and conventional approaches that generated expressed sequence tags (ESTs) (Adams et al. 1991) utilizing complementary deoxyribonucleic acid (cDNA) clones and automated Sanger sequencing

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technology (Sanger and Coulson 1975) provided the fundamental framework for transcriptome studies, which is demonstrated by the Human Genome Project. The field of RNA sequencing (RNA-seq), which comprises a number of nextgeneration sequencing methods for identifying the sequence and presumably also the abundance of RNA transcripts, has emerged recently (Chu and Corey 2012). The prospect of sequencing the complete transcriptome has emerged as a possibility with the development of RNA-seq (Marguerat and Bahler 2010). When used with sufficient scale of sequence analysis (100–1000 reads per base pair of a transcript), RNA-seq can capture a transcriptome near completely (Martin and Wang 2011). Additionally, RNA-seq enables a comprehensive evaluation of transcripts across the entire genome, with tiers of expression that encompass a dynamic range (Nookaew et al. 2012). In contrast to conventional array technologies, it has implications in the investigation of transcript variants and alternative splicing. As a bonus, it has the potential to be an exploration tool for locating previously unidentified transcripts, such as lncRNAs (Lee et al. 2012). Genes that are differentially switched on or off, in particular cell or tissue types with reference to development, aging, and disease, have been identified using techniques including microarray (Mantione et al. 2014), serial analysis of gene expression (SAGE) (Velculescu et al. 1995), and expressed sequence tag (EST) analysis. These methods have also given scientific evidence for the alternative splice variants (Sultan et al. 2008) and noncoding RNAs (ncRNAs) (Christov et al. 2006) as well as hypothesized gene architectures. Latest RNA-seq techniques, such as nanopore sequencers or single-molecule, real-time sequencing technology (SMRT) (Mikheyev and Tin 2014), are delivering longer amplicons of up to several kilobases, which allows them to sequence a full transcript in a single session, and thus addressing the obstacles that have impeded this from happening before (Maitra et al. 2012). This is because of the rapid progression of machine learning for assembly and advancements in reliability of data. The ability to generate data on transcript levels and cellular localization simultaneously through the integration of RNA-sequencing technology and fluorescent in situ hybridization is another recent development. This is known as fluorescent in situ RNA-seq (FISSEQ) (Lee et al. 2014). DNA “nanoballs” are created through in situ reverse transcription and rolling-circle amplification of cDNA to facilitate subsequent in-cell sequencing via “sequencing by oligonucleotide ligation and detection” (SOLiD) technology. RNA-seq techniques nowadays are accessible for exploring a myriad of RNA biology areas, such as single-cell gene expression, translation (the translatome), and RNA framework (the structurome). Interesting new uses, such spatial transcriptomics, are being developed (spatialomics). Advancements in RNA-seq, along with new long-read and direct RNA-seq technologies, as well as optimized computational tools for data assessment, are assisting to enhance our understanding of RNA biology. This discusses aspects like where and when transcription takes place including the folding and intermolecular interactions that regulate RNA function (Stark et al. 2019). Single-molecule sequencing, also known as third-generation sequencing, or TGS (Cartolano et al. 2016), is gradually becoming prominent. These innovative methods

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eliminate the systemic amplification bias present in SGS (second-generation sequencing) (Montgomery et al. 2010) since they do not call for recurrent PCR amplification prior to sequencing (Stark et al. 2019). New advancements can sequence molecules in real time (Ardui et al. 2018), reducing analysis time and enabling longer read lengths, in addition to their capacity to sequence RNA directly with the associated savings in reagents and labor (Stark et al. 2019). The present commercialization of benchtop equipment, such as the 454 GS Junior (Roche), MiSeq (Illumina), and Ion PGM (Life Technologies), pursuing relatively low resources and energy of assessment (Liu et al. 2012) will boost the demand of RNA-seq in industrial production of value-added goods (Li et al. 2020) in the years to come. These instruments will be added to the high-end sequencers already on the market (Lowe et al. 2017).

6.4.1

Role of Transcriptomics in Industrial Microbiology

Transcriptome profiles can be compared across multiple strains of interest or between cells cultivated in various environments to reveal putative regulatory networks and pinpoint possible target genes that could be altered to improve the strain. Pure-environment culture physiology has already been assessed using DNA microarrays, along with profiling the representation of catabolic genes in complex microbial communities. When analyzing variations in mRNA expression levels in anaerobically cultivated Bacillus subtilis, a DNA microarray revealed that more than 100 genes were adversely impacted by limited oxygen scenarios. Furthermore, using DNA microarrays and statistical approaches, studies examined stress genes in microbial genomes to determine bacterial species, as well as genome-wide transcriptional profiles (Malone and Oliver 2011). DNA microarrays have been the subject of numerous publications detailing their use in obtaining global transcriptome profiles and, by extension, in deciphering cellular gene expression regulations and physiological adaptations in microbial strains with industrial significance.

6.4.2

Studying Ethanol Tolerance in Microorganisms

In a study, transcriptome profiling on two cultures of ethanologenic Escherichia coli exhibited varying degrees of ethanol tolerance (Gonzalez et al. 2002). Analysis of transcriptome profiles revealed several genes and putative mechanisms involved in ethanol tolerance, including loss of FNR function and improved glycine, serine, and pyruvate metabolism. Using this case study, we can see how effective transcriptome profiling can be in deciphering the mechanisms underlying modifications in expression and physiology. Dekkera bruxellensis, the most common wine spoilage organism, expressed drug efflux genes according to data on the transcriptome derived from cells cultured in the

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vicinity of hydroxycinnamic acids (Valdes et al. 2014). A methylotrophic yeast, H. polymorpha DL1, is a microorganism that has been engineered to produce recombinant proteins and ethanol at high temperatures. The RNA-seq study found that in methanol-grown cells, 40 percent of total genome was expressed at very high levels and with little to no regulation (Ravin et al. 2013).

6.4.3

To Assess Toxicity Sensitivity and Osmotic Stress Tolerance

The yeast species Debaryomyces hansenii has been studied for its ability to withstand osmotic stress, accumulate and store lipids, produce an array of chemical agents and enzymes, and tolerate high cobalt concentrations (Gonzalez et al. 2009). RNA-seq analysis was performed to look at the cobalt-induced stress response (Nurcholis et al. 2020). The bacterium Pseudomonas pseudoalcaligenes CECT5344 is the sole organism being studied globally for its potential to degrade cyanide and cyanide-containing industrial contaminants. DNA microarrays have been used to characterize the transcriptomic outcome to cyanide in strain CECT5344 by comparing them to microarrays from ammonium-treated or nitrogen-starved cell cultures (LuqueAlmagro et al. 2015). The large industrial potency of this cyanotrophic strain has been affirmed by transcriptomic, proteomic, and genome sequencing studies. The absence of pathogenicity determinants in P. pseudoalcaligenes CECT5344’s genome and the inclusion of genes needed for the breakdown of different contaminants, including aromatic compounds, s-triazine herbicides, and furan derivatives, expand the strain’s potential applicability in the industry (Cabello et al. 2018).

6.4.4

Food Fermentation

The molecular processes governing intriguing metabolic alterations and functions in fermented food ecosystems are being addressed by high-throughput transcriptomics approaches. Expression profiling in organisms like the yeast Saccharomyces cerevisiae and the lactic acid bacteria (LAB) has provided important insights into cellular processes and organismal responses to their environments (Valdés et al. 2013). Research into the effects of stress during fermentation on the transcriptional response of laboratory or industrial wine, lager brewing, and baker’s yeast strains is one of the most recent microarray applications in this field (Rossignol et al. 2006). The dynamics of gene expression throughout multiple fermentation steps in synthetic media or natural substrates (Penacho et al. 2012) and the transcriptional variations among various strains and mutants are now included in the latest applications of microarray. In conjunction to yeasts, LAB too have industrial importance since they may produce several fermented products, like fermented sausages, dairy products, and sourdoughs, and add the distinctive flavors, textures, and preservative properties to

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it. For the identification of key clusters of genes that are part of crucial functional and metabolic processes, the application of gene expression microarrays has clearly established to be highly beneficial. With the aid of the Illumina RNA-seq technique, many facets of the S. cerevisiae transcriptome structure have been addressed. In their groundbreaking work, scientists discovered variation at the 3′ end in the transcriptome of the yeast along with upstream ORFs, different initiation codons, alternative start codons, and the presence of multiple overlapping genes (Nagalakshmi et al. 2008). Another study employed RNA-seq to shed light on the transcriptome of the mold Aspergillus oryzae, which is used to make a variety of fermented foods in Asia (Wang et al. 2010). The authors emphasize the discovery of novel exons, alternative splicing isoforms, UTRs, upstream open reading frames, alternative upstream start codons, and unique transcripts, through the use of Illumina’s paired-end reading system (Valdés et al. 2013).

6.5

Proteomics

Implementation of technology for identifying and quantifying the total amount of proteins in a cell, tissue, or organism is known as proteomics. It complements other “omics” technologies like genomics and transcriptomics to elucidate the identities of an organism’s proteins and to comprehend the composition and roles of a specific protein. Marc Wilkins coined the word “proteomics” in 1996 to refer to the “PROTein complement of a genOME.” The proteome identifies the majority of a gene’s functional information (Bairoch 1997). Although far more intricate than genomics, proteomics is one of the most important methodologies for comprehending how genes work. Evaluation of the transcriptome or proteome can help distinguish between two biological states of the cell, allowing for the determination of variations in gene expression levels. Identification of the proteins whose presence, absence, or modification corresponds with specific physiological conditions is made possible by the comprehensive examination and comparison of the proteome in multiple metabolic and/or pathological circumstances. Every genome has the ability to produce an unlimited variety of proteomes since the set of proteins that are produced not only differs from cell to cell but also relies on the interactions between the surroundings and the genome at a given time (Ballesté 2018). Chromatography-based methods such as ion exchange chromatography (IEC), size exclusion chromatography (SEC), and affinity chromatography are the traditional methods for protein purification (Aslam et al. 2017). Western blotting (Kurien and Scofield 2006) and enzyme-linked immunosorbent assay (ELISA) (Lequin 2005) can be employed for the analysis of specific proteins. These methods may only be able to analyze a small number of proteins individually, and they are also unable to determine the degree of protein expression (Aslam et al. 2017). Complex protein samples are separated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Wood 1993), two-dimensional gel electrophoresis

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(2-DE) (Issaq and Veenstra 2008), and two-dimensional differential gel electrophoresis (2D-DIGE) (Marouga et al. 2005) procedures. Mass spectrometry (MS) (Yates Iii 2011) has emerged as one of the several proteomic techniques, to sensitively evaluate heterogeneous protein mixtures. Isotope-coded affinity tag (ICAT) tagging, stable isotope labeling with amino acids in cell culture (SILAC), and the isobaric tag for relative and absolute quantification are some of the most recent advances in quantitative proteomic approaches (iTRAQ) (Wiese et al. 2007). Along with this, the two significant high-throughput methods that can help us comprehend the three-dimensional (3D) structure of proteins are nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. High-throughput technologies enable the collection of a significant amount of proteomic data. For 3D structure prediction, protein domain and motif analysis, quick investigation of protein-protein interactions, and data processing of MS, different bioinformatic tools have been built. The techniques for alignment are useful for aligning sequences and structures to find evolutionary relationships (Vihinen 2001; Perez-Riverol et al. 2015). Proteome assessment, which may employ a solitary proteomic approach or a blend of processes, gives a thorough overview of the cell’s organization and function and its feedback strategy to various stresses and treatments (Aslam et al. 2017). The exploration of all the proteins in biological samples is now possible using a myriad of methods in the field of proteomics. Along with 2D electrophoresis, MS-based techniques and bioinformatic tools are frequently used (Timp and Timp 2020). In mainstream proteomics, MALDI-TOF/TOF, as well as LC-MS-MS, plays a pivotal role. Unfortunately, the massive prices utilizing proteomic infrastructure, which encompasses sophisticated technology, database management, and the demand for competent specialists, curtail their accessibility, especially in undereconomically developed regions (Perez-Riverol and Moreno 2020).

6.5.1

Role of Proteomics in Industrial Microbiology

Proteomics is one method that can be used to broadly understand the biological systems of microbes. This method allows us to learn about concentrations, localization, interactions, and dynamics, as well as posttranslational modifications (Jung et al. 2019). The ability of proteomics to address key difficulties in the field of microbiology is inextricably connected to the rate of development of various proteomic technologies (Han et al. 2011). Studies on proteome topics focus on the overall metabolic functions of an organism. Large-scale proteomic technologies are successfully developed as a result. We can identify bacteria genomes and/or quantify their proteins, thanks to proteomic investigations. Proteomic techniques are constantly being improved by researchers, making a wide variety of approaches and uses possible (Khodadadi et al. 2020). Terminologies like “industrial process proteomics” and “industrial proteomics” (Mitchell 2003) and others similar to them have already been used to reflect the

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increasingly vital role of proteomic techniques. The majority of industrially important microbial cells have already had their genomes sequenced, and proteomic techniques can be employed to a number of steps in the development stage (Aslam et al. 2017), beginning from the short-listing and enhancing the functionality of commercial strains, implementation of cellular feature insight attributable to modifications of processing conditions, verification of the processing that takes place downstream, and rigorous classification pertaining to the final product (Min et al. 2017).

6.5.2

Lipid Biosynthesis in Microbes

Lipids from microbes, often known as “single cell oils” (SCOs), have the potential to be a more financially viable source of lipids than fish-derived oils (Blomqvist et al. 2018). Rhodotorula toruloides, a basidiomycete yeast, was first utilized in 1980 as an industrial microorganism for large-scale synthesis of oil alternatives to replace cocoa butter (Ratledge and Wynn 2002). Applying both proteomic (Shi et al. 2013) and transcriptomic techniques (Qi et al. 2017), it has been determined how the molecular physiology of R. toruloides varies during the synthesis of lipids from various sugar sources. Xylose-grown cells showed considerably reduced amounts of ribosomal proteins and translation-related components when proteomic analysis was done. Moreover, in order to optimize xylose metabolism, the proteomic data given here indicates several possible candidate sugar transporters and enzymes involved in peroxisomal beta-oxidation or genetic engineering. The data supplied through this study is beneficial when developing lipid synthesis methods that use R. toruloides on xylose-containing substrates (Tiukova et al. 2019).

6.5.3

Antifungal Production

The fungus Penicillium chrysogenum is one of the industrially important microorganisms which is known to produce the first antibiotic, penicillin, and several commercially important enzymes (Delgado et al. 2017). A range of foodborne toxin-producing fungus, like Aspergillus flavus, which makes aflatoxins (Lie and Marth 1967) found in dairy items and is potentially dangerous, is inhibited by Penicillium chrysogenum’s PgAFP, an antifungal protein (Marx et al. 2008). Upon treating A. flavus with PgAFP in calcium-enriched PDB, a comparative proteome evaluation employing label-free mass spectrometry (MS) revealed an increased presence of 125 protein molecules, many of which were involved in oxidative stress, out of which 70 proteins were discovered at relatively lower concentrations with majority of them being associated with biosynthesis of secondary metabolites and metabolic processes (Delgado et al. 2017).

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Synthesis of Amino Acids

The most important bacteria for the biosynthesis of amino acids in industrial settings is Corynebacterium glutamicum (Kim et al. 2015). L-ornithine is a crucial nonprotein amino acid and a key intermediary metabolite in the urea cycle. The implications of Tween 40 or polyoxyethylene sorbitan monopalmitate (a fatty acid ester surfactant), treatment on l-ornithine production (Jiang et al. 2020) was explored in a study for the first time, and the underlying mechanisms causing excessive l-ornithine production in C. glutamicum were also assessed. L-ornithine plays an important role in a range of biological processes including facilitating wound healing, treatment for disease concerning the liver, and also delaying the aging process (Rathi and Taneja 2018). The enhanced production of L-ornithine was putatively linked to the optimization of the thiamine biosynthesis route and attenuation of the biotin absorption system, which were revealed utilizing a whole proteome study (Jiang et al. 2020).

6.5.5

Production of Recombinant Proteins

Bacillus subtilis is one of the industrially important microorganisms which is used for the commercial production of recombinant proteins and has had its genome, transcriptome, and proteome extensively studied (Mader et al. 2002). The expression of B. subtilis genes related to bioprocesses was analyzed in a study which was followed by the assessment of these microscopic organisms’ global expression profiles using a huge spectrum of nitrogen sources in closed industrial fermentations. It was shown that the global expression patterns of cells at various stages of the production process as determined by genomic, transcriptomic, and proteomic analyses present a highly useful tool to investigate the limiting factors of a production strain (Jurgen et al. 2005).

6.5.6

Bio-mining

Metals like copper, gold, and uranium can be extracted from their ores with the help of acidophilic, chemolithotrophic microbes that can oxidize iron and sulfur. This recognized biotechnology offers considerable benefits over conventional mining methods. Leptospirillum sp., Thermoplasma sp., Acidithiobacillus sp., Sulfolobus sp., Ferroplasma sp., and Acidianus sp. are all examples of microbes that may survive in both mesophilic and thermophilic environments. During aerobic oxidation, a biofilm is formed by an assemblage of mineral-degrading bacteria (Valenzuela et al. 2006). Genomic, proteomic, and metabolomic studies of naturally occurring microbial biofilms containing microbes that are used in mineral extraction are discussed in a number of influential works (Lo et al. 2007). These research works can serve as a springboard for more proteome research into these economically significant bacteria for the purpose of fine-tuning bio-mining techniques.

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6.5.7

141

Studying Immobilized Cells in Biofilms

Biofilms, which can be either naturally occurring or synthetically produced, are commonly used to immobilize microbial cells for use in biosynthesis and bioconversions that yield a variety of products. Immobilized cells also have several important uses in the winemaking and brewing processes, analysis of food quality, biosensors for environmental monitoring, and regulation of the fermentation process (Junter et al. 2002). The prospective application of proteomics in the analysis of synthetic (gel-encapsulated) and natural (biofilm) cells employed in biotech was studied (Junter and Jouenne 2004). Key information regarding the behavior of biofilmforming microbes during industrial processes, host infection, symbiosis, and their resistance to antimicrobial treatments can be gleaned from proteomic analysis of these microorganisms (Bao et al. 2021). Thus, proteomic investigations of biofilmforming microbes provide crucial insight into their activities in a variety of contexts, including industrial processes, symbiosis, resistance to antimicrobial treatments, and human infection. These results highlight the promising future of proteomic technologies in biotechnology process development. Together with genomic and metabolomic data, the proteome survey of industrially relevant microbes provides a foundation for improving fermentation and downstream processing, as well as characterizing the end product (Josic and Kovac 2008).

6.6

Metabolomics

In addition to genomics, transcriptomics, and proteomics, “metabolomics” is an evolving and expanding area of omics technology which offers a clear picture regarding the metabolic aspects of lifeforms (Yang et al. 2019). Applying highthroughput analytical techniques, metabolomics studies the complete set of molecules in an organism, known as the metabolome, in order to identify and measure modifications in intrinsic and extrinsic small compounds associated with disease (Tyagi et al. 2021). Some important applications of metabolomics include comparing mutants, assessing responses to environmental stress, exploring the global impacts of genetic alteration, comparing different growth stages, toxicology, drug discovery, nutrition, cancer, diabetes and the discovery of natural products (Shulaev 2006). The term “metabolomics” was coined by Professor Jeremy Nicholson in 1999 (Nicholson et al. 1999). Following in the footsteps of genomics and proteomics, metabolomics provides a method for the comprehensive quantitative analysis of all metabolites in organisms, with the goal of elucidating the association among metabolites and pathological and physiological variations (Yang et al. 2019). The metabolome encompasses all of the small molecules (9.5), and find extensive uses in the production of food, biodiesel, surfactants, and cosmetics. To learn more about the metabolic changes that occur in Bacillus subtilis throughout peak lipase production in both reduced and elevated nitrogen states, a study analyzed the levels of various extracellular chemical compounds (organic acids, glucose, amino acids, organic acids) throughout the process (Yuan et al. 2019).

6.6.4

Biofuels

Because of their clean-burning and sustainable qualities, biofuels are being utilized more frequently as substitutes for fuels derived from petroleum. The metabolic impacts of the blockers such as acetic phenol, furfural, and acetic acid, formed on biomass growth and biofuel synthesis, have already been better understood using metabolomics, which also offers metabolic engineering hints for strains that are more stress-tolerant (Zhao et al. 2019). Due to its efficient production rates along with excellent resistance to ethanol content, the yeast Saccharomyces cerevisiae is the optimal bio-producer of ethanol, which is utilized most extensively in industry. By using comparative metabolic

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profiling, the alcohol synthesis by S. cerevisiae during the co-utilization of glucose along with xylose in the vicinity of inhibiting agents was studied. The findings showed that xylose catabolism and energy supply were the two factors limiting xylose consumption in the presence of inhibitors including acetic acid, phenol, and furfural (Wang et al. 2014). Bio-fermentation for butanol production is gaining interest all over the world because of its remarkable biological traits, high energy content, high calorific value upon combustion, and low vapor pressure, making it a next generation of sustainable source of energy after ethanol. Integrated omics platforms were used to study implications of cytotoxic inhibitors in the process of butanol fermentation in Clostridium acetobutylicum. The metabolic processes were evaluated using metabolomics research, indicating cell death rankings proportional to dose in the order of methanoic acid, phenol, and lastly furfural which additionally enables to cultivate strains of C. acetobutylicum that are more resistant to these inhibitors (Liu et al. 2019).

6.6.5

Antibiotics

Microbes produce antibiotics, a generic secondary metabolite, typically at a rather slow rate of growth (μ) (Wang et al. 2018). The relationship between metabolite level, flux dispersion, and antibiotic synthesis has recently been explored using metabolomics in order to identify the rate-limiting component (possible biomarkers) and further optimize the production. A strain of Saccharopolyspora erythraea is an important industrial microorganism which is mostly used for the synthesis of erythromycin which is a common broad-spectrum antibiotic. In order to construct and test the procedures for analyzing sugar phosphates, organic acids, and acyl-CoAs found inside the cell, a preliminary metabolomics investigation was conducted in Saccharopolyspora erythraea. Additionally, a positive correlation was observed between specific erythromycin synthesis and the size of the intracellular propionyl-CoA pool through this metabolomic study (Hong et al. 2017). In order to gain an insight on the mechanism of rapamycin overproduction, an essential macrolide antibiotic having antifungal, anticancer, and immunosuppressive properties, a study compared 86 metabolites using GC-MS, and 22 were identified as putative biomarkers linked with its elevated yield. The role of amino acids in rapamycin biosynthesis was also highlighted by the metabolic route (Wang et al. 2015). To check the growth of Streptomyces and the synthesis of candicidin, a recently discovered antibiotic with great antifungal activity, a metabolomic study was conducted showing the gradual control of pH in the process (6.8 → 7.8) was beneficial over the constant regulation of pH at 6.8 and 7.8. The metabolomics of wild-type and pFAdpa-mutant Streptomyces ZYJ-6 strains were compared in a study to investigate the role of AdpA which is a known regulatory factor in terms of candicidin synthesis. Together with data from metabolomics and flux assessment,

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the findings demonstrate that methylmalonyl-CoA was essential for the formation of candicidin as methylmalonyl-CoA-encoding gene, methB, was substantially transcribed in the vicinity of adpA (Liu et al. 2019). For efficient evaluation of genotype-phenotype interactions, metabolomics is now being merged with other omics technologies that we discussed, which significantly improves our knowledge of the overall system. The fundamental objective of synthetic biology is to streamline the transformation of substrates into products and eliminate unused metabolic processes. Machine learning and big data investigation is attracting considerable interest as a solution to this problem because it has the potential to significantly advance present knowledge pertaining to cellular metabolism and in concert with conventional mechanistic models to computerize biological methods and intellectual manufacturing of biomaterials (Zhao et al. 2019).

6.7

Metagenomics

While the field of metagenomics is still in its infancy, it has already proven instrumental in providing insights into microbial diversity that were unavailable from more conventional approaches to the study of microbes. Overriding the necessity to separate and cultivate specific types of microbes, metagenomics has become the most effective and consistent procedure for genomic study of the entire community of microbes (Arrial et al. 2009). It is extremely promising for finding new organisms for experiments, antibiotics against numerous pathogenic microorganisms, and novel enzymes for industrial applications (Yadav et al. 2019). As of late, we have witnessed a meteoric rise in the application of metagenomics, which has been developed to counteract the limitations of the culture-based approach. When conducting metagenomics, DNA is extracted straight from an environmental sample as opposed to being cultured in a lab. The analysis of the richness in the variety of microorganisms using DNA yields a valid and thorough result. Pace and his team first extracted environmental DNA in the year 1985 (Stahl et al. 1985). However, a researcher by the name of Handelsman coined the new word “metagenome” in 1998 (Handelsman et al. 1998). Community genomics (DeLuca et al. 2012), environmental genomics, and population genomics are additional terminology for metagenomics. Roughly 99 percent of all microbes in environmental specimen can be assessed using this method (Yadav et al. 2019). The principle of metagenomics emerges in microbiology research, broadening researchers’ perspectives and enabling the identification of novel biochemical components that can be found in the environment and can be applied to industry. Metagenomic analyses of complex microbial communities have become feasible because of the sharp progress of high-throughput next-generation sequencing even for small research laboratories. Two fundamentally distinct methods for metagenomic analysis addressing the entire DNA extracted from the environment could be used. One of them is the functional metagenomics which is centered on

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generating an expression library by cloning arbitrary community DNA pieces into big insert-holding vectors (cosmids, fosmids, etc.) and finally evaluated to get a specific response with a particular substrate (Lorenz and Eck 2005). Along with this, it also assesses relevant metabolic and biochemical activities (Schmidt et al. 1991). The ecosystem chosen for investigation in this case enables for the detection of enzymes with the requisite properties (Hutcheon et al. 2005); for instance, it is logical to assume that the metagenome of a hot spring will have certain genes that encode for enzymes that are thermostable. Functional metagenomics has important and significant applications in detecting different antibiotic resistance genes, enzymes involved in hydrolysis and antibiotics, and gene characterization which encode for enzymes with specific activity and have completely different and novel types of sequence with no resemblance to previously known sequence types (Prayogo et al. 2020). Sequencing-based metagenomics (Jimenez et al. 2012) is another approach that is employed other than the functional metagenomics. This process utilizes the environmental DNA which is put into sequencing at a large scale after which the bioinformatic tools are applied for functional annotation and gene search, typically via homology to the sequences that are already defined. As this approach provides more insight into the prominent activities of the microbes from the metagenome instead of a restricted number as seen in the case of screening during functional metagenomics, it is more beneficial and effective. It is also easier to scale and can be used independently without the requirement of high-throughput systems for screening. This method is based on already available gene annotations; hence, its ability to find exclusively new and unique genes that encode for specific proteins gets constrained (Boolchandani et al. 2019). The advancement of more powerful bioinformatic techniques along with the growth of significant new data in genomic databases encourages the application of these techniques as a tool for finding significant functional activities. Shotgun sequencing and targeted metagenomics are the two next-generation sequencing methods used in metagenomics (Scholz et al. 2012). The most typical application of targeted metagenomics is to determine a sample’s status of phylogenetic diversity and distribution. It is mostly employed to examine small ribosomal RNA (rRNA) diversity (16S/18S rRNA) in a sample. It is frequently used to comprehend how a pollutant affects the makeup of the microbial community. Source retrieval of environmental DNA is done for the targeted metagenomic analysis. Following amplification of the target gene with PCR primers, next-generation sequencing is used to study the resulting amplicons. Targeted metagenomics has constraints due to the nature of the PCR primers used in the study, which efficiently measures variation within a single gene of interest (Eloe-Fadrosh et al. 2016). On the other hand, genome sequencing is utilized to examine the genomic makeup of an environmental community in shotgun metagenomic sequencing. To assess the complete genetic content of an environmental sample, this procedure involves extracting DNA from the sample, fragmenting it to create sequencing libraries, and then sequencing the libraries. The depth of the sequencing usually limits shotgun sequencing.

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Present advances in sequencing technology have led to the development of nanopore-based sequencers and the PacBio RS II (Sahlin and Medvedev 2021). These technologies facilitate the individual molecules of DNA to be sequenced without the requirement for amplification which finally generates nucleotide reads that are several thousand long. These are anticipated to help the composite genomes to be reconstructed from the sequences of metagenome. The cultivation of microbes that is genome-enabled is one of the most beneficial results of decoding the genomes of uncultivated microorganisms. In the upcoming future, research will focus on integrating the culture-independent techniques with the pure culture characterization employing the complete set of genomic, microbiological, and biochemical approaches to gain more insights into the lineage of microbes that have not yet been cultured (Mardanov et al. 2018).

6.7.1

Industrial Importance

Microbes, as we all know, are crucial to human survival and play a pivotal role in the industry sectors that constitute the core of the present global economy. A new foundation for basic research and a new tool for application in the environmental, agricultural, human health, bio-industrial, and other fields will be made possible by directly connecting to the biological makeup of the microbiota of the entire ecosphere. Exploring new areas for potential resource use is one way to make the most of our genetic stock. Metagenomic techniques have been used to produce highly specialized, custom-made, new microbial metabolites for environmental and industrial efficiency. Since microbes are relatively simple to monitor by functional analysis using a metagenomic approach, industry sectors are switching their interest to investigating uncultured organisms. Furthermore, published works show that the bacterial clade is the most diverse. As a result, many sectors are motivated to take advantage of the rich and varied ecosystem to explore and generate new microbes. The destiny of today’s modern industries depends on advancements in bioengineering, which is receiving overwhelming acceptance around the world. Therefore, it is significant to identify new enzymes and create processes and products that can make use of them (Dhanjal and Sharma 2018).

6.7.2

Industrial Enzymes

Enzymes such as cellulase, protease, and others are crucial for industry. The metagenomic technique has emerged as one method that can address these growing demands since the need for these enzymes for industrial applications is rising (Coughlan et al. 2015).

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Cellulase is a crucial enzyme which plays an important role for industry like cotton, detergents, and paper and has the capacity to catalyze polymers of cellulose into simple sugars (Escuder-Rodriguez et al. 2018). Apart from the Aspergillus sp., metagenomic methods have been used to explore other cellulolytic microorganisms such as Tolumonas, Exiguobacterium, Acetivibrio, Cloacibacterium, Clostridium, and Paludibacter which were discovered in the bamboo paper manufacturing facilities, in an environment with high cellulose content (Cui et al. 2019). The enzyme protease is used in several industrial sectors such as pharmaceutical, detergent, beverage, and food (Tavano et al. 2018). Several organisms including animals, plants, and microorganisms are sources of this enzyme among which Bacillus sp. is one of the well-known industrial producers (Razzaq et al. 2019). Alternative organisms that are more productive and effective are now being looked for applying the metagenomic techniques. A new gene coding for protease was discovered in a microbe which shares 69% similarity with Desulfobacter postgatei 2 ac9 (Biver et al. 2013). Organic sludge yielded the protease-encoding gene Prt1A, which is most potent at 55°C (Devi et al. 2016). The most widely known manufacturers of bacterial lipases in the present time are from the Bacillus spp. bacteria such as B. licheniformis, B. subtilis, B. pumilus, and B. alcalophilus (Hassan et al. 2018). The lipase enzyme is employed in the food, bioremediation, detergent, and biodiesel industries for its ability to catalyze the breakdown of ester bonds between alcohol groups and carboxylic acids through hydrolysis. In a view to explore and find new organisms with the ability to synthesize lipase efficiently, researchers are investigating in the present times. Using a functional metagenomic strategy, the gene h1Lip1 was discovered which shares a 54% similarity with the lipase of Pseudomonas putida, and the lipase enzyme from this gene has an optimal activity at low temperature of 35°C (Hardeman and Sjoling 2007) which is an ideal temperature in detergents used for cold washing (LopezLopez et al. 2014).

6.7.3

Antibiotics and Bioactive Compounds Obtained

The metagenomic library was used to find novel antimicrobial compounds like turbomycins A and B that validated the efficacy of a metagenomic concept (Gillespie et al. 2002). To study bioactive substances, the metagenomic strategy is paired with homology- or functional-based methodologies. Homology-based screening is employed for studying heterologous expression where novel molecule-encoding sequences that are unique are synthesized (Banik and Brady 2010). In one of the studies, homology-directed metagenomic library screening was effectively executed in targeting, isolating, and expressing a biosynthetic gene cluster heterologously encoding for indolotryptoline compound, belonging to extremely unusual group of naturally occurring compounds that exerts a compelling influence on tumor cell lines (Chang and Brady 2013).

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Therefore, it is necessary to create an expression system that operates on a highthroughput program and is extremely selective, precise, and sensitive in order to thoroughly examine the metagenomic libraries.

6.7.4

Bioremediation Facilitated by Biosurfactant

We can use metagenomics to identify clones from DNA libraries made from petroleum-contaminated samples that can generate biosurfactants. These molecules are amphipathic, implying they have both hydrophilic and hydrophobic groups that differentiate positively at the interface between two liquids with varying degrees of hydrogen bonding and polarity, such as water and oil or water and air. Due to the toxicity and low biodegradability, chemical surfactants that have been utilized for bioremediation are not used. As a consequence, biosurfactants have become a safer alternative to chemical surfactants in addition to being more eco-friendly (PacwaPlociniczak et al. 2011; Kennedy et al. 2011). For the purpose of identifying metagenomic clones that have the capacity to generate biosurfactant, various screening methods were developed, such as the atomized oil assay, where on the surface of an agar plate, a small amount of oil is dropped and the production of biosurfactant is instantly observed as halos close to the metagenomic clone (Burch et al. 2010). In another method, a halo that is emulsified is used to detect the presence of clones producing biosurfactant in an oil-coated agar plate (Kennedy et al. 2011). Hemolytic cell lysis is also used as an indicator for clones producing biosurfactant. It is thus anticipated that we can find unique gene clusters that is meant for biosurfactant production, by employing the aforementioned screening techniques, and with this the bioremediation processes will also develop that involves the application of biosurfactants. The enzyme, alkane hydroxylase, which is utilized for breaking down of hydrocarbons was discovered using metagenomic techniques (Paul et al. 2005). Another enzyme which also catalyzes the hydrocarbon breakdown was discovered from polluted agricultural soil, known as the bleomycin resistance dioxygenase (BRPD), thus serving the purpose in the process of bioremediation (Sharma et al. 2020).

6.7.5

Other Enzymes from Metagenome Source

The metagenome library also contains many enzymes, many of which have special biochemical characteristics that make them useful for commercial processes and thus have potential for their industrialization. The 53D1 gene which encodes the chitinase enzyme was found by metagenomic study which showed promising results as an insecticide when used for controlling pests like Bombyx mori (Berini et al. 2019). AHL-lactonase (Fan et al. 2017), transaminases (Leipold et al. 2019) employed in the pharmaceutical business, and toxoflavin-degrading enzyme (Choi et al. 2018), in the agricultural sectors, are some of the other enzymes that have been discovered from metagenomic research. Integrating metagenomics and environmental

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methodologies makes it simple to identify important microorganisms that can thrive in harsh ecosystems, highly contaminated soil, fields that are polluted by heavy metals, tissues and cells that are affected by certain disease, water sources polluted by oil, etc. For several industrial applications such as growth promoters of plant, bioremediation, drug discovery, exploring novel enzymes, identification of new microorganisms, and many more, the knowledge obtained from sequencing the environmental specimen proves highly beneficial. Thus, it is anticipated that application of metagenomic techniques will have a beneficial impact on the advancement of new technology that will also be fruitful for the society and humankind (Prayogo et al. 2020).

6.8

Challenges in Omics for Industry

The linking of numerous crucial biological entities has been greatly aided by the accessibility of cutting-edge instruments for high-throughput screening techniques. Deciphering a multitude of intricate molecular and cellular pathways has given scientists access to a wealth of knowledge about them as well as tools to regulate them. Thus, in many circumstances, the merging of different omics techniques has been beneficial in offering the best possible solution. The potential of omics technologies, according to several research organizations in the scientific community, has been exaggerated for both commercial and academic reasons. Extensive industrialization of omics technology has been opposed by several organizations due to the inadequacy of consistency and accuracy. In a similar vein, no single omics strategy can answer all the scientific problems. The process reliance is negatively impacted by the incorrect choice of interpretive and assessment mechanisms. The lack of stability in many of the biological entities becomes a major drawback. Proteome, for example, gets deteriorated as time progresses, and this in turn creates a hindrance in replicating the experiment. Such inefficient approaches are typically not applied when formulating and optimizing plans for industrial applications. Furthermore, in refining these methods for the genetic modification of economically viable organisms, the impact of numerous extraneous variables must be considered. Different experimental conditions must be applied for confirming and validating before reporting any findings because many experiments do not necessarily yield the same outcomes when different experimental setup is applied. A primary cause owing to the ineffectiveness of omics methods implemented in the biotech industry in its entirety is perhaps the barrier between academic and industrial research. The concept of a successful genetic manipulation in an academic and that in an industrial environment is distinct, as for the latter a beneficial commercial product as an outcome is expected. Furthermore, many industries do not invest much for the development of suitable omics infrastructure because of monetary limitations. Hence, as a consequence to obtain the data after experimental intervention, they mostly depend on organization that does the job on a contract

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basis, as such the reliability of the information gathered through such sources might not be valid enough. Before introducing any altered microorganisms into an industrial setting, necessary research must be done on the incorporation of the omics technology under different biological and environmental parameters. The development of effective strategies for metabolic engineering of microbes will depend on the future successful combination of multiple omics tools in direct proximity to computation and statistical strategies (Babar et al. 2018).

6.9

Sequencing Methods

Nucleotide sequencing technologies are one of the contemporary and cornerstone techniques to decipher molecular biology. Sequencing methods, over the decades, proliferated exponentially and touched every nook and corner of cellular and molecular aspects of life (Fig. 6.2). Next-generation sequencing (NGS) approaches can sequence DNA molecules of a very high number concomitantly, in a costeffective way (Koboldt et al. 2013). Numerous high-throughput NGS techniques are leveraged by the researchers to generate and disseminate crucial domain knowledge, i.e., genetics, developmental biology, microbiology, etc., for organisms of interest (Lelieveld et al. 2016). Genome sequences of a given organism can be determined by sequencing and therefore assembling millions of overlapping DNA sequences called reads into larger molecules, i.e., contigs, scaffolds, or even up to chromosomal level. Statistical and computational methods are utilized in those processes to handle the sequencing biases and errors that are often incorporated in the sequencing reads during the sequencing experiments. With present technological advancement, now microbial whole-genome sequencing (WGS) projects can be completed in a span of days to hours. Complementing WGS analysis, different types of NGS approaches are often applied to elucidate whole transcriptome (RNA-Seq) and exome (whole exome sequencing) of an organism in different biological conditions (i.e., physiological or altered cellular conditions) (Furey 2012). Over the past few decades, a good number of sequencing techniques have been developed to advance the understanding of DNA and how it translates into every biological phenomenon we have ever encountered (Table 6.2). We discuss some of them here:

6.9.1

First-Generation Sequencing

Techniques like PCR and molecular cloning techniques were already developed by the time the initial DNA sequencing techniques were developed to sequence the genome of bacteriophage φX174 in 1977 (Sanger et al. 1977). These particular sequencing techniques became the yardstick for the anatomization of DNA for those years. Two techniques have been said to be first-generation sequencing techniques:

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Fig. 6.2 Overview of sequencing methods from the three generations of sequencing

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Table 6.2 Major sequencing techniques developed throughout the years Generation 1

Developed in year 1977

1

1977

3

Chain termination Roche 454 genome sequencing

2

1996

4

Illumina

2

1998

5

Ion torrent

2

2011

6

SOLiD (small oligonucleotide ligation and detection) tSMS (True SingleMolecule Sequencing) SMRT (singlemolecule realtime) sequencing Nanopore sequencing

2

2005

3

Sl. no 1

Sequencing technique Chemical degradation

2

7

8

9

6.9.2

Developed by Allan Maxam and Walter Gilbert Fredrick Sanger Stockholm Royal Institute of Technology Solexa (purchased later by Illumina) Jonathan M. Rothberg et al. George Church

Maximum read length (bp) 350–650 bp

2003

Stephen quake et al.

20–70 bp

3

2010

Pacific Biosciences

~40 kb

3 or 4

2010

Oxford Nanopore Technologies

Can vary depending on whether the library prepared has one- or two-dimensional reads

350–650 bp 400 bp (single end)

150–300 bp (paired end)

200–400 bp (single end) 75 bp (single end), 50 bp (paired end)

Chemical Degradation

This method was fleshed out in 1977 as there was a need of moving on from physical methods of profiling nucleic acid to chemical methods. The method is dependent on random chemical modification of both strands and alkali degradation of the strand backbone at distinct points. The method effectuates fragments of different lengths (Maxam and Gilbert 1977). This method is now considered obsolete because of the development of the chaintermination method, which is much safer, as the chain degradation method uses toxic chemicals and is less economically feasible.

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Chain-Termination Method

The chain-termination method, or popularly known as the Sanger sequencing method, has been the most widely used approach so far in the field of sequencing. This method determines the genomic sequence with the help of dNTPs and ddNTPs. The ratio between the amount of dNTPs and ddNTPs is roughly 10:1. Since the ddNTPs stop the chain reaction going on during the DNA amplification, the sequence can be deduced by arranging the fragments of various lengths according to their size and then identifying the ddNTP at the 3′ end of each fragment. This technique is applied for the quick inspection of specific genomic elements. Sanger sequencing was crucial for decoding the first microbial genomes.

6.9.4

Second-Generation Sequencing Methods

A lot of progress has been made in the last 10–15 years for the NGS methods. It all started when the company 454 Life Sciences established the first massively parallel sequencing instrument, marking the beginning of the second generation of sequencing techniques. Thereafter, the development in the field of technology connected to this area of research dramatically lowered the negative economic impact of DNA sequencing (Costessi et al. 2018).

6.9.4.1 Roche 454 Roche 454 genome sequencer was developed by Stockholm Royal Institute of Technology in 1998 and then launched in 2005. This approach utilizes emulsion PCR to intensify DNA molecules attached to beads in a clonal manner. Roche 454 sequencing has been utilized to sequence the genomes of Neanderthals, barley, and Helicobacter pylori (Rothberg and Leamon 2008). Roche 454 genome sequencer became the first NGS instrument to be developed. 6.9.4.2 Illumina Illumina has been the most widely used sequencing method so far. It was developed by Solexa in 1998; however, it gained its name when the Illumina Inc. bought Solexa. This method uses reversible termination to perform sequencing by synthesis. It is a synthetic method which gives long reads.

6.10

Third Generation of Sequencing Methods

The third generation of sequencing methods focuses on long reads. Long-read technologies can generate continuous sequences, and the lengths of these sequences have a range from 10 KB to a few MB, straight from raw samples of DNA molecules. There are three major techniques classified to be as the third-generation sequencing methods:

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6.10.1 True Single-Molecule Sequencing (tSMS) Helicos BioSciences came up with the method of tSMS. This method has a shortened sample preparation time and can be used to sequence corrupted molecules also. The technique is more rigorous, as the PCR amplification step is a nonnecessity, but the time required to achieve the objective is longer than that of other techniques.

6.10.2 Single-Molecule Real-Time Sequencing (SMRT) Pacific Biosciences, in 2010, came up with the idea of combining nanotechnology with molecular biology. This process happens at elevated speeds as ~10 nucleotides can be introduced per second. This particular sequencing technique can produce read lengths of up to 40 Kb but at a lesser accuracy contrasted with the second-generation sequencers, which is an aftermath of detection of nucleotide remaining inside the active site for an elongated amount of time, even if the nucleotide is not actually incorporated inside the DNA strand. It is presently the most accepted and employed long-read platform across the world for mapping out the sequences of DNA molecules (Ambardar et al. 2016; Mardis 2013).

6.10.3 Nanopore Sequencing The principle of nanopore sequencing is that there will be a change in the electrical conductance of the nanopore (biological or synthetic) whenever a nucleotide is passing through them; the change will be unique for each nucleotide, and it will do so in real time. This method is now amidst the most refined sequencing techniques available in the present times. It does not require any intermediate step of PCR amplification or chemical labeling, and this method allows for sequencing right after the DNA sample is isolated (Ambardar et al. 2016). Nanopore sequencing is inexpensive since we do not have to add any fluorescent markers or modify the bases.

6.11

Annotation

The emergence of high-throughput sequencing methods exponentially increased the number of genomes recovered from different conditions. Genomes obtained from cultured bacteria, clinical isolates, and metagenome assembled genome (MAGs) provide qualitative and quantitative insights on novel genes, taxonomy, and biochemical pathways (Wilkinson et al. 2020). Therefore, a mandatory and critical step after a successful genome/metagenome assembly is genome annotation. Often genome annotation could be very useful to measure the completeness to the genome assembly by comparing the number of predicted genes with a predefined marker gene set (Fig. 6.3). Tools like BUSCO and checkM are often employed in microbial

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Fig. 6.3 Genome annotation workflow

genomics to assess the quality of genome/metagenome assembly. In simpler terms, genome annotation refers to the process to predict or identify genes (protein coding or noncoding RNA) from genome sequence (Richardson and Watson 2013). Several databases have been developed to store information regarding structures related to nucleic acids, proteins, etc., which have been vital for the credibility of genome annotation (Table 6.3). Moreover, Medigue and colleagues defined genome annotation into two conceptual frameworks: static view of genome annotation and dynamic view of genome annotation (Medigue and Moszer 2007). In the static view of annotation, functions of predicted gene products are assigned based on known

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Table 6.3 Data repositories useful for genome annotation Types of biological information Nucleotide

Protein

Databases The international nucleotide sequence database (INSDC); RefSeq; Genbank

Protein domain

UniProt; SwissProt (https://www.expasy. org/resources/uniprotkb-swiss-prot); TrEMBL (http://www.bioinfo.pte.hu/ more/TrEMBL.htm) InterPro (https://www.ebi.ac.uk/interpro/ ); PFAM; Prosite

Orthology

Cluster of orthologous groups (COG)

Metabolic pathway

Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome. jp/kegg/); BioCyc

Protein interaction Protein structures

STRING (string-db.org) Protein data bank (PDB; www.rcsb.org)

Additional information INSDC is a collaborative approach operated by NCBI, DDBJ, and EMBL-EBI. Home page: https://www.insdc. org/ Expertly curated component of UniProtKB; TrEMBL is a computer-annotated protein sequence database InterPro provides analysis of proteins from the functional point of view by categorizing them into families and foretells domains by utilizing information from more than ten member databases The present update of COG database includes complete genomes of 1187 bacteria and 122 archaea that map into 1234 genera The KEGG pathway is a set of pathway maps obtained from the information on molecular interaction, metabolism, etc. Database of known and predicted protein-protein interactions Global database for the threedimensional structures for large biological molecules such as DNA

sequence similarity information. However, relatively small-sized genes with atypical base composition are often missed by gene prediction algorithms, and results of similarity-based functional annotation are often spurious (Huntemann et al. 2015). Therefore, manually curated genomic information of model organisms is highly necessary to improve the performance of functional annotation in the static approach of gene annotation. The contrasting and more prudent process of annotating nucleotide sequence would be supplementing the information of regulatory activity, metabolic processes, and protein-protein interactions. For instance, for accurate assignment of orthology, signals like gene order, gene fusion events, co-localization, and co-expression of genes in closely related organisms are very useful (Smith et al. 2012). Genome annotation has three branches: (1) annotation related to structural genes, (2) annotation describing functional genes, and (3) annotation giving information

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about noncoding sequences. The first branch of annotation identifies the coordinates of genes in a genome and finds features of DNA such as transposons, exons, and introns. Structural annotation now has extended its search to pseudogenes. The second branch of annotation prognosticates gene functions, such as finding out which gene family a gene belongs to and its characteristics. The third branch of annotation, related to noncoding sequences, is the newest category of genome annotation, as many reports have compelled researchers to extend annotation into the noncoding regions too (Dong et al. 2021). Here, we describe in brief some recently developed annotation tools: Apollo is an open-source annotation editing platform which focuses on structural annotation (Lueder et al. 2021). MicrobeAnnotator is a fully automated and a userfriendly pipeline for the functional annotation of microbial genomes that combines results from several protein databases and outputs the matching annotations together with relevant metadata. It is an open-source software and implemented in Python 3.x (Ruiz-Perez et al. 2021). Manual Annotation Studio was created to enhance the efficacy of the procedure of handwrought functional annotation of prokaryotic genomes and is excellent for annotation projects which involve multiple people. It helps in providing structural to functional annotation projects (Lueder et al. 2021). There are some annotation tools that can do both structural and functional annotations, such as DFAST. DFAST is an open-source package that can be run in Python 2.x and 3.x on MacOsX and Linux systems. It was developed to counter two specific issues – NCBI’s annotation service called Prokaryotic Annotation Pipeline (PGAP) is available only for GenBank submitters, and the online server Microbial Genome Annotation Pipeline (MiGAP) requires a high amount of manual curation (Tanizawa et al. 2018).

6.12

Summary and Future Outlook

Studies indicate that all areas of biological sciences have advanced tremendously since the revolution and the rise of omics. Despite their relevance, many industrial environments have struggled to implement them because of resource, regulations, and technological limitations. Numerous studies have used omics technology tools, both individually and in conjunction, to better comprehend microbial processes and bio-based products. Making a significant contribution to our understanding of fundamental systems, they aid in the interpretation of intricate biological mechanisms at various biomolecular dimensions. Although if research-related benchmark sequences are derived, projects involving population-scale genetic analysis to this day widely use Illumina short-read techniques, not long-read sequence analysis. For the time being, short-read sequencing still provides advantages in terms of time and efficiency, two crucial factors in commercial strain development initiatives. Even though numerous technological advancements in this area prove that these methods work effectively in a wide range of contexts, further research is still

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required to uplift their selectivity, precision, expenditure, and overall performance because many industrial environments have struggled to implement them because of resource, regulations, and technological limitations. Extremely precise layout in strain development will become more efficient and straightforward as a consequence of present breakthroughs in bioengineering with the implications of whole-genome strategy. The assessment of metabolic processes and the application of the information for generalizing different experimental constraints in silico prior to actually their wet-lab screening can be facilitated by the implementation of effective approaches to merge standard strategies of all the omics branches which will subsequently help to achieve measurable enhancements in the performance of processes applied in the field of industrial microbiology.

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New Developments in the Production and Recovery of Amino Acids, Vitamins, and Metabolites from Microbial Sources Priya Shukla, Pradeep Srivastava, and Abha Mishra

Abstract

In the food, chemical, and pharmaceutical industries, macromolecules like amino acids, vitamins, and metabolites generated by microorganisms using renewable feedstocks are significant due to their low cost and sustainability. In addition to their conventional uses, these are also employed in emerging research fields like the production of bioplastics and aesthetic surgeries. These macromolecules can be manufactured commercially using chemical and biological processes, which is an efficient and environmentally benign technique. Microbial solid cell factories with exceptional resistance to extreme pH conditions, large concentrations of metabolites, and lignocellulosic inhibitors are required for an economically feasible fermentation process. Strain improvement and metabolic engineering are two techniques that can be used to create strains with high productivity. This chapter discusses the biosynthetic pathways, substrates, strain improvement, and upstream techniques for methionine L-glutamate, L-lysine, vitamins B2 and B12, coenzyme Q10 or ubiquinone, lactic acid, itaconic acid, and hyaluronic acid as well as their market perspective and budding challenges. Keywords

Amino acids · Vitamins · Metabolites · Microorganisms · Fermentation · Purification

P. Shukla · P. Srivastava · A. Mishra (✉) School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, India e-mail: [email protected]; [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_7

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Abbreviations AA ALA AMM CoQ ET FAD FMN GDH Glu HA IA LA MEP MVA NL ODHC RF SSF UQ UTR UV

7.1

Amino acid Aminolevulinate α-Methyl-DL-methionine Coenzyme Q Ethionine Flavin adenine dinucleotide Flavin mononucleotide Glutamate dehydrogenase Glutamate Hyaluronic acid Itaconic acid Lactic acid 2-C-methyl-D-erythritol 4-phosphate Mevalonate Norleucine 2-Oxoglutarate dehydrogenase complex Riboflavin Solid-state fermentation Ubiquinone Untranslated regions Ultraviolet

Introduction

The procedure of microbiological growth and product generation in aerobic, microaerobic, or anaerobic settings is called “fermentation.” It has become a more sustainable option for traditional chemical reaction-based production. Compared to the chemical approach of manufacturing, the chiral selectivity of the final products becomes more practical when using microbial biosynthetic pathways. Improvements are being made to generate several metabolites with critical industrial uses of microbial origin. Among fundamental substances with various industrial applications encompass AAs, vitamins, and metabolites. Since the beginning of MSG manufacturing in 1907, AA demand has increased (Sano 2009). AAs are utilized in several commercial pertinence as abundant biochemicals to create a variety of commodities, including flavor enhancers for human food, additives for animal feed, and components for cosmetic and pharmaceutical products. Additionally, the essential function AAs play as intermediaries in the building of peptides and the regulation of crucial metabolic processes and activities is required for the growth and sustenance of organisms (Cesari et al. 2005; Wu et al. 2009a). They significantly enhance health by enhancing food

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utilization, reducing dyslipidemia, managing the metabolism of mitochondrial biogenesis, and modulating the body’s natural development and resilience (Yamane et al. 2007; Wu et al. 2009a; Weinert 2009). A lack of AAs has been linked to significant illnesses in humans and animals (Paul et al. 2014; Wu et al. 2004). As a result, there has been a noticeable rise in interest in researching and creating new methods to create them more efficient and environmentally friendly in recent years. Several processes could be used to manufacture AAs, including chemical modification, enzyme- and fermentation-based processes, and excavation from protein hydrolysates. The fermentation procedure is progressively establishing itself as a mighty significant boost for the commercial availability of AAs due to the revolutionary genetic modification methodologies employed to boost the output, precision, and efficacy of the target molecules (Ikeda and Nakagawa 2003). Animals that cannot manufacture enough vitamins or synthesize insufficient amounts to meet their demands depend on vitamins for healthy growth and good health (Capone and Sentongo 2019; Suter 2020). Either chemical synthesis or fermentative generation is the basis for the procedures used to produce vitamins (Yuan et al. 2020). More than 20 of at least 30 compounds alluded to as “vitamins” are understood to be necessary for physiological well-being. Both water- and fat-soluble vitamins are obtainable. A water-soluble vitamin readily dissolves in water but does not disintegrate in polar solvents. Most of these proteins are eliminated with urine after absorption, and very few are stored by the body (Berdanier 2015). Vitamins that can dissolve in fat but not in water are known as fat-soluble vitamins and are kept in the liver or fatty tissues for later use. All living things require vitamins as necessary nutrients, yet many plants and microbes can synthesize them spontaneously. Contrarily, to maintain good health, people and other animals must consume enough vitamins through their diets or supplements (Blake and Konings 2005). Genetic variation and metabolomics, which can be done through biologic or chemical techniques, have historically been employed to boost vitamin generation variants (Yang and Xu 2016). The primary chemical strategies are chemical modification, N+ particle beam utilization, UV irradiation gene editing, and laser point mutations. The fundamental biological methodologies include establishing and recombining the original culture, genetic manipulation, bioengineering, substrate and cultivation environment refinement, biofilm reactor development, and so on (Nie et al. 2013; Song et al. 2014). Tiny molecular end products are the primary source of primary microbial metabolites during the exponential growth phase. Primary metabolites are also intermediate products of metabolic processes. AAs, purines, pyrimidines, nucleotides, alcohol, and vitamins are the most significant products. The pentose pathway and Krebs cycle are the sources of the other main metabolites. These primary metabolites are typically used in fermentation and foods, drinks, fine chemicals, flavors, biodegradable polymers, and solvents. Secondary metabolites are synthesized whenever a microbial growth phase transforms from the second to the third phase (exponential to stationary). Infinitesimal secondary metabolites encompass, for instance, antibiotics, carotenoids, toxins, ecological antagonism, commensal agents, pheromones, immunomodulating representatives, receptor

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antagonists and agonists, insecticides, anticancer prescription drugs, and growth promoters for both plant and animal species (Demain 1998). Most microbes do not squander energy (nutrient sources) producing secondary metabolites, but some microorganisms will manufacture a lot of secondary metabolites to support their growth in specific conditions. Different microbial metabolites are produced industrially nowadays through fermentation, which covers a wide range of markets. The bioprocessing techniques of various commercially produced products with significant commercial applications, including L-glutamate, L-lysine, vitamins B2 and B12, coenzyme Q10, or ubiquinone, LA, IA, and HA, are summarized in this chapter. Their metabolic pathways in various microbes have been studied to comprehend the potential interventions for increased production. The limits of the production process have been examined concerning the demands for pH, medium, temperature, agitation, and aeration. The question of whether agricultural waste can be utilized as a feedstock to incorporate the fabrication system into a sustainable society has been researched. The purifying procedure consumes a sizeable fraction of the total capital required. As a result, it has been examined to identify the significant problems with process efficiency.

7.1.1

L-Methionine

Together with cysteine, methionine has been among the two AAs that have been essential for the synthesis of protein and contains sulfur. This could function in biological systems as a precursor to cysteine. Since this sulfur atom builds disulfide bonds that stabilize tertiary protein complexes, cysteine seems primarily prevalent in structural proteins, including feathers, skin, nails, collagen, or keratin throughout the hair. Among the water-soluble proteins (globulins), albumins, mainly egg albumin, have the most amazing methionine content, at roughly 5%. L- and D-methionine are methionine’s two isomers, with L-methionine predominating in the natural world. Since 1943, numerous studies have demonstrated that utilizing L- or D-methionine in poultry diets has no effect (Goodson et al. 2014). Numerous bacteria, fungi, and plants could biomanufacture methionine utilizing inorganic or organic nitrogen, carbohydrates, and sulfur sources. Animals, together with humans, however, rely on methionine sources that are delivered from outside. An all-vegetable diet may fall short of nutritional requirements because plant proteins usually lack methionine. Toxemia, infantile hair loss, rheumatic fever, depression, muscle paralysis, Parkinson’s liver degeneration, schizophrenia, and stunted growth are still only some of the disorders and physiological abnormalities that have been attributed to methionine inadequacy (Rose 1938). There is a tremendous interest in creating a biotechnological procedure for the commercialization of methionine because various AAs can now be acquired at reasonable prices attributable to fermentation techniques (Chay et al. 1992; Umerie et al. 2000; Odunfa et al. 2001).

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7.1.1.1 Biosynthetic Pathway for Methionine Production Isoleucine, threonine, lysine, and methionine are AAs that belong to the aspartate family. The methods for this family’s AA biosynthesis have been described by various researchers (Stadtman et al. 1961; Patte et al. 1967; Morinaga et al. 1983). Studies have been done on methionine biosynthesis specifics (Jetten and Sinskey 1995; Malumbres and Martin 1996). There are significant similarities among the methionine biosynthesis routes seen in different bacteria. Although microorganisms use multiple biochemical pathways, most fungi and bacteria produce methionine. Conforming to this route, aspartate kinase transforms aspartate onto 4-phospho-Laspartate, at which point aspartaldehyde dehydrogenase eventually oxidizes to produce aspartate semialdehyde (Fig. 7.1). Homoserine dehydrogenase converts aspartate semialdehyde to homoserine. O-succinylhomoserine is manufactured in E. coli using homoserine O-succinyltransferase, consolidating succinyl CoA with homoserine. O-acetylhomoserine is employed as an intermediary rather than O-succinylhomoserine, according to most fungi and a couple of extra bacteria, such as Bacillus and Corynebacterium (Flavin et al. 1964). There are two potential metabolic routes by which O-acetylhomoserine can be converted into methionine. According to the first, some unknown bacteria and some fungi, like Neurospora crassa, produce homocysteine from cystathionine. In the second, O-acetylhomoserine (thiol)-lyase directly transforms acetylhomoserine into homocysteine (Kerr and Flavin 1970). Methionine is produced when homocysteine S-methyltransferase methylates homocysteine.

Fig. 7.1 Biosynthesis of methionine in Corynebacterium glutamicum (Rückert et al. 2003)

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7.1.1.2 Methionine-Producing Microorganisms To successfully implement a methodology for the commercially viable microbiological creation of methionine, a slightly elevated microorganism should always be found or generated (Table 7.1). Because methionine’s production is tightly controlled, wild-type strains typically cannot produce considerable amounts of it (Rowbury and Woods 1961; Somerville and Herrmann 1983). Methionine production by microorganisms has been attributed to about 16 distinct species. Among them, Corynebacterium or Brevibacterium species has substantially more straightforward regulation systems. Thoughts about the possible evolutionary causes of this simplification are yet unresolved. It is necessary to isolate mutants with disrupted pathways since the methionine production route is tightly regulated. The microorganisms reported are Arthrobacter, Bacillus megaterium, Candida boidinii, C. lilium, Hansenula polymorpha, Kluyveromyces lactis, Micrococcus glutamicus, Pichia pastoris, Pseudomonas sp., Pseudomonas putida, Serratia marcescens var. kiliensis, and Torulopsis glabrata (Gomes and Kumar 2005). In conjunction with Corynebacterium and Brevibacterium, other complex species, including specific Lactobacilli and yeasts, have been assessed for synthesizing lysine and methionine. Comparable to this, submerged fermentation has been implemented to try and produce methionine (Kumar et al. 2003). 7.1.1.3 Substrates for Methionine Production The structure of the medium has a significant influence on commercial fermentation. The components needed for developing and producing a product must be present in the medium used in fermentation in the proper concentrations, which can be changed through media optimization. Biotin is a necessary component of all growth media for Corynebacterium species. Achieving high production is typically associated with particular physiological patterns, and the physiology of microbes is significantly influenced by the medium’s constitution (Ward 1989). It is necessary to establish a medium to produce an environment suitable for creating a specific product even Table 7.1 Methionine-producing microorganisms with their yield Microbial strain Bacillus megaterium B71 Ustilago maydis Lactobacilli isolated from Cassava pulp Corynebacterium lilium = C. glutamicum NTG Streptomyces sp. Bacillus cereus C. glutamicum KY10574 Kluyveromyces lactis IPU126 Pseudomonas FM 518 Brevibacterium heali

Yield (g/L) 0.072 6.5 1.35 2.3 3.7 1.9 1.45 14.2 0.8 1.3

References Swapan et al. (1984) Dulaney et al. (1964) Anike and Okafor (2008) Kumar et al. (2003) Nwachukwu and Ekwealor (2009) Dike and Ekwealor (2012) Willke et al. (2010) Kitamoto and Nakahara (1994) Morinaga et al. (1982) Mondal and Chatterjee (1994)

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though different varieties of bacteria have enough capacity to change their patterns of enzyme synthesis to respond to their environment. An essential factor in forming a particular metabolite in fermentation media is the ratio of nitrogen and carbon sources. It has been noted that different strains produce L-methionine using various carbon sources. The far more widespread carbon source is glucose (Kase and Nakayama 1974; Chattapadhyay et al. 1995; Chattopadhyay et al. 1995). Researchers have concluded that maltose seems to be the preferred carbon source for manufacturing methionine (Banik 1975). Several researchers have used methanol and n-alkanes as the primary carbon sources for methionine synthesis (Morinaga et al. 1982; Tani et al. 1988; Ghosh and Banerjee 1986). Harmful exploited carbon sources include molasses, sugarcane juice, coconut water, cassava, and bananas to generate methionine (Pham and Padolina n.d.). Urea, ammonium tartrate, ammonium nitrate, ammonium chloride, sodium nitrate, ammonium acetate, ammonium citrate, ammonium sulfate, ammonium dihydrogen phosphate, and ammonium oxalate have all been documented as inorganic nitrogen sources for the manufacturing of methionine (Kase and Nakayama 1975a, b, c; Yamada et al. 1982; Ghosh and Banerjee 1986). Although yeast extract has been investigated in several experiments as a nitrogen source for the generation of methionine, adopting organic nitrogen sources is not advocated, given that it contains the preponderance of the AAs and raises the probability of revertant development. Fermentation demands minerals or metal ions in conjunction with carbon and nitrogen sources since many metal ions serve as cofactors for various enzymes.

7.1.1.4 Methionine Production Strategies Enzymatic Conversion and Chemical Synthesis The process flow often used to synthesize methionine is chemical polymerization, which primarily requires acrolein, methyl mercaptan, and hydrogen cyanide (Lüssling et al. 1981; Pack 2004). The methodology has been implemented at Evonik Degussa in Germany for over five decades, contributing to roughly 60% of said world’s methionine productivity, with around one million tonnes annually. Enzyme-mediated transformation of methionine to N-acetyl-methionine upon acetylation is the most widely known and frequently employed technique. The L-amino acylase then catalyzes the enzymatic conversion of the L-isomer to L-methionine. Additionally, techniques for immobilizing complete enzyme-producing organisms (such as Aspergillus oryzae and Pseudomonas sp.) in gelatine pellets with a half-life of up to 70 days have already been developed (Yuan et al. 2002). The method’s non-transformed D-N-acetyl-methionine is racemized with acetic anhydride and then circulated (Wöltinger et al. 2005). Through this method, Rexim® in Nanning, China, a subsidiary of German company Evonik, produces several hundred tonnes of pharmaceutical-grade L-methionine annually. Fermentation Another technique for enantiomerically producing methionine is the fermentative transformation of biologically or chemically manufactured precursors. The

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enzymatic bifurcation of 5′-monosubstituted hydantoin derivatives manufactures the enantiomerically unalloyed AAs. For a hydantoinase-based method that produces optically pure L-methionine utilizing D-5-(2-methylthioethyl) hydantoin as a precursor, Degussa attempted to genetically optimize enzymes through directed evolution in the late 1990s (Wagner et al. 1996; May et al. 2000, 2002). Additional CJ Intellectual property describes the enzymatic metamorphosis of the precursor O-acetylhomoserine (Willke 2014). Under the right circumstances, many bacteria and yeasts can overproduce AAs. Few strains can synthesize meaningful amounts of methionine due to the L-methionine syntheses’ extremely complicated regulatory structure. As a result, they typically need to go through numerous iterations of process improvement, genetic manipulation, or mutation and selection. Methionine can also be directly manufactured by aspartate using one ATP and two NADPH. Including oxidized inorganic sulfate also necessitates the supply of 1 GTP, 2 ATP, and 4 NADPH. This demonstrates the sulfur source’s significant influence. If reduced sulfur is utilized, immediate absorption of these sulfur resources to methionine may promote energy equilibrium. There is proof that using this route (shortcut) could significantly increase methionine production (Lievense 1993; Kiene et al. 1999; Krömer et al. 2006; Bolten et al. 2010). Chitin, the far more ubiquitous biodegradable polymer on the planet, may be broken down to generate glucosamine, a potential biological nitrogen source for methionine fermentation (Himmel et al. 2007). Screening for Strains and Enhancement Numerous experiments have been conducted using traditional screening techniques to determine whether naturally occurring bacteria or yeast produce excess methionine, either inwardly retained or expelled into the medium. The findings of the investigations have led to increased efforts to expedite screening. Until it was discovered that Met analogues, such as DL-ethionine (ET), α-methyl-DL-methionine (AMM), and DL-norleucine (NL), can operate as negative controllers without interfering with other crucial cellular processes, they were often frequently utilized as markers to find Met overproducers (Rowbury and Woods 1961; Lawrence et al. 1968). Due to flaws in the feedback regulation, organisms that thrive in the presence of Met analogues are visibly resistant and, as a result, should produce an abundance of methionine. A single gene (NCgl2640) that encodes for a carboxylate-amine ligase was discovered, and this gene was responsible for the resistance to ethionine in the first venture to appreciate the inhibitory mechanism of ethionine in C. glutamicum. Ethionine resistance was provided by the knockout of NCgl2640 (Gomes and Kumar 2005). It has been demonstrated that protoplast fusion effectively transfers valuable industrial traits from yeast, such as osmotolerance or substrate consumption (Legmann and Margalith 1986; Pina et al. 1986). It is challenging to control the genes that are part of the complex, highly regulated methionine pathway. To augment the methionine concentration of seedlings, bacteria or yeasts were introduced to plants genetically modified in the middle of the 1980s (Altenbach et al. 1989). In 2003, after the nucleotide sequence of C. glutamicum had already

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been sequenced, meticulous and systematic genome modification was accomplished, and it provided quality products by systems biology techniques (Willke 2014; Kalinowski et al. 2003).

7.1.2

L-Glutamate

L-glutamate

(Glu), among the most frequent AAs in animal and food structures, has sparked the curiosity of nutritionists due to its essential participation in metabolic and physiological activities (Blachier et al. 2009; Brosnan and Brosnan 2013; Hu et al. 2017; Wu 2017). Recently, it has been recognized that Glu is a nourishingly necessary AA for newborn intestinal and overall body homeostasis (Hou and Wu 2017; Rezaei et al. 2013). Considering that glutamate is recognized as a nonessential AA, sentient beings may manufacture enough of it. It must be produced in vivo. This is proven by thorough balance experiments conducted on the intestines of various animals, which show that enterocytes, primarily, are responsible for the almost quantitative metabolism of dietary glutamate within the intestine. Proteins receive a negative charge from glutamate, which might be essential for preserving the structure of the protein. For instance, ion pairs comprising glutamyl residues are necessary to stabilize the leucine zipper configuration of the transcription factor GCN4 (Matousek et al. 2007). The outside of globular proteins frequently contains charged residues, like glutamate. In addition to being integrated into proteins during synthesis, glutamate can be added as a posttranslational modification through polyglutamyl tails. The glutamate affinity for calcium could be significantly increased by a vitamin K-dependent carboxylation that happens post-translation and introduces α-carboxylated glutamyl residue within proteins. In proteins, albeit very sparingly, glutamate binds cations (Berkner 2005). L-Glutamate production by utilizing the fermentation process has increased with the demand for this AA in the commercial market.

7.1.2.1 Biosynthetic Pathway of L-Glutamate It was formerly believed that C. glutamicum produces so much L-glutamate that it possesses unique L-glutamate biosynthetic pathways. Amination of 2-oxoglutarate results in the production of glutamate. GDH, glutamine synthase, and glutamine synthetase are interrelated enzymes that produce amination (Ertan 1992; Kimura 1962; Sung et al. 1984, 1985). While the GS/GOGAT system or GDH can support cell development, GDH causes glutamate overproduction (Sung et al. 1985). Glycolysis, a step in the Krebs cycle, converts carbon from sugar to 2-oxoglutarate. Despite the complexity of the flux surrounding oxaloacetate, the role of the contributing enzymes has been identified. These enzymes’ characteristics and regulation have also been documented (Mori and Shiio 1985; Shiio et al. 1977; Delaunay et al. 2004; Eikmanns et al. 1995; Sato et al. 2008; Shiio and Ujigawa 1978). The crucial branch point is 2-oxoglutarate, from which glutamate is formed by amination and degraded by oxidative decarboxylation to succinate with the help of a complex

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Fig. 7.2 Biosynthetic pathway of L-glutamate (Abbreviations: LDH lactate dehydrogenase, PFL pyruvate-formate lyase, PEPC phosphoenolpyruvate carboxylase, CS citrate synthase, ACN aconitase, GDH glutamate dehydrogenase, ICDH isocitrate dehydrogenase, GS glutamine synthase, GOGAT glutamine oxoglutarate aminotransferase/glutamate synthase)

called 2-oxoglutarate dehydrogenase (ODHC). The center of attention for flux control is ODHC (Fig. 7.2).

7.1.2.2 Glutamate-Producing Microorganisms Numerous species, including Brevibacterium lactofermentum, Corynebacterium lilium, Corynebacterium callunae, Brevibacterium flavum, etc., were asserted to be glutamate-producing microbes. Most were gram-positive, rod-shaped, nonsporeforming, aerobic, nonacid-fast, and biotin that required bacteria. Through careful taxonomic analysis, these “novel” species were later included in the species of Corynebacterium glutamicum (Liebl et al. 1991; Rytter et al. 2014; Yamada and Komagata 1972). The amount of GA generated by C. glutamicum is tremendous, and the strain of C. glutamicum that is no more AAs is produced by the wild type. Thus, these bacteria are referred to as C. glutamicum moving forward. Within a few years after the bacteria’s discovery, it was widely accepted that, given the right circumstances, the bacterium synthesized glutamate at a yield of more than 40% against input sugar. There have also been reports of bacteria unrelated to C. glutamicum that can synthesize glutamate from sugar. According to Chao and Foster, Bacillus megaterium produced 13.5 g/L of productivity from 3% glucose (Chao and Foster 1959). It is intriguing that this strain also produces biotin. It has also been observed that some strains of Streptomyces (Chatterjee and Chatterjee 1982) and Arthrobacter (Roy and Chatterjee 1982) synthesize glutamate. It was recently revealed that Pantoea agglomerans, Klebsiella planticola, Enterobacter

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Table 7.2 GA-producing microorganisms with their yield Microbial strain Bacillus subtilis CCTCC202048 Bacillus amyloliquefaciens C1 Corynebacterium glutamicum Bacillus megaterium B. subtilis (natto) B. subtilis IFO 3335

Yield (g/L) 83.61 g/kg of dry substrate 0.0437 g/g of substrates 85 13.5 35 20

B. licheniformis ATCC 9945 Brevibacterium divaricatum

23 3.86% based on the weight of residues 2

Brevibacterium lactofermentum 2256

References Jian et al. (2005) Yong et al. (2011) Delaunay et al. (1999) Chao and Foster (1959) Ogawa et al. (1997) Kunioka and Goto (1994) Troy (1973) Jyothi et al. (2005) Momose and Takagi (1978)

agglomerans, and E. coli all produce a significant amount of glutamate from metabolic engineering (Hashimoto 2016; Imaizumi et al. 2006) (Table 7.2).

7.1.2.3 Substrate for Glutamate Production Glucose, fructose, and sucrose are quickly metabolized by C. glutamicum, and they are first employed as a carbon source. After identifying a different method of biotin constraints, molasses, a somewhat more commercially enticing source, were usually employed since it contains an excessive amount of biotin. Furthermore, it has been asserted that the bacterium produces more when introduced to ethanol and acetic acid. Recently, it was shown that by expressing heterologous genes, the range of useable sugar in C. glutamicum can be increased (Schneider et al. 2011). Petrochemically produced carbohydrates were investigated as an alternate carbon source. Researchers have identified numerous bacteria capable of converting paraffin into glutamate (Shiio and Uchio 1969). The productivity of C. hydrocarboclustus was reportedly relatively high. The manufacture of n-alkane has been described by researchers by the Serratia marcescens strain (Ghosh and Banerjee 1986). Additionally, benzoate and other aromatic compounds have been researched as potential carbon sources (Hong et al. n.d.; Nampoothiri and Pandey 1995; Revillas et al. 2005). Since the 1970s, glutamate fermentation utilizing methanol has been endeavored (OKI et al. 1973; Ju 1976). Different bacteria, including Methylobacillus glycogenes (Motoyama et al. 1993), Methanomonas methylovora (OKI et al. 1973), and B. methanolicus (Brautaset et al. 2010), have been investigated and employed because C. glutamicum is unable to assimilate methanol. It indicates that the strain B. methanolicus M168-20(pHP13) produces the most from utilizing methanol. Algae have also been used to study glutamate production during photosynthesis (when the carbon source is CO2) (Bagchi and Rao 1997; Matsunaga et al. 1988).

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7.1.2.4 Glutamate Production Strategies Fermentation It is intriguing how coryneform bacteria produce too much L-glutamate. These bacteria need biotin to develop, and when too much biotin is present during culture, L-glutamate does not build up in the media. However, in a biotin-restricted medium, these bacteria create a sizable amount of L-glutamate (Shiio et al. 1962). Some types of detergents can cause glutamate overproduction even when there is an excess of biotin. Introducing a surfactant, polyoxyethylene sorbitan monostearate or polyoxyethylene sorbitan monopalmitate, causes coryneform bacterium to ramp up the production of L-glutamate (Takinami et al. 1965; Duperray et al. 1992). Specific beta-lactam antimicrobial agents, notable penicillin, are known to promote L-glutamate production similarly to surfactants (Nunheimer et al. 1970). Variants auxotrophic for glycerol or fatty acids frequently generate excessive proportion of Lglutamate in the fermentation broth (Kanzaki et al. 1967; Okazaki et al. 1967; Nakao et al. 1970). Analysis of enzyme activity has suggested the significance of flow variations. Investigations into the connection between L-glutamate and 2-oxoglutarate dehydrogenase complex (ODHC) activity productivity (Shingu 1971) have shown that the particular ODHC activity is decreased when L-glutamate mass production is brought on by biotin deficiency. Using the Krebs cycle, the synthesis of L-glutamate and energy generation are connected metabolically, and this is where ODHC operates. The metabolic flow is assumed to be biased toward the synthesis of L-glutamate when ODHC activity is decreased (Kawahara et al. 1997). Gene Modifications Since more than 30 years ago, coryneform bacteria have been investigated for their propensity to synthesize excessive amounts of AAs, and as early as the middle of the 1980s, reports of gene modification techniques were made. To study and control the metabolic flow of AA synthesis, the fundamental plasmids cloning vectors and tools, gene expression systems, and gene transfer strategies for coryneform bacteria have been created (Jetten and Sinskey 1995). Almost all the genes encoding the enzymes that produce L-glutamate have been cloned, apart from those involved in sugar metabolism and energy. DtsR1 and DtsR2 are homologous, while AccBC is thought to represent a DtsR1 counterpart (Jäger et al. 1996). It appears that AccBC and DtsR1 create a complex with molecules of biotin that is necessary for coryneform bacteria to synthesize fatty acids. Metabolic Flux Perusal of Glutamate Overproduction The synthesis of AAs, particularly L-lysine, has gradually grown over the past few decades due to advancements in fermentation processes and modifications to bacterial strains made possible by genetic engineering. However, the metabolic restrictions connected to the synthesis of L-glutamate have not been thoroughly defined. In order to study the glucose metabolism in C. glutamicum, 13C NMR spectroscopy was used. Additionally, the distribution of carbon flux via C. glutamicum’s key metabolic pathways during the development of fructose was

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investigated. According to NMR studies utilizing fructose, glycolysis was the primary path during the exponential development of fructose, and the contribution of the pentose route was significantly lowered. Throughout fructose development, an enhanced flux across glycolysis was associated with a higher NADH/NAD+ ratio (Dominguez et al. 1998). Approximately 80 g/L of L-glutamate was formed, whereas other compounds like trehalose, lactate, and dihydroxyacetone were also present. When the synthesis of L-glutamate decreased, the intercellular ratios of glycolytic intermediates and the NADH/NAD+ ratio dramatically raised (Gourdon and Lindley 1999). Metabolic flux research has recently shown that the loss of ODHC activity does have the most considerable influence on flux allocation at the metabolic junction point of 2-oxoglutarate glutamate (Kimura 2005).

7.1.3

L-Lysine

To fulfill the dietary requirements of both people and animals, diets should contain enough vital AA lysine. Livestock feed, which frequently consists of wheat, corn, or barley, is deficient in lysine. This replenishment is accomplished through the direct addition of lysine, and as a corollary, the trade has grown significantly over the last 10 years. Over 600,000 metric tonnes of lysine are reportedly produced each year, and the industry is predicted to rise by 7–10% yearly due to the emergence of new applications in pharmaceuticals, cosmetics, and polymer materials (Félix et al. 2019). Because it provides a somewhat more affordable option, lysine has been predominantly introduced to retail as a flavor enhancer. Nevertheless, adding AA supplementation is possible to lower the protein content of feed even while satisfying the animals’ nutritional demands. It enhances the growth potential and protein synthesis in the tissues of fish, poultry, and pigs (Eggeling and Bott 2015; Hamid et al. 2016; Nguyen and Davis 2016). Since only the L-isomer of lysine is suitable as a supplementary feed, all fabrication procedures involve fermentative synthesis (Leuchtenberger 2001). Japan is the birthplace of biotechnological lysine manufacturing. A significant screening campaign was started in Japan around half a century ago in response to the rising demand for AAs, particularly glutamate. Due to this, the glutamate-emitting bacterium Corynebacterium glutamicum was discovered in 1956 (Wittmann and Becker 2007; Udaka 1960). The first C. glutamicum mutants that excreted lysine were accessible and used for large-scale manufacturing within a few years (Kelle et al. 2005; Shukuo et al. 1961). Since the significant demand for this molecule needed today has been successfully met by manufacturing lysine from renewable energy resources (De Graaf et al. 2001; Eggeling and Sahm 1999; Hermann et al. 2001; Wittmann and Becker 2007), it has been accomplished by ongoing processes and producing strain refinement.

7.1.3.1 Biosynthetic Pathways of L-Lysine Two distinct pathways can manufacture lysine in microorganisms: aspartate via the diaminopimelate pathway or 2-oxoglutarate via the aminoadipate pathway (Velasco

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Fig. 7.3 Biosynthetic pathway of L-Lysine

et al. 2002). Higher fungi and archaea like Thermus thermophilus and Thermoproteus neutrophilus exhibit two variations of the aminoadipate pathway. Lysine can be produced from aspartate in bacteria and plants via one or more diaminopimelate pathway types (Fig. 7.3). These pathways’ variations split at the tetrahydrodipicolinate common intermediate (Schrumpf et al. 1991; Born and Blanchard 1999; McCoy et al. 2006). Succinylated intermediates are present in one of these processes, acetylated intermediates are present in the acetylase pathway, and dehydrogenase pathway immediately converts tetrahydrodipicolinate to DLdiaminopimelate (Schrumpf et al. 1991; Wehrmann et al. 1998). Tetrahydrodipicolinate is converted to diaminopimelate-11 via the aminotransferase pathway, which has recently been shown to function in Chlamydia (McCoy et al. 2006). Most bacteria only have one of these routes (Bartlett and White 1985; White 1983). Although gram-negative and gram-positive microorganisms use the succinylase route, some Bacillus species are now the only ones using the acetylase variant (Bartlett and White 1985; Born and Blanchard 1999). Only a few organisms, such as the genus Corynebacterium and Bacillus macerans, are known to have two

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lysine biosynthetic routes that work in concert (Bartlett and White 1985; Schrumpf et al. 1991; Malumbres and Martin 1996).

7.1.3.2 L-Lysine-Producing Microorganism The most significant organism for commercial lysine synthesis is Corynebacterium glutamicum, including its subspecies, Corynebacterium lilium, Corynebacterium efficiens, and Brevibacterium divaricatum, Brevibacterium flavum, and Brevibacterium lactofermentum (Liebl 2005). Recombinant E. coli strains are the only other species employed for synthesizing lysine (Imaizumi et al. 2005; Imaizumi et al. 2006). In the 1950s, it was discovered that C. glutamicum could secrete AAs (Shukuo et al. 1961; Wittmann and Becker 2007). It is an aerobic, rod-shaped, grampositive, nonmotile microbe. Its genome has a GC content of 53.8% (Kalinowski et al. 1991; Kalinowski et al. 2003). The genomic sequencing was recently decoded (Wittmann and Becker 2007; Ikeda and Nakagawa 2003; Kalinowski et al. 2003; Tauch et al. 2005), and extensive biochemical studies over the past few decades have given rise to a detailed understanding of the central metabolism and pathway of lysine biosynthesis in this microorganism (Table 7.3). 7.1.3.3 Substrate for Lysine Production Carbon feedstock is the primary source of marginal costing in the large-scale manufacturing of AAs. Starch and molasses are the most suitable substrates for the industrial synthesis of lysine. Their primary carbon sources are glucose, fructose, and sucrose. Phosphoenolpyruvate-dependent phosphotransferase systems take these chemicals up (Dominguez and Lindley 1996; Dominguez et al. 1998; Malin and Bourd 1991; Moon et al. 2005). Considering that 3% upwards of unhydrolyzed maltodextrins are present in maize or wheat hydrolysate glucose syrup, utilizing these maltodextrins to produce L-lysine boosts economic viability. Table 7.3 Lysine-producing microorganisms and their yield Microbial strain Corynebacterium glutamicum Brevibacterium lactofermentum Bacillus laterosporus Bacillus methanolicus MGA3 Escherichia coli Methylophilus methylotrophus B. lactofermentum AJ12592 E. coli W3110tyrA/pCABD2 C. glutamicum H-8241

Yield (g/L) 4.0 0.343 g/g 5.67 11.0 1.8 11.3 120.5 12.23 48

References Becker et al. (2011) Yao et al. (2001) Umerie et al. (2000) Jakobsen et al. (2009) Ye et al. (2020) Gunji and Yasueda (2006) Shiratsuchi et al. (1995) Ikeda (2003) Pandey et al. (2020)

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7.1.3.4 Lysine Production Strategies Fermentation In the large-scale commercial manufacturing of lysine, there are operations for upstream procedures, fermentation process, and purification processes. Production procedures comprise testing raw resources, their supply and preservation, development of medium from the building ingredients, and generation of the inoculum for production. The predominant economic carbon sources used to make lysine include sucrose, dextrose, cane molasses, and beet molasses; the latter is produced by hydrolyzing starch (Ikeda 2003). The primary inoculation together in a bioreactor that has already been filled with growth media and the ultimate extraction of the biomass or products beneath regulated pH and temperature parameters are two hallmarks of batch culture. Batch cultivation of microorganisms frequently results in catabolic suppression or osmotic stress because of the expensive and extensive quantities of carbon and nitrogen supplies needed for AA synthesis (Sassi et al. 1998). A fascinating alternative is fed-batch culture conductivity, which reduces yield losses imposed by hazardous antecedents or catabolic suppression. It necessitates a rise in biomass concentration and bulk density manufacturing because the presence of carbon or nitrogen resources during the brewing process may alter the culture’s physiological condition and kinetic parameters (Sassi et al. 1998; Skjerdal et al. 1995; Kawahara et al. 1990). Continuous culture seems to be another alternative for avoiding problems brought on by osmotic stress since this functions with continual nutrient input and product removal and is capable of reaching a stable state that would raise yield about processes in fed-batch or batch (Sassi et al. 1998; Kelle et al. 1996). Genetic Engineering The theoretical highest productivity of a C. glutamicum cell that has the capacity to produce lysine is essential because it estimates the amount of room for further process improvement in an operational industrial process and offers guidance to process or genetic engineers. It is simple to grow, simple to operate genetically, widely accepted as safe, and highly resilient to fluctuations in oxygen and substrate availability that are common in industrial scale fermentations (Buchholz et al. 2014; Käß et al. 2014). All of this helps make C. glutamicum an impeccable microorganism for studying new notable biochemical properties within the Corynebacteriales and being a producer and platform organism in biotechnology. Three enzymes— lysine exporter (LysE), dihydrodipicolinate synthase, and aspartate kinase (LysC)— were altered to produce more lysine. Because it is susceptible to feedback regulation by lysine, aspartate kinase is an essential enzyme for the energy metabolism of the lysine cascade (Kalinowski et al. 1991; Malumbres and Martin 1996). Various point mutations in the lysC gene, particularly within the region expressing its regulatory subunit, have been found to free the enzyme from feedback control and enhance lysine production (Cremer et al. 1991; Follettie et al. 1993; Kalinowski et al. 1991; Sugimoto et al. 1997).

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Riboflavin (Vitamin B2)

The isoalloxazine ring produces a group of yellow chemical compounds with pteridine bases called flavins. All flavins with biological significance originate from riboflavin (RF), also known as vitamin B2. This was identified as a yellow milk pigment, and its elemental composition was uncovered in the beginning of the twentieth century (Abbas and Sibirny 2011). The functional domains in flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are required by most flavoproteins and flavo-coenzymes. These flavo-coenzymes are necessary for several physiological activities, including redox homeostasis, AA oxidation, fatty acid oxidation, DNA repair, protein folding, and choline metabolism (Mansoorabadi et al. 2007; Walsh and Wencewicz 2013; Macheroux et al. 2011; Frerman 1988; Crane III et al. 1995). Flavins are rarely discovered as free RF despite being abundantly dispersed through tissues. The bulk, however, is linked to flavoproteins, principally as FAD and in abate as FMN (Joosten and van Berkel 2007; Lienhart et al. 2013). The primary industries that use commercialized RF are those in the food, feed, and medicinal sectors. The possibilities for small-scale RF production by probiotic LA bacteria could enable the development of cereal- and dairy-based healthy ingredients for the in situ delivery of RF to individuals (for example, Lactobacillus Plantarum) (Thakur et al. 2016a, b; Wu and Zhang 2019; Thakur and Tomar 2016; Levit et al. 2017; Burgess et al. 2006). Night blindness, impaired iron metabolism, and a higher risk of cardiovascular disease are all possible effects of RF deficiency. In situations of malnutrition or as a medication for labialis, a circumstance characterized by lesions in the corners of the mouth, around the nose and eyes, and on the lips, flavins are used as general health supplements (Yatsyshyn et al. 2009). Currently, RF is produced industrially only by biotechnological fermentation without the help of chemical modification. Starting with D-glucose or D-ribose, the fundamental synthesis method of RF comprises six to eight steps.

7.1.4.1 Biosynthetic Pathway of RF Most bacteria, as well as all fungi and plants, can produce RF (Abbas and Sibirny 2011). The enzyme that the RF synthesis operon codes for catalyzes a seven-step enzymatic reaction to produce RF from ribulose-5-phosphate and GTP. The primary stage in manufacturing RF is the hydrolysis of GTP into 2,5-diamino-6ribosylamino-4 (3H)-pyrimidinedione 5′-phosphate (Schwechheimer et al. 2016). The bifunctional enzyme produced by the ribA gene functions as both 3,4-dihydroxy-2-butanone 4-phosphate synthase and GTP cyclohydrolase in bacteria like B. subtilis and E. coli (Ren et al. 2005). A bifunctional pyrimidine deaminase/reductase encoded by ribD deaminates and reduces the intermediate DARPP (2,5-diamino-6-ribosylamino-4(3H)-pyrimidinedione) to create ArPP (5-amino-6ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate). The second stage of the dephosphorylation reaction, which transforms ArPP into ArP (5-amino-6ribitylamino-2,4(1H,3H)-pyrimidinedione), is catalyzed by hydrolases. The intermediate products DHPB and ArP are combined to form the immediate precursor of

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Fig. 7.4 Biosynthetic pathway of RF. Ribu5P ribulose-5-phosphate; ArP 5-amino-6-ribitylamino2,4-(1H,3H)-pyrimidinedione; DARPP 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinedione phosphate; ribo5P ribose-5-phosphate; DarPP 2,5-diamino-6-ribitylamino-4-(3H)-pyrimidinedione-5′-phosphate; DRL 6,7-dimethyl-8-ribityl-lumazine; ArPP 5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione-5′-phosphate; DHBP 3,4-dihydroxy-2-butanone-4-phosphate

RF DRL (6,7-dimethyl-8-ribityllumazine), which is then processed by RF synthase to produce the end product RF (Fig. 7.4).

7.1.4.2 RF-Producing Microorganism Today, microbial fermentation is the only commercially used method to synthesize RF [192]. Most of the microbes utilized in commercial RF production are from the flavinogenic fungi A. gossypii and the bacteria B. subtilis. But other species, such as C. ammoniagenes, C. famata, Pichia guilliermondii, E. coli, and Eremothecium gossypii, are also manipulated to produce RF. RF typically has a titer of 26–30 g/L in industrial processes (Schallmey et al. 2004; Lee et al. 2006). Usually, numerous rounds of genetic engineering were combined with mutagenesis to get the industrial RF production strains. The mutagenesis procedure includes several screening procedures for antimetabolite resistance. In assessing RF mass-producing B. subtilis mutants, the structural analogues of RF, such as roseoflavin; various purine analogues, such as 8-azaxanthine, thioguanine, 8-azaguanine, and decoyinine; and similarly glutamine antagonist methionine sulfoxide have all been successful (Perkins et al. 1999; Pedrolli et al. 2014). A. gossypii has been treated effectively using antimetabolites of oxalate and itaconate in addition to inhibitors of isocitrate lyase while not always being responsive to structural analogues of purines or RF (Schmidt et al. 1996a, b; Park et al. 2007; Sugimoto et al. 2010; Tajima et al. 2009). Analogous mutagenesis was also successful in isolating the RF mass manufacturer A. gossypii (Table 7.4).

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Table 7.4 RF-producing microorganisms and their yield Microbial strain Clostridium acetobutylicum Bacillus subtilis RB50 Bacillus subtilis KCCM 10445 C. ammoniagenes B. subtilis RH44 Lactobacillus fermentum KTLF1 Corynebacterium glutamicum KCCM 1223P A. gossypii Eremothecium ashbyii

Yield (g/L) 0.097 15 26.8 15.3 16.36 2.36 245 5.0 2.85

References Leviton (1946) Sauer et al. (1996) Lee et al. (2006) Koizumi et al. (2000) Wu et al. (2007) Thakur et al. (2016b) Tajima et al. (2009) Szcześniak et al. (1971) Kalingan and Liao (2002)

7.1.4.3 Substrate for RF Production Fungi like A. gossypii use oil as a carbon source, whereas bacteria like E. coli and B. subtilis utilize carbohydrates as a carbon source. A. gossypii breaks down the oil into glycerol and fatty acids via extracellular lipase. Additionally, fatty acids undergo oxidation to produce acetyl coenzyme A, which is then transformed into glucose-6-phosphate (G6P) by the Krebs cycle, gluconeogenesis, and glyoxylate cycle. The pentose phosphate pathway (PPP) in bacteria is an efficient mechanism for glucose to be converted to G6P. The purine, pentose phosphate (PP), and RF fabrication processes, which are comparable in bacteria and fungi, are then used to convert G6P to RF. An advantage of using plant oil as a feedstock is decent production. Triglycerides provide a high feedstock charge in the bioreactor due to their osmotic neutrality and adaptability as an oil or solid fat. 7.1.4.4 Production Strategies for RF Chemical Synthesis Most industrial RF synthesis is still produced chemically. To start the procedure, 3,4-xylidine and D-ribose are combined in methanol. The hydrogenation of the riboside results in the production of N-(3,4-dimethylphenyl)-D-1-ribamine. The newly formed azo compound couples with a phenyl diazonium halogenide to create RF in a cyclo-condensation with barbituric acid. Aniline is removed in this process. Chemically produced RF has a footprint of traces of aniline. The procedure’s primary drawbacks include a maximum yield of just around 60%, which results in much waste, the need for organic solvents, and the need for 25% more energy than the Bacillus approach (Stahmann et al. 2000). Biotechnological Production RF could be manufactured in bulk amounts by both chemical synthesis and fermentation. The fermentation process makes it possible to produce vitamin B2 in just one step at a low cost. On the other hand, chemical procedures need many steps and are expensive. As a result, the chemical synthesis of RF is no longer used because fermentative production is more practical from an economic and environmental

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standpoint (Schwechheimer et al. 2016). A significant portion of the RF presently available on the market—roughly 70%—is used as vitamin B2, a feeding supplement manufactured through fermentation using genetically engineered microbes. International industries produce RF from the Candida famata var. flareri, Ashbya gossypii, and Bacillus subtilis, reaching titers of up to 14–20 g/L (Lim et al. 2001; Revuelta et al. 2016). Even though fermentation is the only method utilized for industrial RF synthesis, most microbes used in this sector were obtained by mutagenesis, which left their genetic backgrounds unclear. Through a variety of methods, including screening of transposon-tagged mutants (Tännler et al. 2008) and transcriptome analysis (Shi et al. 2009), similarly most recently delineated transcriptome sequence analysis, and integrated whole-genome (Wang et al. 2018) and metabolic flux analysis (Schwechheimer et al. 2018; Jeong et al. 2015), studies have identified the genes and genetic components accountable for the RF mass manufacture phenotype. Metabolomics, a novel “omics” approach, may help elucidate the specifics of the flavin overproduction mechanism. Genetic Modifications Several research teams successfully accomplished the creation of genetically altered strains of species like Escherichia coli, B. subtilis, Candida spp., and Corynebacterium ammoniagenes over the past few decades using metabolic engineering techniques. In turn, this has enhanced strain efficiency with the yield of the commercialized fermentation output. The amplification of regulatory and structural genes responsible for forming its precursors or RF itself becomes a more prevalent psychological effect of such approaches (Perkins et al. 1999; Koizumi et al. 2000; Taniguchi and Wendisch 2015; Wang et al. 2015). Because the bulk of the economically successful RF overproducers was developed utilizing a combination of genetic engineering and conventional mutagenesis, it is imperative to pinpoint the genetic features that give rise to RF overproduction. Omics approaches, transposon-tagged mutagenesis, and metabolic flux analyses have been used to identify the genetic features that are associated with the RF overproduction phenotype. Furthermore, certain innovative methods have been opportunely used to strengthen RF production. To comprehend the genetic alterations in a B. subtilis strain that produces excessive amounts of RF, transcriptome analysis was also used (Shi et al. 2009). It was discovered that the overproducer had downregulated levels of the pur operon and other PurR-regulated genes. According to the research, co-overexpression of the genes for ribose-5-phosphate isomerase B and PRPP synthetase increased the RF titer by 25%. RF-overproducing B. subtilis underwent transcriptome sequence analysis and a second integrated whole genome, which identified positive mutations in the genes ribD + (G + 39A), ribC (G199D), ccpN (A44S), purA (P242L), and yvrH (R222Q) (Wang et al. 2018). Notable is the ability of the yvrH (R222Q) mutation to deregulate the purine route for enhanced RF generation.

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Vitamin B12

The cobalamin family of substances, including the vitamin B12 molecule cyanocobalamin, comprises an upper and a lower ligand and a corrinoid ring. The upper ligand can be the adenosine hydroxy, cyano, or methyl group (Roth et al. 1993). Prokaryotes produce vitamin B12, which prevents mammals from developing pernicious anemia. In bacteria and archaea, the aerobic or anaerobic pathway is used for the microbial de novo production of vitamin B12. Certain strains have a salvage mechanism that enables them to produce cobalamin by consuming corrinoids. In many bacterial species, there is a complex interactional and interdependent link between the tetrapyrrole molecules cobalamin, heme, and bacteriochlorophyll, which are all produced from δ-aminolevulinate (ALA) (Yin and Bauer 2013). To maintain consistent amounts of vitamin B12, a cobalamin riboswitch is in the 5′ untranslated regions (UTRs) of the necessary genes. Finding foods with high levels of vitamin B12 is crucial to prevent vitamin B12 deficiency in high-risk groups, including vegetarians and older adults. Only a few types of bacteria and archaeans can produce vitamin B12; neither plants nor animals can. Through microbial contact, the produced B12 is distributed and accumulates in animal and plant tissues. Humans can get adequate B12 from the meat and milk of herbivorous ruminant animals. Through a symbiotic interaction with body bacteria, ruminants obtain the vital B12 nutrient. Therefore, we also rely on B12-producing bacteria found in the stomachs of ruminants. While edible plants and mushrooms infrequently have a significant amount of B12, this is primarily because of associated bacteria on their aerial surfaces and in the soil.

7.1.5.1 Biosynthetic Pathway for Vitamin B12 Production Adenosylcobalamin is made from tetrapyrrole molecules via the salvage pathways or de novo, while ALA is made from them via the C4 or C5 pathway. The enzymes used in the manufacture of adenosylcobalamin come from the organisms. In the process that results in tetrapyrroles, ALA is the first committed precursor. ALA can be produced using the C5 or C4 pathways. The C4 pathway uses the enzyme ALA synthase to catalyze the conversion of glycine and succinyl-CoA into ALA. Three enzyme activities turn glutamate into ALA in the C5 pathway (Avissar et al. 1989). The enzyme porphobilinogen synthase transforms two molecules of ALA into monopyrrole porphobilinogen, which is then polymerized and cyclized to form uroporphyrinogen III. This process is catalyzed by the enzyme’s uroporphyrinogen III synthase and porphobilinogen deaminase. Precorrin-2, a common precursor of siroheme, cobalamin, and coenzyme F430, is created when uroporphyrinogen III is methylated at locations C-2 and C-7 (Zappa et al. 2010; Martens et al. 2002). CobA catalyzes this methylation process in P. denitrificans. The fusion enzyme CysG is shared by siroheme and cobalamin production in E. coli and S. typhimurium. Dehydrogenase/ferrochelatase activity is present in CysG’s N-terminus, and uroporphyrinogen III methyltransferase activity is present in the C-terminus. MET1p serves as a uroporphyrinogen III methyltransferase in S. cerevisiae (Raux et al. 1999). Precorrin-2 is where the anaerobic and aerobic routes split off, and coby

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(II)rinic acid a,c-diamide is where they come together. During de novo cobalamin biosynthesis, eight peripheral methylation processes occur in the same temporal and geographical order in both the anaerobic and aerobic pathways. There is a lot of sequence similarity among the methyltransferase enzymes involved in these processes (Escalante-Semerena and Warren 2008). The final phase of vitamin B12 production has two opposing theories. According to one theory, cobalamin synthase catalyzes the addition of α-ribazole as the final process in the biosynthesis of AdoCbl. The final reaction, however, in S. typhimurium would be the dephosphorylation of AdoCbl 5′-phosphate to AdoCbl, which is carried out by an AdoCbl-5-P phosphatase. This is because in this organism, α-ribazole 5′-phosphate is added to AdoCbi-GDP (Zayas and Escalante-Semerena 2007). The final reaction in the production of vitamin B12 which has been defined is the nucleotide loop assembling route. FMNH2 can be converted to DMB by the reduced version of the nicotinamideadenine dinucleotide (NADH)/flavin mononucleotide (FMN)-dependent nitroreductase family, particularly encompassing BluB from S. meliloti. (Taga et al. 2007). Enzymes expressed in the bza operon convert 5-aminoimidazole ribotide to DMB in the anaerobic microbe E. limosum (Mehta et al. 2015); as a result, CobT can activate a variety of lower ligand substrates, including DMB, which determines carbamide diversity (Hazra et al. 2013) (Fig. 7.5).

7.1.5.2 Microorganisms Producing Vitamin B12 The commercial manufacture of vitamin B12 only uses biosynthetic fermentation procedures since the chemical mechanisms for making vitamin B12 are extraordinarily complex and expensive. There are two distinct methods for producing vitamin B12 by microorganisms: aerobic fermentation and anaerobic fermentation. Some industrial bacteria, such as Pseudomonas denitrificans, Propionibacterium freudenreichii, Propionibacterium shermanii, and others, have been effectively used for the industrial synthesis of vitamin B12 by utilizing the techniques of random mutation and genetic engineering (Survase et al. 2006; Kang et al. 2012). Because Propionibacterium strains often produce vitamin B12 anaerobically, their bioprocesses must be regulated in environments with low oxygen concentrations (Wang et al. 2012b). The aerobic P. denitrificans fermentation procedures outperformed the anaerobic Propionibacterium fermentation processes regarding cell proliferation and vitamin B12 output. Propionibacterium freudenreichii subsps. shermanii is a probiotic and is frequently used to manufacture vitamin B12 (Gardner and Champagne 2005). Despite a long history of usage in cheese manufacturing, dairy propionibacteria are also used as a biosynthesis host for enzymes, antimicrobials, and nutraceuticals like propionicin (Jan et al. 2002) (Table 7.5). 7.1.5.3 Substrate for Producing Vitamin B12 The only source of VB12 is microbes. Propionibacterium and Pseudomonas species are used in the fermentation process to manufacture VB12 (Martens et al. 2002; Kośmider et al. 2012). Propionic acid and acetic acid are two extracellular products produced by Propionibacterium freudenreichii (P. freudenreichii) during the fermentation process used to create food-grade VB12 (Hajfarajollah et al. 2014).

Fig. 7.5 The process by which adenosylcobalamin is made. Black represents the E. coli endogenous enzymes. Orange represents the enzymes from aerobic bacteria like B. melitensis, S. meliloti, R. palustris, and R. capsulatus. Blue indicates S. typhimurium enzymes. Adenosyl is referred to by the abbreviation “ado” (Fang et al. 2018)

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Table 7.5 Vitamin B12-producing microorganisms and their yield Microbial strain Propionibacterium freudenreichii Pseudomonas denitrificans MB2202 Pseudomonas denitrificans B. megaterium ATCC 13639 E. aerogenes P. denitrificans Pseudomonas denitrificans

Yield (g/L) 1.7 mg/L 14.7 13.7 5.04 μg/100 g of substrate 4.35 μg/100 g of substrate 0.2 7

References Piao et al. (2004) Lago and Demain (1969) Lago and Demain (1969) Chung and Fields (1986) Chung and Fields (1986) Marie Sych et al. (2016) Li et al. (2008b)

Among its many uses are as a mold inhibitor, a preservative in food for humans and animals, and a substrate for synthesizing herbicides, perfumes, pharmaceuticals, and cellulose fibers (Suwannakham et al. 2006; Zhang and Yang 2009). However, the production of these acids inhibits cell growth, which in turn prevents the production of VB12 (Wang et al. 2012a). The manufacturing of VB12 is an overprized task, similar to that of many other biomaterials, as a result of the need for synthetic culture media and the high cost of its purification from the broth medium because of the vitamin’s low concentration, which is typically less than 10 mg/L (Kośmider et al. 2012). It may be possible to cut costs using economic raw materials like agricultural, food, or industrial wastes like molasses, frying oils, and starch-rich wastes. Futurescale manufacturing of biomaterials like vitamins might use these wastes as a starting point. Propionibacteria have the ability to manufacture VB12 from a range of carbon sources, including tomato pomace (Haddadin et al. 2001), sucrose (Li et al. 2008a), home sugar (Quesada-Chanto et al. 1994), molasses (Quesada-Chanto et al. 1994), and glycerol (Kośmider et al. 2012). One of the least expensive substrates for forming several different chemicals has been demonstrated to be molasses (QuesadaChanto et al. 1994).

7.1.5.4 Production Strategies for Vitamin B12 Microbial Production of Vitamin B12 A gram-negative bacterium called P. denitrificans employs the aerobic biosynthetic route to create vitamin B12. P. denitrificans is currently the primary source of vitamin B12 used by industrial producers despite the lack of Generally Recognized as Safe (GRAS) classification. The culture procedure is crucial in obtaining high concentrations of the desired compounds from a well-characterized strain. As a result, numerous studies have been conducted to increase ALA output. For instance, the cultivation medium was typically supplemented with glycine and succinate, the precursors for the synthesis of ALA, to boost ALA production. As ATP is an energy carrier comprehended in the C4 pathway, it was supplemented with glucose, a suitable carbon source, to improve ATP supply, ultimately improving ALA production. Additionally, yeast extract was enhanced for cell development and ALA formation due to its abundance of vitamins, AAs, and trace minerals (Nishikawa et al. 1999). ALA dehydratase would catalyze the conversion of ALA to

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porphobilinogen (PBG) in the natural world (encoding by hemB gene). To increase the efficiency of ALA accumulation, levulinic acid (LA), a competitive inhibitor of ALA dehydratase, was utilized in the medium (Miyachi 1998; Saikeur et al. 2009). A successful R. sphaeroides CR720 strain was created by combining several techniques and having the ability to accumulate ALA up to 27.5 mM (Kamiyama et al. 2000). Even though the amount of ALA that R. sphaeroides produces has significantly increased, there are still many drawbacks, including the need for light illumination and poor cell growth. The effects of various supplements, pH control, dissolved oxygen management, and trace element addition to media have all been studied. In this regard, it has been noted that the presence of Zn2+ significantly improves the synthesis of PBG and ALA, two of the significant cobalamin precursors, whereas the addition of DMBI and Co2+, the base inserted into the nucleotide loop, favorably influences cobalamin synthesis (Li et al. 2008c). In P. denitrificans, oxygen transfer rate (OTR) optimization has also been a critical focus. While lower OTRs in later stages were found to be essential for greater yield, higher OTRs during the early stages of culture stages enhance cell growth (Wang et al. 2010). E. coli Cell Enzyme Transformation With the introduction of recombinant technology, a substantial focus has been on building recombinant cell factories due to the several drawbacks of photosynthetic bacteria. Consequently, due to its numerous benefits, including rapid growth, ease of cultivation, and ease of genetic manipulation, E. coli has been chosen as a target microbe and is thought to be the original replacement website for ALA biotransformation (Sasaki et al. 2002). The R. sphaeroides ALAS genes (hemA and hemT) were cloned and overexpressed in E. coli (Neidle and Kaplan 1993). Where to increase the expression and activity of the heterologous peptide is crucial for enzyme conversion. A sizable titer of ALA (39 mM) was manufactured adopting transgenic E. coli and a comprehensive factorial methodological approach following the alteration of ALAS expression (Xie et al. 2003b). E. coli Rosetta (DE3), a rare codon optimizer strain, was utilized to produce ALA and express hemA heterologously to increase ALAS’s expression and activity (Fu et al. 2007). Hem optimization of uncommon codons needs to be yet another option for enhancing ALA. The mutant of ALA dehydratase in E. coli appears to be advantageous for ALA assemblage, but rather than increasing, ALA synthase activity decreased by 50% (Xie et al. 2003a). The cultivation procedure was further investigated to produce recombinant strains with significantly better potential. It was discovered that glucose, the principal carbon source, inhibited ALA dehydratase, which was advantageous for ALA buildup (Choi et al. 2008; Liu et al. 2010). The R cultivation method is similar. To increase ALA accumulation, glycine, succinate, and sphaeroides were added to LA as ALA dehydratase inhibitors and precursors (Qin et al. 2006). Several factors were also examined for the generation of ALA, including culture pH, cultivation temperature, and inducer concentrations (Fu et al. 2008). After hemA overexpression and culturing were optimized, the greatest concentration of ALA up to 56 mM was attained (Lin et al. 2009).

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Coenzyme Q10

Coenzyme Q (ubiquinone) molecules are contained in biomembranes and situated in the phospholipid bilayer’s hydrophobic portion (Battino et al. 1990; Lenaz et al. 1999). Coenzyme Q, sometimes called ubiquinone (UQ), is a natural coenzyme that develops when a benzoquinone ring conjugates with an isoprenoid strand with a hydrophobic molecular structure that fluctuates in length regardless of the species. In eukaryotes, electrons are transported through membrane-bound dehydrogenases to complex III of the electron transport chain in the mitochondrial inner membrane (Lenaz et al. 2007; Klingen et al. 2007; Zhu et al. 2007). UQ also functions as an antioxidant, preventing lipid peroxidation and protecting proteins and membrane phospholipids by either directly scavenging free radicals or replenishing tocopherol levels (Lass and Sohal 1998; Yoshida et al. 2006; Martin et al. 2007). UQ also modulates the number of 2-integrins upon the surface of blood monocytes, safeguards the physicochemical characteristics of the cellular membrane, governs the permeability transition pores in the mitochondria, activates the mitochondrial uncoupling proteins, and alleviates endothelial dysfunction likely to be greatest by enhancing nitrogen oxide, oxidizing sulfide (in yeast), and instituting disulfide bonds (in bacteria). UQ also participates in the biological processes of sulfur oxidation, controlling the mitochondrial permeability transition pore, and transferring protons and Ca2+ across biomembranes (Lagoutte et al. 2010; Bogeski et al. 2011). The extent of UQ deficit correlates with the severity of the condition, and in humans, UQ inadequacy has already been connected to a range of conditions that impact the growth of the muscles and the nervous system (Quinzii et al. 2007). Conditions like central nervous system myopathy, dysfunction, or cardiomyopathy are just a few examples of how these disorders may present themselves (Sharma et al. 2016; Mancuso et al. 2010; Gempel et al. 2007; Rötig et al. 2000). Numerous clinical characteristics have also been linked to the oxidative damage brought on by reduced UQ function. UQ is particularly significant in the biomedical and health supplement scene due to its therapeutic relevance.

7.1.6.1 Biosynthetic Pathway of Coenzyme Q10 A similar metabolic route connects cholesterol with UQ. The UQ biosynthetic pathway was proposed based on the genetic analysis of mutant S. cerevisiae and E. coli strains (Choi et al. 2005; Cluis et al. 2007; Kawamukai 2002; Meganathan 2001). The production of a quinonoid ring and the modification of the quinonoid ring make up the two main steps in the pathway for UQ; the production of decaprenyl diphosphate, albeit prokaryotes and eukaryotes, requires different precursors. The first stage of the production of UQ is the conversion of chorismate to 4-hydroxybenzoate. The chorismate pyruvate-lyase enzyme, produced by the ubiC gene in E. coli, catalyzes this process. Tyrosine, an essential AA, is converted to 4-hydroxybenzoate in animals because they lack the shikimate pathway. The ubiquitous metabolic precursors IPP and its isomer dimethylallyl diphosphate act as the fundamental building blocks for synthesizing all isoprenoids. The conventional mevalonate (MVA) system and the more recent 2-C-methyl-D-erythritol 4-phosphate

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Fig. 7.6 The metabolic process that produces UQ. UQ is created when decaprenyl diphosphate, which was created by DPS, mixes with pHBA and proceeds through a series of modification processes. 4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, MECD 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, and DCME 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate

(MEP), often known as the non-MVA pathway, are the two different systems that make IPP (Rohmer et al. 1993). IPP synthesis through the MEP route begins in E. coli, probably in most other bacteria, and green algae (Orihara et al. 1998; Rohmer et al. 1996). This condensation process between pyruvate and glyceraldehyde-3phosphate (GA3P) results in IPP and DMAPP. Streptomycetes and plants utilize both methods (Hamano et al. 2002; Lichtenthaler et al. 1997) (Fig. 7.6).

7.1.6.2 Coenzyme Q10-Producing Microorganisms Microbes like Agrobacterium tumefaciens, Paracoccus denitrificans, and Rhodobacter sphaeroides are naturally occurring high UQ producers (Yoshida et al. 1998). In contrast to the quinones formed by E. coli, which also create quinones such as demethylmenaquinone and menaquinone, UQ is notable as being the only class of quinone found in all these strains (Collins and Jones 1981). Due to UQ’s complex structure, numerous attempts have been made to synthesize it using bacteria, which is a more advantageous method than chemical synthesis (Barker and Frost 2001; Chen et al. 2003). By adjusting culture media and conditions, UQ-containing bacteria such as A. tumefaciens, Cryptococcus laurentii, P. denitrificans, Sporobolomyces salmonicolor, Rhodobacter sphaeroides, and Trichosporon sp. have been employed in fermentation processes for the manufacture of UQ (Kaplan et al. 1993). Chemical mutagenesis has also been used to produce strains and identify mutants with increased CoQ10 productivity (Yoshida et al. 1998). Additionally, the DPS gene from P. denitrificans or A. tumefaciens has been used to turn E. coli, which

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Table 7.6 Coenzyme Q10-producing microorganisms and their yield Microbial strain Agrobacterium tumefaciens ATCC 4452 A. tumefaciens AU-55 Paracoccus denitrificans ATCC 19367 R. sphaeroides Co-22-11 R. sphaeroides KY8598 Rhodobacter sphaeroides FERM-P4675 Escherichia coli BL21/pACDdsA

Yield (mg/L) 89.7 110 27.6 347 770 97.2 25.5

References Yoshida et al. (1998) Yoshida et al. (1998) Yoshida et al. (1998) Yoshida et al. (1998) Sakato and Tanaka (1992) Yoshida et al. (1998) Park et al. (2005)

can only make CoQ8, into a source of UQ (Lee et al. 2004; Takahashi et al. 2003) (Table 7.6).

7.1.6.3 Substrates for Coenzyme Q10 Production The final UQ production potential is directly impacted by the N source, C source, and carbon-to-nitrogen (C/N) ratio, which all play vital roles in PNSB growth in terms of the required nutritional supplies (Alloul et al. 2019; Meng et al. 2019; Wang et al. 2019). Glucose, fructose, molasses, malic acid, and sodium acetate are all excellent dietary sources for PNSB and are suitable carbon sources for UQ production in purple non-sulfur bacteria fermentation methods (Lu et al. 2019). The amount of C source present is crucial in fostering the growth of PNSB biomass. According to researchers, when the introductory total sugar was increased to 80 g/L, the biomass of R. sphaeroides and the production of UQ increased by 287% and 321%, respectively. However, a 100 g/L total sugar content prevented R. sphaeroides from growing and producing UQ (Wang et al. 2019). Using ammonium sulfate, yeast extract, and corn steep as N sources is preferable while making UQ in PNSB. Nitrogen sources are crucial for the growth of R. sphaeroides. Additionally, compound N sources were superior to single N sources in their ability to produce UQ. Studies have shown that when used together as the compound N source, yeast extract, and ammonium sulfate, R. rubrum biomass generation was 12.2% and 44.6% higher than when used individually. Production of UQ was then increased by 15.3% and 59.9%, respectively (Tian et al. 2010). Furthermore, prior research has demonstrated that a healthy C/N ratio can encourage the production of CUQ. 7.1.6.4 Production Strategies for Coenzyme Q10 Chemical Synthesis Methods There are two types of chemical fabrication techniques: full-chemical generation and semichemical generation whose starting ingredients are geraniol and solanesol, respectively. First, polymerize the solvent to produce the side chain from solanesol or geraniol, which is then coupled with the mitigated quinone ring to produce UQ (Lipshutz et al. 2002). Several processes, including reduction, methylation,

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oxidation, coupling, and Friedel-Crafts, are frequently used in chemical synthesis (Oh et al. 2012). A low overall yield of UQ is caused by these lengthy and complex reaction processes (Mu et al. 2011). The chemical synthesis process is very costly and unfriendly to the environment due to the necessity for catalysts for chemical reactions, the high cost of raw materials (up to 5–200 kg/USD), and the quantity of chemical waste (such as petroleum ether, trichloromethane) that is generated (Takahashi et al. 2010). These drawbacks restrict the use of the chemical synthesis method. Biotechnological Production Methods for Coenzyme Q10 CoQ10 is created via microbial synthesis through fermentation. The CoQ10containing microorganisms are grown in a fermentation tank. PNSB, Paracoccus denitrificans, Agrobacterium tumefaciens, Schizosaccharomyces pombe, Sphingomonas paucimobilis, Escherichia coli, and Saccharomyces cerevisiae can all produce UQ under the right fermentation circumstances. Saponification is done following filtering or centrifugation. Solvent extraction or ultrasonic extraction is additionally added in order to get the crude product from cells containing UQ and other contaminants (such as pigments and esters) (Bule and Singhal 2012; Deng et al. 2019; Wu and Tsai 2013). The purity of UQ could be intensified through the utilization of column chromatography, crystallization, or macroporous resin adsorption techniques (Cao et al. 2006). Microbial synthesis of UQ has distinct benefits over chemical synthesis and tissue extraction from animals and plants. First off, microbes are widely accessible and reasonably priced. Second, compared to the amount extracted from animal and plant tissues, the UQ level of natural strains obtained by fermentation techniques can range from 0.68 to 88.8 mg/g. Additionally, since microbial fermentation does not require catalysts, it is simple to scale up and results in less environmental impact. Therefore, microbial fabrication is the preferable and recommended technique to create UQ (Wu and Tsai 2013). There are currently two methods for UQ microbial synthesis: heterologous host fermentation and natural strain fermentation. For example, PNSB (R. sphaeroides, R. capsulate, R. rubrum, and R. palustris), A. tumefaciens, P. denitrificans, S. pombe, and Sphingomonas sp. are natural strains, which are naturally occurring bacteria that can manufacture UQ (Chen et al. 2019; Tokdar et al. 2015). These are all non-phototrophic bacteria, except PNSB. Strategies for enhancing UQ production in natural strain fermentation have been investigated, including strain development for high UQ yields and microbial fermentation process modification (Zhu et al. 2017). Genetic Modification Host fermentation on heterologous organisms is another method of UQ microbial production. The heterologous hosts are suited for large-scale fermentation even though they lack UQ synthesis, have a distinct genetic history, and only require primary culture conditions. This method uses molecular biology to introduce crucial genes for UQ production from native strains into the heterologous hosts. Following the expression of these genes in heterologous hosts, new strains capable of

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producing UQ are created. Hosts like E. coli and S. cerevisiae are frequently employed heterologous ones (Martínez et al. 2015; Payet et al. 2016). According to studies, E. coli fermentation can result in up to 70.5 g/L of biomass production (Choi et al. 2009; Park et al. 2005). However, compared to wild strains (14.12–770.0 mg/L), E. coli produced less UQ (0.78–99.4 mg/L). The reduced UQ level of the newly created strains compared to the wild strains may be the root cause. Additionally, since E. coli may synthesize CoQ8 and generate various CoQ products, its UQ is probably of low quality (Bule and Singhal 2011; Qiu et al. 2012; Martínez et al. 2015). Therefore, the preferred strains for producing UQ continue to be naturally occurring.

7.1.7

HA

Regarding biopolymers, HA belongs to the family of polysaccharides, which are non-sulfated glycosaminoglycans. HA contains recurrent units (2000–2500) of β-Dglucuronic acid and β-N-acetylglucosamine (Schiraldi et al. 2010). Many bacterial extracellular matrices, including Streptococcus, contain HA as a crucial component and facilitate adherence and protection. Additionally, it can fool the host’s immune system while infected by acting as a molecular spoof (Wessels et al. 1991). HA’s molecular weight (MW) contributes to its predominant role in its biological activities and utility (Jagannath and Ramachandran 2010); HA with an MW higher than 10 kDa is preferable for applications in orthopedics, engineering of tissues, cosmetics, and ophthalmology (Allison and Grande-Allen 2006; Fagien and Cassuto 2012; Kogan et al. 2007). Meanwhile, MW under 5 kDa helps form the products essential in angiogenesis and obstruct tumor furtherance (Jagannath and Ramachandran 2010; Tammi et al. 2008). Due to its biological functions, which include biocompatibility and angiogenic and immunostimulatory provinces, HA has a wide variety of utilization in medicines encircling plastic surgery, osteoarthritis (OA) treatment, targeted drug conveyance, skin moisturizers, ophthalmic surgery, and wound alleviation; it also has some practical applications in drug formulations and targeted drug delivery (Chong et al. 2005; Goa and Benfield 1994; Kogan et al. 2007). Extraction of HA from rooster combs and other sources is a challenging, costly, and contamination-prone process (Boeriu et al. 2013) with many technical impediments. To avoid this, contamination-free microbial production of HA is being practiced nowadays using the bacteria Streptococcus zooepidemicus (Liu et al. 2011). It gives enhanced productivity and more adequate recovery processes with the lowest chances of viral contamination (Yamada and Kawasaki 2005). S. zooepidemicus is a gram-positive, catalase, and oxidase-negative, facultative anaerobic coccus but also an aerotolerant species (Chong and Nielsen 2003). Because of the remarkable productivity of HA using bacterial strains, fermentation is more suitable for mass manufacturer. Although biotechnological manufacturing has many advantages, it must be cost-effective compared to HA extraction from animal sources. However, this production process is associated with simultaneous production of other secondary metabolites including LA, which further complicates

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the downstream processing. To solve this problem, researchers sought genes accountable for HA biomanufacturing, and bacteria such as Agrobacterium, Lactococcus, E. coli, and Bacillus were used as a tool for genetic modification. Genetic modifications were done in these bacteria to articulate HA genes, so they synthesize an astounding amount of HA (DeAngelis et al. 1993, 1998; Mao and Chen 2007; Wessels et al. 1991).

7.1.7.1 Biosynthetic Pathway of HA A significant trend in finding the probability of production of HA via genetic processes is having a vicious knowledge of the biosynthetic pathway of HA. Even though there is no elaborate work on the genetics of biosynthesis of HA on the establishment of the pathway’s each step in Streptococci, the progression of all the biochemical events could be assembled to obtain the formation of two precursors of HA, gathered from the studies on different microorganisms. The leading pathway for HA production is divided into two different sets of reactions, while in the foremost synthesis of glucuronic acid, N-acetylglucosamine is formed in the second set, both acting as building blocks of HA. A single enzyme then utilizes both sugars to form HA (DeAngelis and Weigel 1994). While the site for most of the glycosaminoglycan synthesis is the Golgi network, HA synthesis occurs in the plasma membrane (Prehm 1984; Chavaroche et al. 2013). The sugar backbone for the HA is synthesized with the help of fructose-6-phosphate and glucose-6-phosphate. The whole process of agglutination of HA is divided into two parts. With the help of α-phosphoglucomutase, during the initial set of the reaction, glucose-1-phosphate is produced by the metamorphosis of glucose-6-phosphate. A uridine-5-triphosphate is appended to G1P (glucose-1-phosphate) with the help of uridine diphosphateglucose pyrophosphorylase to synthesize UDP-glucose. In the final step of the first set of reactions, the first precursor of HA, i.e., UDP-glucuronic acid, is formed with the help of UDP-glucose dehydrogenase as it oxidizes the primary alcohol of UDP-glucose. In the second group of reactions, the amido group is transferred to fructose-6-phosphate from glutamine with the help of the enzyme glutamine fructose-6-phosphate amidotransferase to generate GlcN6P (glucosamine-6-phosphate). Rearrangement of the phosphate group via phosphoglucosamine mutase produces GlcN1P (glucosamine-1-phosphate). It is then acetylated via phosphoglucosamine acetyltransferase in the next step. In the final step of the second group of reactions, with the addition of UTP, the intermediate is activated with the help of enzyme N-acetylglucosamine-1-phosphate pyrophosphorylase, thereby producing the second HA precursor, UDP-N-acetylglucosamine. In the final step, the precursor combines to form HA with the help of hyaluronate synthase. 7.1.7.2 Microorganisms Producing HA Obtaining HA from rooster comb extraction and other sources is a challenging, costly, and contamination-prone process (Boeriu et al. 2013) with many technical impediments. To avoid this, contamination-free microbial production of HA is being practiced nowadays by using the bacteria Streptococcus zooepidemicus (Liu et al. 2011) as it gives enhanced productivity and more adequate recovery processes with

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the lowest chances of viral contamination (Yamada and Kawasaki 2005). S. zooepidemicus is a gram-positive, catalase, and oxidase-negative, facultative anaerobic coccus but also an aerotolerant species (Chong and Nielsen 2003). Because of the remarkable productivity of HA from bacterial strains, as shown in Table 7.2, the process of fermentation is more suitable for large-scale production. Although microbial production has many advantages, it must also be cost-effective when compared with HA extraction from animal sources. However, during fermentative production of HA, other secondary metabolites like LA are simultaneously produced, reducing HA production and complicating its downstream processing. To overcome this problem, genes responsible for the production of HA were sought, and bacteria such as Lactococcus, E. coli, Agrobacterium, and Bacillus were used as a tool for genetic modification. Genetic modifications were done in these bacteria to express HA genes and synthesize a good amount of HA (DeAngelis et al. 1993, 1998; Mao and Chen 2007; Wessels et al. 1991) (Fig. 7.7; Table 7.7).

7.1.7.3 Substrates for Production of HA Nitrogen and carbon sources perform a critical role in the production of any of the secondary metabolites. The central carbon source used in the production of HA through Streptococci is glucose. By using glucose as a primary carbon source in the aerobic fermentation process, the yield of HA ranges between 5% and 10% (Chong et al. 2005; Gao et al. 2006; Duan et al. 2009), which is remarkably more prominent than other complex polysaccharides in LA bacteria (Zhang et al. 2006). Complex carbon sources such as sucrose, starch, dextrin, molasses, and lactose (Im et al. 2009) can also be used for the fermentative production of HA. Carbon sources like lactose and starch are cheaper options and hence affect the economics of the entire production cost. The biosynthesis of HA in Streptococci needs a considerable sum of energy as it tussles with the growth of the microbial cell for glucose. The highest bacterial growth can be perceived during unlimited availability of glucose and ideal cultivation conditions, i.e., pH 7 and 37 °C temperature; at the same time, topmost molecular weight and output of HA were achieved when the growth conditions were suboptimal, for the reason that when the growth rate of the organism is slow, carbon and energy resources are not entirely involved in growth process only (Armstrong et al. 1997). HA yield and molecular weight decline under glucose-limiting conditions (Chong et al. 2005). 7.1.7.4 Production Strategies for HA Extraction Production of HA on the commercial scale is achieved by mainly two procedures, first through extraction from animal tissues and second by using fermentation technique with the help of bacteria. Both processes yield high-molecular-weight HA in cosmetics and biomedical applications (Shiedlin et al. 2004; Šoltés et al. 2002; Harmon et al. 2012). The extraction process from animal waste was the first to be used in commercial-scale HA production and is still a crucial technology for mercantile products, but it has several drawbacks and technical limitations. The

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Fig. 7.7 Biosynthetic pathway of HA (Shukla et al. 2022)

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Table 7.7 HA-producing microorganisms and their yield Microbial strain Streptococcus thermophilus Streptococcus zooepidemicus Lactobacillus acidophilus Streptococcus zooepidemicus Streptococcus equi Lactococcus lactis Pichia pastoris Escherichia coli

Yield (g/L) 1.2 g/L 0.589 1.7 g/L 3.67 g/L 0.87 g/L 6.09 g/L 1.7 g/L 29.98 mg/mL

References Izawa et al. (2011) Don and Shoparwe (2010) Chahuki et al. (2019) Vázquez et al. (2010) Gedikli et al. (2018) Sunguroğlu et al. (2018) Jeong et al. (2014) Woo et al. (2019)

indispensable degradation of HA is one of the significant flaws induced by stringent conditions of the extraction process and the breaking of the HA chain length due to enzymatic hydrolysis. Although the protocols for the extraction process have been developed over the years, lower product yield is still a problem as HA is present in a meager amount in the tissues. Chances of contamination with proteins, nucleic acids, and viruses are exceptionally high, but they can be reduced by using healthy animal tissues and a detailed purification process. However, there is still a potential risk of viral and bovine protein contamination, which diverted researchers’ attention toward the fermentation production process. Fermentation The chemical-defined medium (CDM) used for the growth of Streptococci can also produce HA, in which glucose and peptone are used as significant sources of carbon and nitrogen, which are essential components for both cell growth metabolism and HA synthesis. Streptococci are characterized as fastidious LA bacteria incapable of synthesizing some AAs (Armstrong et al. 1997). To attain better cell growth and HA production, adding some AAs like arginine and lysine is beneficial (Liu et al. 2009b). Carbon sources like molasses, sucrose, and maltose, and nitrogen sources like steep corn liquor and casein hydrolysate, are also favorable for HA production (Amado et al. 2017). Fermentative production of HA requires a controlled microbial culture environment, for instance, pH, temperature, aeration and agitation rate, dissolved oxygen, and type of bioreactor used. The optimum pH and temperature for Streptococci cell growth and production of HA are usually set at 7 and 37 °C (Kim et al. 1996; Johns et al. 1994). Agitation, aeration rate, shear stress, and dissolved oxygen influence HA production and its molecular weight (Johns et al. 1994; Kim et al. 1996; Huang et al. 2006; Duan et al. 2008; Liu et al. 2009b; Liu et al. 2009a; Duan et al. 2009; Wu et al. 2009b). A higher HA titer and molecular weight are attained in aerobic culture conditions than in anaerobic (Johns et al. 1994; Armstrong et al. 1997). A controlled aeration rate can increase acetyl-CoA accumulation in such a manner that more divulgence of acetyl-CoA from the central carbon metabolism pathway to HA synthesis can be achieved (Wu et al. 2009b). An increase in the production rate of HA can be achieved with an increase in aeration and

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agitation rate, but extensive agitation speed can cause the rupture of cells, and the concentration of HA is retarded (Hasegawa et al. 1999). The first fermentative production of HA by using Streptococci was reported to be 60–140 mg/L (Kendall et al. 1937). Ever since, various efforts have been carried out to increase production, including optimizing traditional extraction processes, customizing the fermentation culture media, and selecting high-producing strains. In the past three decades, production of HA has raised from 300–400 mg/L (Holmström and Řičica 1967) to 6–7 g per liters (Kim et al. 1996), which is the most practical demarcation line because of mass transfer demarcations (Chong et al. 2005). Comparing production of HA in different groups of Streptococci, group C Streptococci (nonhuman pathogenic) were found to be having high productivity than group A Streptococci and also these have high productivity than animal pathogenic bacterium P. multocida. S. thermophilus strains with strong synthesizing rates of exopolysaccharides together with HA were segregated from dairy food products (Izawa et al. 2009), providing a safe microorganism for producing HA. By using recombinant technology, S. thermophilus was constructed which culminated in the production of 1.2 g/L of HA, and average molecular weight was also commensurate to the wild-type one (Izawa et al. 2011). Genetic Modification Enhanced HA-yielding microbial strains were assessed through uncalculated mutagenesis experiments. Then, a screening of the resultant strains (mutant) was adapted to the rebate of HA-depolymerizing enzymes authoritative of reduction in molecular mass of HA amid fermentation and streptolysin’s decrease, the exotoxin creditworthy of beta hemolysis (non-hemolytic strains). Molecular mass ranging from 3.50 to 5.90 million Dalton and HA production of nearly 6–7 g/L were achieved with the help of fermentation of non-hemolytic and hyaluronidase-free mutants (Kim et al. 1996; Im et al. 2009; Han et al. 2009). In strains of Streptococci, mutagenesis has further emanated, which yielded HA with outstanding increased molecular weight parameters of 6–9 million Dalton but with limited production of 100–400 mg/L (Boeriu et al. 2013). Genetic engineering of the microorganism is required for the added augmentation of HA production with high molecular weight. Genetic engineering is performed in the metabolic pathway of HA production. Two ratios that are most important for determining the molecular weight of HA are the proportion between uridine diphosphate-glucuronic acid and uridine diphosphate-Nacetylglucosamine and the second between the substrates and HA synthase. In Streptococcus zooepidemicus, individual overexpression of each of the five genes of has operon was done by Chen et al. It was discovered that there was a decrease in molecular weight due to overexpression of hasA, hasC, and hasB (hasA engaged in the biosynthesis of HA while hasC and hasB in uridine diphosphate-glucuronic acid). At the same time, an enormous enhancement in the molecular weight was observed because of the overexpression of hasE, engaged in the biosynthesis of UDP-GlcNAc. No effect was observed due to hasD overexpression; however, the molecular mass was increased when it was united with the hasE overexpression (Chen et al. 2009).

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Moreover, with the proportion of the two precursors, the prominence of the proportion between HA synthase and UDP-GlcUA played a critical role in determining the molecular weight of HA. It was illustrated for the L. lactis, a heterologous host; the construction of two plasmids encompassed one of the two hasB or hasA from S. zooepidemicus with the NICE and lacA, inducible expression promoters. A substantial increase in the molecular mass of HA was observed at a ratio of below 2 of hasA/hasB, hinting that higher availability of UDP-GlcUA per HA synthase raises the molecular weight of HA (Sheng et al. 2009).

7.1.8

Lactic Acid

Due to its numerous uses, primarily in the pharmaceutical, chemical, food, and cosmetic industries, lactic acid (LA) is of great importance. Because of its significant function in producing yogurt and cheese, the food processing industry employs approximately 70% of the total LA generated (Martinez et al. 2013). Due to inherent hygroscopic and emulsifying properties, certain LA derivatives, including lactate esters, can also be used as emulsifiers and enhancement emissaries in food commodities (Gao et al. 2011). Additionally, polyLA (PLA) polymers have a considerable production potential and are frequently utilized as raw materials in the packaging in addition to fibers and foams. Nonetheless, when compared to petrochemical raw materials on an industrial scale, PLA can be seen as a relatively young technology, primarily because LA, which serves as the basis for PLA, is expensive to produce. Even though the use of LA as a medium for PLA manufacturing has expanded relatively dramatically, the quantity of PLA generated from it is also smaller than the average amount of petrochemical materials used to manufacture plastics (Wang et al. 2016). LA can be created through either chemical processing or fermentation (LA fermentation). Typically, dl-LA is synthesized chemically in a racemic mixture. On the other side, once the right microbe is chosen as the LA manufacturer, fermentative generation systems have the advantage of using inexpensive renewable feedstock, minimal production temperatures, minimal power consumption, and biosynthesis of enantiomerically pure D- or L-LA. All the global LA comes from the fermentative production pathway (Abdel-Rahman et al. 2011). LA producers, fermentation substrates, and operating modes are the key determinants of how effectively LA fermentation processes work. Different microbial species, such as bacteria, fungi, yeast, microalgae, and cyanobacteria, can create LA from renewable resources.

7.1.8.1 Biosynthetic Pathway of LA Through carbohydrate fermentation and substrate-level phosphorylation, LA bacteria produce energy. The two critical biochemical reactions for hexoses are homofermentative metabolism, which yields LA as the primary byproduct, and phosphoketolase route, which yields a variety of products for export including CO2, acetic acid, propionic acid, ethanol, and others (heterofermentative metabolism) (Kandler 1983). Researchers proposed a thorough glycolytic model for

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Fig. 7.8 Biosynthetic pathway of LA

L. lactis based on information from the literature on enzyme kinetics (Papagianni et al. 2007). The final enzyme in the pathway that converts sugar to lactate in L. lactis is lactate dehydrogenase (LDH). Most of the pyruvate is diverted to mixed-acid products when the corresponding gene (ldh) is disrupted (Papagianni and Avramidis 2011; Raftari et al. 2013). LA bacteria can create either the L- or the D-isomer of LA. While D-LA is poisonous to humans, L-LA is chosen for use in food and medicine and as a building block for biopolymers. As a result, research on metabolic engineering has concentrated on how homofermentative LAB produces pure L-LA (Fig. 7.8).

7.1.8.2 Microorganisms for the Production of LA Numerous microorganisms can produce LA, including bacteria, yeast, fungi, cyanobacteria, and algae. Each biocatalyst has outperformed the others in one or more ways, such as a broader range of substrates, increased efficiency and production, decreased nutrient needs, or greater LA optical purity. Mixed strains may provide advantageous permutations of biochemical activities for using complex substrates utilized in fermentation, which would improve the generation of LA (Cui et al. 2011; Kleerebezem and van Loosdrecht 2007; Nancib et al. 2009). Wild-type and modified producers of LA are both types of generating bacteria. The four leading producers of these species are Escherichia coli, LA bacteria (LAB), C. glutamicum, and Bacillus strains. LA can be created by LAB, a diverse

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Table 7.8 LA-producing microorganisms and their yield Microbial strain Lactobacillus pentosus Lb. delbrueckii NCIMB 8130 Lb. delbrueckii mutant DP3 Lb. delbrueckii sp. bulgaricus 5085 Lb. rhamnosus ATCC 7469 Pediococcus acidilactici PA204 Bacillus coagulans Lb. bulgaricus ATCC 8001, PTCC 1332

Yield (g/L) 0.66 g/g stover 90 77 16 73 1.28 2.59 24.6

References Hu et al. (2016) Kotzamanidis et al. (2002) Abdel-Rahman et al. (2013) Abdel-Rahman et al. (2011) Marques et al. (2008) Zhang et al. (2020) Alexandri et al. (2020) Fakhravar et al. (2012)

collection of gram-positive bacteria with exceptional efficiency and yield. LA is an anaerobic byproduct of glycolysis. The various growth requirements differ considerably according to the manufacturers because these bacteria can flourish in the pH range of 3.5–10.0 and temperature range of 5–45 °C. Most LAB, particularly Lactobacilli, are recognized as benign for commercial LA manufacturing because they possess a rich history of industrial output without exhibiting detrimental health consequences on the consumers or assembly-line workers. Important and abundant LAB strains like Lactobacillus strains are incredibly beneficial due to their superior acid endurance and capability to be changed for the preferential synthesis of D- or LLA (Benthin and Villadsen 1995; Kylä-Nikkilä et al. 2000; Lapierre et al. 1999). Nevertheless, most LAB species need complex nutrients, like AAs, nucleotides, vitamins, and peptides, for their growth. This makes recovering LA more difficult and drives production expenses (Hofvendahl and Hahn–Hägerdal 2000; Lapierre et al. 1999; Litchfield 2009; Reddy et al. 2008; Singh et al. 2006). Some Bacillus species, for instance, Bacillus coagulans, Bacillus stearothermophilus, Bacillus licheniformis, and Bacillus subtilis, have also been shown to produce LA. Finding superior LA manufacturers from natural sources takes time and effort. Because E. coli strains quickly metabolize pentose and hexose sugars, have basic dietary needs, and are straightforward to modify genetically, the latest researchers have utilized engineering methods to improve fermentation efficiency. Wild-type E. coli frequently manufactures a concoction of ethanol and various organic acids to acclimatize the reducing equivalents produced during glycolysis (LA, acetic acid, formic acid, and succinic acid) (Clark 1989; Chen et al. 2014; Zhou et al. 2003) (Table 7.8).

7.1.8.3 Substrates for Production of LA The commodities’ prices are a significant factor affecting how LA may be manufactured economically. It is propitious to achieve a pure LA generation and reduce pretreatment and recuperation costs when employing edible crops or pure sugars as the substrates for LA biosynthesis. Since the cost of the substrate cannot be minimized by process scale-up, numerous studies are being carried out to explore new substrates to produce LA. The byproducts of the food and agriculture sectors and naturally underutilized biomasses, including whey, yogurt, glycerol, and algal

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biomass, have all been explored as appealing alternative substrates and renewable resources. Depending on the affordability of substrate on the trading market, breakthroughs have been seen in the manufacturing of LA from various carbohydrates, including starchy and lignocellulosic biomasses (Litchfield 2009). Whey is produced in huge quantities by dairy companies worldwide when milk is processed in various manufactured goods. The dairy industry’s biggest pollution issue is the disposal of whey, a byproduct of the wastewater released during the cheese-making process. Lactose, lipids, protein, water-soluble vitamins, mineral salts, and other crucial elements for microbial development make whey a potent and ideal raw material for the synthesis of LA (Panesar et al. 2007). Recently, microalgae have been utilized as fermentative LA production substrates. Microalgae are less lignin-rich than lignocellulosic biomass, making it easier to convert them into fermentable sugars (Nguyen et al. 2012a, b).

7.1.8.4 Production Strategies for LA Co-culture Techniques A fermentation methodology known as co-culture involves the cultivation of two or more cell populations to generate a particular product with a specific amount of interaction (Eş et al. 2017). When one culture cannot thrive on its own and requires the existence of another cellular population to encourage growth, co-culture is primarily required. Although there is little research on LA creation through co-culture, neoteric studies have shown that this strategy is beneficial. Due to their heterofermentative nature, some LA bacteria (LAB) producer strains generate remarkable proportion of byproducts, such as acetic acid and ethanol, which reduce productivity and raise overall costs because they require more processes for purification and separation. By forcing competing cultures to follow a different metabolic pathway to metabolize xylose into LA, the inclusion of a homofermentative bacteria that can outpace other strains in the consumption of glucose would also aid in preventing the accumulation of byproducts. Researchers co-cultured the L. brevis ATCC 367 and L. plantarum ATCC 21028 strains to synthesize LA from biomassderived sugars (Zhang and Vadlani 2015). As a sole heterofermentative strain, L. brevis could eat glucose and xylose simultaneously with a relatively meager LA production of 0.52 g/g. When L. brevis and L. plantarum were co-cultured, the yield increased significantly (0.80 g/g), and there was less byproduct accumulation. However, for simultaneous and sequential co-fermentation, the greatest productivities were between 0.43 and 0.59 and 0.43 and 0.51 g/L/h, respectively. In a prior study of a similar nature, 0.70 g/L/h was found to have the highest output (Cui et al. 2011). However, L. rhamnosus and L. brevis were co-cultured in the works of these writers. This outcome demonstrates that the LA co-culture production is strain dependent. Genetic Engineering In fermentation technology, metabolic engineering is a sophisticated approach for developing microbes that generate LA with increased productivity, cheaper cost, and

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less byproduct production (Upadhyaya et al. 2014). The low acid magnanimity of microbes and their restricted capacity to use a variety of feedstocks are the most frequent issues in LA production. The majority of LAB naturally create LA as the main metabolite. However, LA’s chemical and optical purity can differ since they consume substrate through ineffective paths. This cutting-edge technology is mostly employed in this context to improve acid sufferance, reroute routes to use substrate more effectively, and create purer LA. Each gene drives an inevitable metabolic process when expressed in a host-microbe. For the technique to be successful, a comprehensive examination of a transferred gene, as well as the response of the host microorganism, is required. Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293’s highly stereospecific D-lactate dehydrogenase gene was expressed in S. cerevisiae by Baek et al. (2016). Some host microbes may consume the final product to proliferate after glucose depletion. D-LA consumption was successfully stopped by removing the genes that code for monocarboxylate transporter and Dlactate dehydrogenase. Additionally, certain microbes create byproducts like ethanol through heterofermentative metabolism (Elshaghabee et al. 2016). The genes for glycerol-3-phosphate dehydrogenases can be removed to stop ethanol production (GPD1 and GPD2). More crucially, the pH of the fermentation medium is reduced by LA formation. Thus, S. cerevisiae’s acidic adaptation was increased by the overexpression of a transcriptional activator accountable for a modest acid stress response. After all these exact changes, the host variant produced LA at a rate of 2.2 g/L/h. In a different research, the same team employed the S. cerevisiae strain JHY5610 (Baek et al. 2017). At pH 3.5, the host-microbe produced LA at a yield of 1.50 g/L/h, a 32% decrease from the prior output. This finding demonstrates that genetic alterations cause some host microorganisms to respond less effectively than others. Design of an Immobilized Bioreactor for LA Production Since many years ago, non-immobilized biocatalytic processes have been the subject of extensive research. Along with the rapid expansion of the global population, there has also been a rise in the need for foods of higher quality and greater quantity (Misra et al. 2017). LA has also become a viable fermentation byproduct that significantly improves food quality. Given this critical benefit of LA fermentation, much research has focused on developing modern biotechnologies to meet demand. Throughout this situation, it seemed desirable to use immobilized biotransformation mechanisms (Eş et al. 2015). To create a more flexible and effective microbial metabolism, similar immobilization approaches must be applied in large-scale industrialization. As a result, the significance of immobilized bioreactor systems, which were developed to boost LA fermentation productivity, has increased noticeably. According to (Sirisansaneeyakul et al. 2007), immobilization is the way to secure biocatalysts (enzymes or cells) chemically or physically in a specific location to conduct out catalysis of a specified reaction. Immobilization’s primary goal is to prevent the valuable biocatalyst from being lost to activity and enable recurrent usage. Some biocatalysts have been genetically and metabolically modified to produce LA more effectively. Because they make up a sizeable portion of the overall cost, reuse of

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these biocatalysts is crucial to maintaining low manufacturing costs. The kind of immobilization technology and support material employed affect the productivity of LA fermentation. LA fermentation has been examined using a variety of biocatalyst immobilization approaches, including adsorption, covalent bonding, trapping, encapsulation, and cross-linking (Martinez et al. 2013).

7.1.9

IA

One of the top 12 value-added biomass products, according to the US Department of Energy (Werpy and Petersen 2004), is IA. It is frequently used to produce chemical intermediates, including styrene, 2-methyl-1,4-butanediol, and 3-methyl tetrahydrofuran, as well as polymers like poly-IA and acrylic replacement fibers (Geilen et al. 2010; Klement and Büchs 2013; Magalhães et al. 2017). Additionally, IA has some antibacterial properties (Cordes et al. 2015). Aspergillus terreus is mainly used in fermentation to make IA (Willke and Vorlop 2001). IA is currently produced globally at a rate of 40,000 tonnes annually, with an estimated cost of $2 per kg (Klement and Büchs 2013; Steiger et al. 2013; Cordes et al. 2015). IA is produced industrially in five main phases, with a yield of around 80%: fermentation, filtering, crystallization, decolorization, and drying (Okabe et al. 2009). Industrial methods for isolating IA have additionally included crystallization, membrane separation, liquidliquid extraction, precipitation, and adsorption (Magalhães et al. 2017). The traditional producing organism, A. terreus, presents several challenges for effective IA fermentation. Continuous oxygenation is necessary, which leads to excessive NADH production, which inhibits the activity of essential enzymes, and vigorous stirring, which harms the mycelia (Cordes et al. 2015). Numerous studies have concentrated on streamlining manufacturing procedures to meet the rising demand for IA. The IA production route has also been introduced into different hosts to increase IA titers.

7.1.9.1 Biosynthetic Pathway of IA After discovering that a filamentous fungus could make IA, (Kinoshita 1932) named this species A. itaconicus; it was unclear for a long time how IA was produced, whether it came through a process involving the Krebs cycle, an alternate pathway involving citramalate, or the condensation of acetyl-CoA. The movement of intermediary metabolites across various intracellular compartments is necessary for synthesizing carboxylic acids, such as IA and citric acid, and utilizes the distinct collection of enzyme resources found in each compartment. In the instance of IA, fractionated cell extracts were used to study the compartmentalization of the pathway and separate the enzymatic activity of a mitochondrial enzyme from that of a cytosolic enzyme. Citrate synthase and aconitase are the enzymes that come before CadA in the pathway; however, it has been discovered that CadA is situated in the cytosol rather than the mitochondria (Jaklitsch et al. 1991). However, the cytosolic fraction also exhibits residual aconitase and citrate synthase activity. According to the hypothesized mechanism, the malate-citrate antiporter carries cis-aconitate into

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Fig. 7.9 Biosynthetic pathway of IA

the cytosol (Jaklitsch et al. 1991). However, it still has not been established whether cis-aconitate travels from the mitochondria to the cytosol using the mitochondrial malate-citrate antiporter or another mitochondrial carrier protein (Fig. 7.9).

7.1.9.2 Microorganisms for IA Production Along with A. terreus, it is also known that Candida sp.(Tabuchi et al. 1981), Rhodotorula sp. (Blumhoff et al. 2013), U. zeae (Haskins et al. 1955), and U. maydis (Klement et al. 2012) generate IA (Kawamura et al. 1981). The fundamental principles of the reactions causing the generation of IA in these species have not been further studied. However, new research (Strelko et al. 2011) suggests that the general mechanism for synthesizing IA in nature comprises CadA activity. IA was recently discovered in mammalian cells, specifically in cells generated from macrophages (Strelko et al. 2011). The native strain A. terreus NRRL 1960, also known as IAM 2054, DSM 826, CBS 116.46, IMI 44243, QM 6856, IFO 6123, and WB 1960, is the one that is most frequently employed in the manufacturing of IAs. According to research (Kuenz et al. 2012), this strain can use glucose to generate up to 91 g/L of IA. Other fungi besides A. itaconicus and A. terreus have also been utilized to produce IA. During the investigation of various Ustilago strains for the production of ustilagic acid, it was found in the fermentation broth of an Ustilago zeae strain (Haskins et al. 1955). Later, using glucose as a substrate, IA by U. maydis was detected at a concentration of 53 g/L by the Iwata Corporation (Japan) (Tabuchi and Nakahara 1980). In shake flask cultures (Levinson et al. 2006), through one culture of the basidiomycete Pseudozyma antarctica, 30 g/L of IA was recovered.

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Table 7.9 LA-producing microorganisms and their yield Microbial strain Aspergillus terreus A. terreus DSM23081, NRRL1960, NRRL 1963 A. terreus M-8 A. terreus NRRL1960 A. niger (genetically engineered) Ustilago maydis MB215 Pseudozyma antarctica

Yield (g/L) 48.7 86.2 55 18.4 2.5 20 30

References El-Imam et al. (2013) Kuenz et al. (2012) Tsai et al. (2001) Petruccioli et al. (1999) Li et al. (2012) Klement et al. (2012) Levinson et al. (2006)

Similar to this, after screening and subsequent mutation, a yeast strain known as Candida was obtained, and it generated about 53 g/L IA in 5 days (Tabuchi et al. 1981). Similarly, Yarrowia lipolytica produces about 200 g of citric acid per hour and may be used to manufacture IA (Blazeck et al. 2015). Additionally, IA has been engineered into Saccharomyces cerevisiae (Blazeck et al. 2014), Corynebacterium glutamicum (Otten et al. 2015), and Escherichia coli (Vuoristo et al. 2015). Due to low quantities of endogenous multifunctional CAD, amplifying the cadA (cis-aconitic acid decarboxylase) gene from A. terreus was necessary. These modified strains nevertheless exhibited poor IA titers. Subsequently, a few methods for boosting IA production were suggested (Table 7.9).

7.1.9.3 Substrates for IA Production Although most mono- and disaccharides support good growth in A. terreus, only a few can be converted into IA (Eimhjellen and Larsen 1955). The most common substrate for A. terreus to metabolize and produce IA is glucose (Reddy and Singh 2002). Another often utilized substrate is sucrose. In addition to being tested for IA production, arabinose, xylose, and lactose also produced poor yields. With varied degrees of success, sorghum, starches from sago, wheat, sweet potatoes, potatoes, and cassava have been used to make IA (Petruccioli et al. 1999; Eimhjellen and Larsen 1955). The utilization of these complex substrates presents several significant obstacles, including the following: (1) Their extremely varied composition between substrates and even among multiple sampling of the same substrates. (2) In comparison to sugars, crude substrate yields are often lower. (3) The growth and productivity of said fermented microorganism may be hampered, consequently reducing yields, by possibly hazardous compounds in the substrate, whether these are naturally abundant or are the consequence of pretreatment. (4) The cost of purifying processes can increase due to impurities in the final product. It is noteworthy that IA biosynthesis has also used citric acid as a precursor. This approach might be costeffective because citric acid is far less expensive than IA (Bressler and Braun 2000). IA fermentation is susceptible to trace metal concentrations and nitrogen sources, just like many other fungal fermentations. Nitrogen and trace levels of Zn2+ and Fe2+ ions considerably increase IA generation, but phosphate ions should be restricted once mycelial development prevails to prevent carbon from diverting into creating more mycelia (Lockwood and Reeves 1945).

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7.1.9.4 Production Strategies for IA Fermentation Techniques For IA fermentation, a wide range of fermentation periods have been investigated, spanning from 2 to 14 days (Bressler and Braun 2000; Kautola et al. 1991). However, numerous authors have claimed that the ideal production period is 7 days (Kocabas et al. 2014). IA production has been reported at various temperatures but is best stored at about 37 °C (Willke and Vorlop 2001). Successful efforts have been undertaken to boost this optimum, and a mutant strain at 40 °C was shown to produce five times as much IA as a parent strain. The fermentation of IA is only aerobic. Researchers (Park et al. 1993) studied the effects of agitation speed and dissolved oxygen (DO). The productivity per unit of consumed glucose was found to be at its highest when DO was around 20% of the point of saturation at an impeller frequency of 0.94 m/s, despite the fact that the percentage yield rose with DO. While studies (Riscaldati et al. 2000) observed that IA generation increased as the agitation rate was increased to 400 rpm (corresponding to an impeller frequency of 1.57 m/s), researchers (Rychtera and Wase 1981) discovered that the optimal aeration was occurring at a 0.71 m/s impeller speed. Because citrate synthase and phosphofructokinase, two enzymes involved in creating IA, are less active when oxygen levels are low in A. terreus, less ATP is produced. This decreases the amount of IA that can be manufactured. As a result, the generation of IA necessitates constant aeration, high power input, and vigorous stirring, which increases mechanical stress and damages mycelia (Willke and Vorlop 2001; Klement and Büchs 2013; Cordes et al. 2015). This injury was reduced by adopting an airlift bioreactor where the mycelia were present as pellets instead of the pulp produced by shear stress in stirring fermenters. Immobilization Technique Investigations on IA biosynthesis using immobilized microbes have also been conducted. Polyacrylamide gel was the first matrix utilized in IA synthesis (Horitsu 1991). Since then, numerous different matrices have been employed, including the structural fiber network of pawpaw trunk wood, silica-based material, a porous disc reactor system, polyurethane cubes, alginate, and silica-based material (Iqbal and Saeed 2005; Kautola et al. 1991). According to Vassilev et al. (He et al. 2012), the porous structure of the polyurethane foam transporter had no impact on how quickly A. terreus loaded, and the carrier had an average yield of 15.1 g/L IA. Solid-state fermentation (SSF) has been used to manufacture IA successfully. The best substrates for SSF are agricultural wastes, and this process has been utilized to create a variety of significant metabolites profitably. The agricultural waste may already possess the preponderance of the nutrients needed for the fermentation, or it might simply serve as the individual’s supporting structure or anchorage, where more nutrients may be added. Aspergillus terreus CECT 20365 was used by Vassilev et al. (Nikolay et al. 2013) to produce 44 g/L IA from dried olive wastes and beet pressmud, while a mutant of A. terreus, A. terreus M8, was used in a patented SSF method to produce 55 g/L IA from sugar adsorbed on sugarcane pressmud (Blazeck et al. 2015). The substrate bed must be heated to the ideal

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temperature to ensure spore germination and product formation because IA bioprocessing requires slightly higher than average temperatures. Aeration will have two effects: it will give the cells oxygen and circulate the heat to prevent hotspots. In many biochemical production processes, it is widely accepted that SSF can offer yields that are superior or at least comparable to those produced from submerged fermentation. As a purely aerobic process that occurs at high temperatures and uses a filamentous fungus, the IA production process theoretically seems to be ideal for SSF conditions. SSF has recently attracted more attention, particularly when using agricultural waste as substrates. However, there is still room for improvement in the fermenting conditions. Genetic Engineering Microorganisms convert glucose into IA in industrial operations at a rate of up to 85 g/L (Okabe et al. 2009; Kuenz et al. 2012). Unfortunately, IA fabrication is considerably inefficient when contrasted to citric acid, whereby 200 g/L has been accomplished (Magnuson and Lasure 2004). The overall titer of IA has increased thanks to genetic engineering. IA buildup is influenced to varying degrees by the overexpression of the associated critical genes cadA, mttA, and mfsA (Li et al. 2011). In the example, co-overexpression of the genes cadA and mfsA increased IA synthesis by 8.7% (Huang et al. 2014a, b). Additionally, in U. maydis, overexpression of either MTT1 or a particular transcription factor (RIA1) increased IA titers (Geiser et al. 2016). Additionally, these researchers discovered an inverse relationship between IA synthesis and cytochrome P450 monooxygenase gene cyp3 expression (Geiser et al. 2016). Citrate and ATP both interfere negatively with the production of IA by inhibiting the activity of the crucial enzyme 6-phosphofructo-1kinase (PFK1) in glycolysis during cellular carbon metabolism. A. terreus PFK1 was genetically shortened to address this inhibition, leading to a ternary increase in enzyme metabolism and a twice increase in IA synthesis (Tevž et al. 2010). The levels of IA increased by around 27% in A. terreus when the Alg3 gene, which encodes Man(5)GlcNAc-PP-dolichyl mannosyltransferase, was deleted or when LaeA (loss of aflR expression A) was overexpressed. Additionally, the LaeA gene was overexpressed in the Alg3 mutant, which enhanced the synthesis of IA by approximately double (Dai and Baker 2018).

7.1.10 Conclusions Based on examining the microbiological biosynthesis of AAs, vitamins, and organic acids completed in this chapter, a proactive strategy must be created for each organic acid. The fabrication method employed for refinement at the lab scale must be commercially scalable. The metabolic pathways can be analyzed and engineered to address the bottlenecks in each process. It is essential to do a techno-economic examination of these items, and additional efforts are needed to incorporate them into a circular economy. There is a large market for these low-value, high-volume

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products. Therefore, greater research into process development for increased productivity is necessary in the future.

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Exploring Plant-Microbe Interaction Through the Lens of Genome Editing Upasna Chettry , Sunita Upadhaya, Amilia Nongbet, Nikhil Kumar Chrungoo, and S. R. Joshi

Abstract

Dynamic advances in genetic engineering and plant studies leading to unprecedented plant yield have been largely offset by unfettered global population growth. It is therefore imperative to further enhance scientific techniques to meet the requirements of food and nutrition security. Interaction of microbes with the soil and their ability to provide essential nutrients make the plantmicrobe (PM) consortia a vital aspect determining plant life, yield and quality. Depending on various factors, their impact may be beneficial or detrimental to the health of the hosts. Therefore, the best strategy to arrive at global food security would be to comprehend the intricate machinations of PM interactions in order for optimal implementation in environmentally friendly, far-reaching and economically sustainable agricultural initiatives. To this end, pan-genome and metagenomic studies through molecular research would aid in creating a

U. Chettry Department of Botany, Royal School of Life Sciences, Royal Global University, Guwahati, Assam, India S. Upadhaya Department of Botany, School of Life Sciences, North-Eastern Hill University, Shillong, Meghalaya, India A. Nongbet Department of Botany, School of Biological Sciences, University of Science and Technology Meghalaya (USTM), Shillong, Meghalaya, India N. K. Chrungoo Royal School of Life Sciences, Royal Global University, Guwahati, Assam, India S. R. Joshi (✉) Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_8

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systematic road map to widen the scope of PM engineering in agriculture using contemporary technologies. Identification and characterisation of individual plant or microbial candidate genes with special focus on transferring optimum nutrients from different soil compositions to the plant, allied with cutting-edge CRISPR/ Cas-mediated genome editing technology, could be a game changer and force multiplier in the efforts to derive improved, economic and sustainable agronomic traits. Keywords

Plant-microbe interaction · Food security · Pan-genomics · CRISPR/Cas genome editing

8.1

Introduction

Since the advent of life on Earth, inter-species interactions have been the driving factor for the evolution of varied and higher life forms. As these life forms evolved, so did their interactions with other species, so much so, any higher life form today is an amalgamation of a varied cocktail of microorganisms that carry out a number of essential functions. These interactions may be evident or innocuous—depending upon the result, scope or visibility of the same. Millions of years of evolution have resulted in a multicoloured palate of plant-microbe interactions, which are a continuous and dynamic process ranging from the beneficial to the pathogenic (Kroll et al. 2017). These interactions may vary from the simple to the complex and are dynamic yet continuous processes that are not only as old as plant colonisation on Earth, but also fascinating in their evolution over the ages. They need not be looked at in isolation, as the way of evolution has been the varied and multifarious interactions between different species and cells along with the elements of the prevalent environment. In any case, it is necessary to realise and accept that plants and microbes exist in concert with one another. There are numerous processes and functions being carried out by the plant and its microbiome that are intrinsically linked to one another (Rosier et al. 2016). That is, a complex and myriad environment exists in and around any plant which is populated by beneficial and pathogenic microbes carrying out millions of interactions that ultimately determine the health and growth of the plant. Any mechanism that assists in transfer of nutrients, fixing nitrogen or stimulation of growth could point towards beneficial interactions (Di Benedetto et al. 2017), whereas conversely, if an interaction causes harm to plants such as disease, infection or destruction of plant cells, then that particular microbe is classified as a pathogen (Lorrai and Ferrari 2021). The identification and subsequent labelling of different phenomena and actors causing the same are essential to our understanding of plantmicrobe interactions. A comprehensive study of complex interactions will have realworld applications on the crop development and agricultural yield—especially with regard to food crops. Understanding these interactions is essential to understanding

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agricultural portfolio of a certain crop in a given region and how best to optimise growth and production in the most ecologically friendly manner. This is critical as the world hangs on the precipice of a widespread food and nutrient shortage that may have devastating effect on the world population—none more than the poorer regions of Africa and Asia that are less developed and have a large population to feed. An exponential growth in the global population since the dawn of the last century fuelled by unprecedented technological advancement, rapid urbanisation, relative peace and economic prosperity has progressively increased the demands on world food production and consequently impacted food and nutritional security across the cross sections of society (FAO 2017). Towards the second half of the twentieth century, the advent of genetic engineering allowed scientists and agriculturists around the world to tweak the very nature of crops, leading to greater yields of staple crops that, for a time, alleviated the stress on the crop production mechanisms across the globe (Bailey-Serres et al. 2019). However, this unprecedented yield was soon offset by burgeoning population growth due to unfettered urbanisation, greater diversity in jobs and a general shift from an agrarian society to an urban environment. The result has been a forever shrinking surface area available to grow crops coupled with an alarming decrease in the available biodiversity. However, the use of chemical fertilisers for over two centuries has yielded temporary gains to farmers but has also left behind ecological scars on the environment—the effects of which are becoming evident in the present day and age (Rahman and Zhang 2018). These chemicals not only are detrimental to the environment but also provide diminishing returns to poor farmers with limited land, solely dependent on these processes. The long-term negative impact on the yield and soil often leads to further distress, while in the grand scheme of things, their negative impact on the biodiversity and ecological balance has far-reaching and long-lasting implications on the planet (FAO 2021). Apropos, there is a concerted effort from around the globe to explore and germinate ideas that can increase crop yield in a sustainable yet economic manner that makes sense environmentally as well. While classical methods have long been used to study plant-microbe interactions (Houlden et al. 2008; Micallef et al. 2009; Prasanna et al. 2009), the advent of new-generation technologies has brought about a fresh and dynamic dimension to these studies, greatly improving the scope and speed of research in the field (Imam et al. 2016). A principal method is through gene manipulation to enhance beneficial plant-microbe interactions to further stimulate absorption of minerals, rekindle growth and increase tolerance to biotic and abiotic stresses. Research on these aspects would primarily focus on the combination of beneficial microbes and their relationships with crops, exploiting CRISPR/Casmediated gene editing technology to enhance the supply and intake of macro- and micronutrients. The research would also study the pathogenic microbes and explore the means to reduce their imprint on the plant and unwanted effects on its growth. Furthermore, the implementation of high-throughput technologies in the manipulation of plant-microbe interactions to improve plant yield as well as the environmental niche can be accelerated. This chapter discusses the different PM interactions and high-throughput tech, specifically CRISPR/Cas, and how they not only have

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revolutionised the field but also aim to inspire the reader to critically analyse further avenues of development and posit future developments and outcomes.

8.2

Plant-Microbe Interactions: A Glimpse into Evolution and Survival

Plant-microbe interactions largely take place at various cellular levels in a wide variety of forms (Vishwakarma et al. 2020). These interactions are borne out of evolutionary needs of both plants and microbes and have resulted in almost every part of a typical plant having some form of interaction with microbes at some point or the other in its life cycle. While these interactions may be characterised as symbiotic or pathogenic, it is a matter of great interest to study how plants differentiate between the two and between different pathogenic microbe species. In interactions where the associated bacteria provide some advantage to the plant, either directly or indirectly, plants provide a safe haven for microorganisms that colonise apoplastic regions, such as the soil near the roots, the rhizosoil (Al-Ani et al. 2020). Plants release chemicals that attract and feed related microorganisms, as also providing a secure area and a future nutrient source upon plant death. In return, the microorganisms produce substances promoting plant growth, increase the plant’s resistance to abiotic or biotic stress or even protect the plant against more harmful microbes (Schirawski and Perlin 2018). In order to maximise plant growth, enhance soil structure and control plant diseases, beneficial plant-microbe interactions are of vital importance. Such symbiotic relationships, where plants provide nutrition for rhizospheric microbes which in turn aid plant growth and reduce stress, are, unsurprisingly, the bedrock of environmentally friendly farming practices. On the contrary, to counter threats posed by pathogens, plants respond to plant-microbe interactions by engaging in a wide variety of defence-responsive activities that include activation of antioxidant status of the plant by reprogramming defencerelated enzymes, modulation of quorum sensing phenomenon and instigation of phenylpropanoid pathway leading to phenolics production, lignin deposition and transgenerational defence response (Mishra et al. 2015).

8.3

Nature’s Grace: The Beneficial Aspects of PM Interactions

As stated earlier, symbiotic interrelations between various species are the bedrock of survivability and evolution of any given species. Genetic evolution has often been linked to the response of cells to challenging environments and/or pathogens. Plantmicrobe interactions have occurred for millions of years and are one of the key factors in the wide biodiversity available on the planet. Consequently, they have been the subject of several biotechnological studies, seeking sustainable development especially in agriculture as well as exploring environmentally friendly alternatives to chemical fertilisation. Although the manipulation of soil microbiomes to optimise crop productivity has been carried out, its mechanistic study in

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heterogeneous communities and diverse locations, soils and hosts is limited (Finkel et al. 2019). The presence of beneficial microbes in the rhizospheric regions helps in partitioning minerals and nutrition (Van Der Heijden et al. 2008), induces tolerance against abiotic stress (Selvakumar et al. 2012) and increases innate immunity of the plant (Zamioudis and Pieterse 2012) and resistance against pathogenic microbes (Mendes et al. 2011). Specific Streptomyces species stand out among the various microorganisms that make their home in the rhizosoil. These active producers of antibiotics protect the plant from attack by more dangerous bacteria; they show filamentous growth and can colonise not only soil but also roots and aerial parts of the plant; they also produce volatile organic compounds that give rise to the distinctive scent of fresh forest soil. Therefore, it would be vital to find strains that act as antagonists of numerous plant diseases, utilising them as biocontrol agents in a variety of cropping systems. These multipurpose Streptomyces species can also be utilised as biofertilisers to stimulate plant growth. In comparison to other microorganisms, the ability to produce spores allied with the propensity to survive harsh soil conditions gives them an inherent advantage over others. Furthermore, they create lytic enzymes, which degrade insoluble organic polymers into usable nutrients that can be immensely beneficial to plants (Vurukonda et al. 2018). Phytoremediation of metal-contaminated soils can also make use of bacteria that stimulate plant growth. Helianthus tuberosus is a high biomass crop used for bioethanol production. It has been demonstrated that treating Helianthus tuberosus with plant growth-promoting bacteria isolated from plants growing in metalcontaminated soil significantly increases the plant’s ability to withstand elevated amounts of cadmium and zinc (Montalbán et al. 2017). There was a remarkable increase in cadmium uptake by the plant after the bacteria were demonstrated to thrive endophytically in the root. The plant’s metal-induced stress decreased, while its growth rate increased when the bacteria were present. It follows that these bacteria can aid in both phytoremediation and sustainable biomass production (Montalbán et al. 2017). Plant growth-promoting bacteria can also induce drought and salt tolerance. Perennial ryegrass (Lolium perenne) is a cool-season perennial grass with high yield and exceptional turf quality, such as a deep root structure, superior tillering, and regeneration ability. Perennial ryegrass’s resistance to drought was significantly increased when the beneficial soil bacterium Bacillus amyloliquefaciens GB03 was combined with a water-retaining agent consisting of superabsorbent hydrogels used for soil erosion control, as reported by Su et al. (2017). Using a novel bacterium isolated from a C4 perennial succulent xerohalophyte shrub with good drought and salt tolerance, He et al. (2018) significantly improved the growth and salt tolerance of perennial ryegrass. In addition, they sequenced the bacterial genome and discovered many genes that are likely involved in growth-promoting traits and promoting abiotic stress tolerance in plants. By introducing the arbuscular mycorrhizal fungus Rhizophagus irregularis CD1 to cotton, Zhang et al. (2018a, b) were able to increase the plant’s resistance to Verticillium wilt and boost its growth. They evaluated 17 different types of cotton for their symbiotic response to R. irregularis. Both

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plant growth and disease resistance against Verticillium dahlia wilt were significantly improved by the presence of the mycorrhizal fungus. These studies and research conclusively demonstrated that microbes can be employed to improve plant growth and make them more resistant to biotic and abiotic stresses like drought and salt. These findings have real-world and real-time implications due to global warming and climate crisis, which are expected to further aggravate the global food and nutrient shortages. Consequently, in order to address future challenges while guaranteeing plant fitness under harsher conditions, our understanding of how these systems operate and how to manage these systems in the prevalent environmental set-up will need to grow and develop accordingly.

8.4

Pathogenic Interactions and the Eco-Friendly Alternatives: Surviving the Apocalypse

While the beneficial microbes are currently thought to be stores of extra genes and characteristics that are important for the host’s growth and development, identification and characterisation of pathobiome are indispensable to protect crop damage and crop loss (Mannaa and Seo 2021). Fungi pose the largest threat among plantpathogenic microorganisms. Despite the fact that many plant-pathogenic fungi are exceedingly selective about the plants they infect, new fungal diseases can arise through host-switching events. Determining the factors that indicate whether hosts are conducive to fungal growth and disease development is thus one of the main areas of research in plant pathology. Borah et al. (2018) provide a comparative analysis of several approaches to the molecular identification of host specificity variables. Fungal plant pathogens that feed on living plant tissue must coexist closely with their hosts in order to successfully evade the host’s defences. Secreting effector proteins that interact with plant proteins to their benefit is one strategy for survival in the hostile plant tissue environment. A genomic analysis of the biotrophic fungus Microbotryum lychnidis-dioicae, the agent of anther smut in the common weed Silene latifolia, was conducted by Kuppireddy et al. (2017). Only 4 of the 50 potential effectors were proven to be secreted proteins. Investigating these ties led to the discovery of a protein in plants that had a lot of sequence similarities with a protein needed for pollen germination. Given that M. lychnis-dioicae only produces spores in the anthers, where pollen is formed, this could provide intriguing new information about how the fungus interacts with its host tissue (Kuppireddy et al. 2017). In addition, effectors were discovered by Gao et al. (2017), albeit in a different pathosystem. Researchers developed and analysed the transcriptome of Fusarium proliferatum, the fungus responsible for a fatal illness that manifests as dark brown necrotic spots on the leaves and stems of infected plants, eventually causing the entire plant to wilt and die. Without a complete genome sequence, scientists used de novo assembly of the sequencing transcriptome to discover 184 effector candidate genes, majority of which showed upregulated expression during plant colonisation (Gao et al. 2017). Wang et al. (2019a, b) examined the kiwi fruit transcriptome after infection with the bacterial canker pathogen Pseudomonas

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syringae pv. actinidiae (Psa). Many genes were found to be upregulated after studying the infected kiwi fruit plant’s gene expression. Among these were a shift in the expression of metabolic processes and genes involved in plant immunity (pathogen-associated molecular pattern-induced immunity and effector-triggered immunity), both of which may play a role in preventing the spread of Psa (Wang et al. 2018a, b, c). An alternative perspective was taken by Adams et al. (2018) who studied the pathogenesis of fungal infections in plants. Researchers discovered that rhodopsin-encoding genes are present in both phytopathogenic and phyto-associated ascomycetes. Even though new techniques have greatly enhanced our understanding of the many interactions between plants and microbes, and thereby furthered our understanding of the varied processes that take place between these organisms, how plant infections occur and what happens when plants are attacked by such pathogens are still mostly unknown. The preceding example shows that it is not uncommon for research to unearth unexpected results that both inspire new investigations and need re-evaluation of existing models of plant-pathogen interactions.

8.5

The Advent of Omics: A Defining Point in PM Studies

Although the classical method has unravelled a plethora of information on plantmicrobe interaction (Houlden et al. 2008; Micallef et al. 2009; Prasanna et al. 2009), the use of multi-omics approach in recent years has opened a multidimensional datum on rhizosphere-driven selection of microbiome on crop improvement (Table 8.1). The use of these methodologies has helped to not only understand the microbiome status in a specific niche, but also evaluate the consequences of perturbations caused by biotic and abiotic stress on the diversity of soil microbiomes and plant-microbial interfaces. Metagenomics from barley rhizosphere samples grown in phosphorus-devoid regime led to the dentification of novel ORF and homologs to P-regulatory and transport genes involved in mineral phosphate solubilisation in these regions (Chhabra et al. 2013). The question thus arises: Can microbe consortia from these areas be an alternative to P biofertiliser or can the Pho regulon gene isolated from these microorganisms be a tool for biofertiliser production? Similarly, comparative metagenomics study carried out in the rhizosphere of willow grown in contaminated and non-contaminated soils by Yergeau et al. (2014) revealed an upregulation of hydrocarbon degradation genes in contaminated soil regions. However, it is imperative to understand the unique mechanism which causes the shift in the biofilm accumulation and its expression. Is it just the microbial biofilms that determine the destiny of plant health or is it the plant exudates that influence the structure of microbial communities? A collaborative approach to understanding the functional capacity of the rhizo-microorganism and the impact of plant development on microbial partnerships that may arise naturally in the rhizosphere, not just for defence but also for other essential plant demands such as nutrition absorption, would open the Pandora’s box in PM-mediated crop improvement programmes. An in vitro analysis of Arabidopsis root exudate composition at different stages of plant development by Chapparo et al. (2013) revealed a spatio-

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Table 8.1 List of PM interaction studies based on omics-based technique (transcriptomics, metabolomics and proteomics) Omics approach Transcriptomics

Metabolomics

Proteomics

Plant Arabidopsis thaliana Arabidopsis thaliana

Microbe (s) Burkholderiaceae

Sample Root

Burkholderiaceae

Lactuca sativa Nicotiana tabacum L. Oryza sativa Nipponbare Panax ginseng cv. Damaya Oryza sativa

Botrytis cinerea

Root, shoot and seedling Whole plant

Saccharum officinarum L. Saccharum officinarum L. Brassica rapa

De Cremer et al. (2013) Liu et al. (2020) Kawahara et al. (2012) Gao et al. (2016)

Paenibacillus polymyxa YC0136 Magnaporthe oryzae

Roots

Cylindrocarpon destructans

Roots

Herbaspirillum seropedicae and Azospirillum brasilense Burkholderia anthina MYSP113

Roots

Enterobacter roggenkampii ED5

Leaves

Guo et al. (2022) Ichihashi et al. (2020)

Leaf blades

Roots

Vitis vinifera

Plasmopara viticola

Root, rhizosphere and soil Leaves

Glycine max

Phytophthora sojae

Hypocotyls

Solanum lycopersicum Arabidopsis thaliana Arabidopsis thaliana Cucurbita pepo Z. Pisum sativum

Tuta absoluta

Leaves

Verticillium dahliae

Seedlings

Kosakonia radicincitans

Roots

Zucchini yellow mosaic virus (P) Bacillus subtilis, Trichoderma harzianum, Pseudomonas aeruginosa and Sclerotinia sclerotiorum Azospirillum brasilense

Young leaves Leaves

Zea mays

References Harbort et al. (2020) Finkel et al. (2019)

Roots

Wiggins et al. (2022) Malviya et al. (2020)

Negrel et al. (2018) Zhu et al. (2018) de Falco et al. (2019) Su et al. (2018) Witzel et al. (2017) Nováková et al. (2015) Jain et al. (2015)

Faleiro et al. (2015)

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temporal regulation of microbiome by the root exudates. Another study carried out by Chaparro et al. (2014) in A. thaliana at seedling, vegetative, bolting and flowering stages unveiled that plants have a core rhizosphere microbiome that expresses and performs diverse tasks at different phases of plant growth. The root exudation of phytochemicals follows a genetically programmed developmental rhythm that aids in orchestrating the rhizosphere microbial assemblage. Such differential profiling of metabolites at different stages of plant development gives cues to identify specific microbial candidates that perform certain microbial functions at the specific time point and to uncover pathways for quorum signalling in the rhizosphere. Metatranscriptomics has also emerged as a key player in the identification of potent microbes, key genes and transcription factor involved in plant resilience and disease resistance (De Cremer et al. 2013; Das et al. 2022). Microarray studies induced by the endophytic plant growth-promoting rhizobacteria Pseudomonas fluorescens FPT9601-T5 in Arabidopsis by Wang et al. (2005) identified a candidate plant growth-promoting microbe. They reported that P. fluorescens imparts plantpromoting effect on tomato by a dynamic upregulation of auxin-regulated genes and nodulin-like genes and downregulation of ethylene-responsive genes—a mechanism mediated by most of the PGPR’s colonising rhizosphere. The universality of its mechanism and impact of its action on different plant systems are, however, yet to be deciphered and lay the emphasis for extensive field performance testing of novel materials across several seasons. De Cremer et al. (2013) revealed an active upregulation of phenylpropanoid pathway and terpenoid biosynthesis pathway following infection in Lactuca sativa by Botrytis cinerea. They identified several pathogen recognition genes such as WAKs, LRR-RLK and BRI-like as putative candidates mediating plants’ responsiveness against B. cinerea infection and early pathogen recognition. Similarly, time-course expressional changes in Panax ginseng following Cylindrocarpon destructans infection using RNA-Seq approach revealed an upregulation of pathogen response unigenes, defence-associated transcription factors such as TIFY1A, TIFY1B and MYB108 (Gao et al. 2016). Comparative transcriptome approach of host Oryza sativa L. ssp. japonica cv. Nipponbare and its pathogen Magnaporthe oryzae helped Kawahara et al. (2012) to identify infectionresponse profile at a spatio-temporal level. While the rice expression profile showed an upregulation of pathogenesis-related, phytoalexin biosynthetic genes, WRKY and NAC-family transcription factors, that of M. oryzae showed an increase of expression of secretory proteins encoding glycosyl hydrolases, LysM domain protein and cutinases. Such contemporaneous evaluation of gene expression profile between the coexisting species serves as a resource-induced marker for quick detection. Furthermore, these technologies have revealed several factors that are essential to understanding genetic variation and functional property of genes with its regulatory control that have resulted in the creation of high yields and stress-resilient plants along with their temporal responses to a dynamic environment. Transcriptome profiling of legumes (M. truncatula and L. japonica) and rice (Oryza sativa) has shown arbuscular mycorrhizal associations and is closely linked with transcriptional switch (Kistner et al. 2005; Liu et al. 2007; Guether et al. 2009; Grunwald et al.

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2009). A common finding from the above research groups was the AM-associated genetic marker, which could be used as a reliable marker for symbiosis. Das et al. (2022) identified phosphate starvation response transcription factor (PHR2) as a potent target for the activation of genes involved in several steps of arbuscular mycorrhiza development in model legume Lotus japonicus. In another analysis carried out by Liu et al. (2020), growth-promoting effect of Paenibacillus polymyxa YC0136 on Nicotiana tabacum L. was observed using RNA-Seq approach. Transcriptome profiles obtained from YC0136-infected tobacco showed upregulation of transcription factors involved in plant stress tolerance (WRKY2, WRKY33, HSFs, MYB and MYC2), genes associated with hormone signal transduction (SAUR, GA2ox, auxin-responsive proteins and SGTI) and genes associated with secondary metabolism (PAL, C4H, 4CL, COMT, CCoAOMT and shikimic acid O-hydroxycinnamoyl transferase), signifying the induction of plant defence response following YC0136 infection. YC0136 infection also resulted in the upregulation of gene coding for laccase, which plays an important role in plant resistance to fungi and insects. Expression of laccase in tobacco post-infection enhances the toughness of its rhizome and increases its resistance to pathogens. Using microarray-based comparative transcriptomics technique, wheat COMT gene TaCOMT-3D was identified by Wang et al. (2018a, b, c), overexpression of which resulted in enhanced resistance of wheat to sharp eyespot disease. These findings point towards the contribution of TaCOMT-3D positively to wheat resistance against sharp eyespot as well as promoting of lignin accumulation leading to mechanical strength of the stem. Combinatorial approach using metatranscriptomics and metaproteomics has rather enhanced the quality of PM interaction research by several degrees. Using geLC-MS/MS proteomic approach, Delmotte et al. (2010) reported the presence of 2315 B. japonicum strain USDA110 proteins in its legume host Glycine max 21 days after infection in Glycine max grown in growth chambers. Transcriptomic analysis of B. japonicum during soybean symbiosis revealed a total of 3587 genes predicted to be B. japonicum, and pathway enrichment showed expression of genes involved in carbon and nitrogen fixation, TCA cycle, gluconeogenesis and PPP. Similar observations were also made under field condition. Using multi-omics approach (transcriptomics, metabolomics and proteomics), Larsen et al. (2016) deciphered the molecular mechanism behind plant-fungus mycorrhizal interaction between Populus tremuloides (aspen tree) and Laccaria bicolor (mycorrhizal fungi). Their study identified a total of nine clusters of co-expressed genes from aspen root transcriptome and five mycorrhizal associated sensor protein complexes for aspen root. They developed a metabolome model for predicting the class of signalling compounds such as terpenoids and signalling molecules such as jasmonic acid and salicylic acid synthesised by Laccaria bicolor after mycorrhizal interaction with Populus tremuloides.

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Genome Editing: Hi-Tech Scalpels

A comprehensive understanding of plant-specific microbiome using metagenomics or metatranscriptomics approach has enhanced our knowledge about PM interactions, while combining these omics approaches with genome editing tools has upscaled the improvement of agronomic traits in model plants like Arabidopsis and tobacco; cereal crops such as rice, wheat, maize and barley; fruits such as apple, banana, grapes, oranges and lychee; and vegetables such as potato, tomato, cucumber and pea (Zhang et al. 2018a, b; Ali et al. 2015; Tripathi et al. 2019; Kis et al. 2019; Huang et al. 2020; Sánchez-León et al. 2018; Liu et al. 2021a, b; Jia et al. 2016; Camerlengo et al. 2022; Kong et al. 2019; Yu et al. 2019; Chandrasekaran et al. 2016; Liu et al. 2021a, b). Thus, genome editing has emerged as a vital component and an essential tool for sustainable discovery and development in this field (Table 8.2). Genome editing refers to a technology or combination of technologies to facilitate gene modification via addition, deletion or alteration of genetic material. Genome editing technology utilises engineered endonucleases to generate a double-strand break (DSB), which then undergoes an endogenous repair mechanism to generate various mutations (Fig. 8.1a). The first approach available exploited meganucleases to achieve targeted double-strand DNA breaks in genomes of eukaryotes that include zinc finger nucleases (ZFNs) and thereafter transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPRs) and CRISPR-associated protein (Cas) (CRISPR/Cas) system. While ZFNs and TALENs are a costly and tedious procedure with long turnover time, CRISPR/Cas system represents a new perspective for genetic engineering in terms of efficiency, specificity, versatility and cost-effectiveness (Brandt and Barrangou 2019). CRISPR/Cas system has therefore emerged as an important tool for genome editing in eukaryotes and often been described as nature’s toolbox for genome engineering (Wright et al. 2016; Sakuma and Yamamoto 2017). CRISPR/Cas system is a part of archaeal and bacterial type II immune system first reported by Ishino et al. (1987) in Escherichia coli while studying the isozyme conversion of alkaline phosphatase through iap gene. Since its discovery, CRISPR/Cas is being employed for improving agronomic traits in cereal crops, vegetables and fruits. A mechanistic approach has been represented in Fig. 8.1. CRISPR/Cas, the RNA-guided DNA/RNA cleavage, occurs through a complex called “effector complex”, which comprises a guide RNA called CRISPR RNA (crRNA) and a set of endonucleases called Cas proteins. CRISPR/Cas has been classified into 2 classes, 6 types and 33 subtypes based on Cas proteins and the nature of the interference complex (Makarova et al. 2020). The most common type used for genome editing is type II Cas9 derived from Streptococcus pyogenes (SpCas9). Type II Cas9 protein is bilobed consisting of a large recognition lobe (REC) and a small nuclease lobe (NUC). The NUC lobe also consists of a protospacer adjacent motif (PAM) interacting domain (PI) and two cleavage domains (RuvC and HNH), which introduces dsDNA breaks in target DNA (Fig. 8.1b:I, ii, iii). Cas protein is activated when an additional accessory

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Table 8.2 Table representing CRISPR applications to study plant-microbe interaction Plant species Rice

Target genes Zep1 Als2 PYL1, PYL4, PYL6 GN1a, DEP1, GS3

GW2, GW5, TGW6 CrtI, PSY GAD3 SBEIIb, SBEI GBSS GBSS IPK1 CrtI, PSY >GBSS OsSEC3A OsMATL OsSWEET13 ALS EPSPS 25604gRNA for 12,802 genes OsPDS, OsMPK2, OsBADH2 OsMPK2, OsDEP1 OsDERF1, OsPMS3, OsEPSPS, OsMSH1, OsMYB5 OsMPK5 OsAOX1a, OsAOX1b, OsAOX1c, OsBEL OsHAK-1 OsPRX2 LAZY1 OsERF922 OsSWEET11,13,14 Pi21

Resulting traits Increased frequency of genetic recombination Generating chlorsulfuronresistant maize Better growth and production Increased grain size and yield, with upright panicles and dense stouter clusters Increased grain density Enhanced carotene level High GABA content Enhanced amylase content Low amylose content Low amylose content Low levels of phytic acid DNA insertion Low amylose content Tolerance against Magnaporthe oryzae pathogen Induction of haploid plants Bacterial blight resistance Herbicide resistance Herbicide resistance Establishing a mutant collection covering the entire genome Involved in a wide range of abiotic stress tolerance Yield under stress Drought tolerance

References Liu et al. (2021a, b) Svitashev et al. (2015) Miao et al. (2018) Li et al. (2016)

Xu et al. (2016) Waltz (2016) Akama et al. (2020) Sun et al. (2017) Huang et al. (2020) Gao et al. (2020) Ibrahim et al. (2022) (Waltz 2016) Andersson et al. (2017) Woo et al. (2015), Xu et al. (2014), Zhou et al. (2014), Meng et al. (2017), Shan et al. (2013), Zhang et al. (2016), Xie and Yang (2013), NievesCordones et al. (2017), Mao et al. (2018)

Abiotic stress tolerance and disease resistance Abiotic stress tolerance and disease resistance Reduced cesium deposition Insusceptibility to potassium deficiency Tiller-spreading Increased resilience to rice blast Lack of sugar metabolites and broad-spectrum resistance Tough resistance

Oliva et al. (2019), Jiang et al. (2013) Li et al. (2019) (continued)

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Table 8.2 (continued) Plant species Oryza sativa L. Japonica Pea Tomato

Target genes eIF4G

Resulting traits Hindrance of viral translation

References Pyott et al. (2016)

Zep1

Increased frequency of genetic recombination Increased frequency of crossover Soybean breeding for resistance to the herbicide glyphosate Phytophthora infestans resistance enhancement Vulnerability to powdery mildew has been decreased Better protection against Botrytis cinerea Superior resistance to the effects of high temperatures Larger and heavier grains

Liu et al. (2021a, b)

RECQ4 ALS RXLR effector gene Avr 4/6 Mlo1 ACET1a, ACET1b MAPK3 GW7 FT2a, FT5a fas, lc SBEI CRTISO SIML01 SIJAZ2 SP5G SIAGL6 SP, SP5G, CLV3, WUS, GGP1 DMR6–1, DMR6–2

Orange Cucumber

SiDMR6–1, SiDMR6–2 Solyc08g075770 (CYCLOPS) CP CsLOB1 promoter eIF4E eIF4E

A greater abundance of pods and seeds Bigger and better fruit Increased amylase content Inter-homologous somatic recombination Resilience against powdery mildew Spotted resistance in bacteria Early harvesting Parthenocarpy Tomato domestication

Mieulet et al. (2018) Dong et al. (2021) Fang and Tyler (2016a, b) Nekrasov et al. (2017) Jeon et al. (2020) Yu et al. (2019) Wang et al. (2019a, b, c) Cai et al. (2015) Rodríguez-Leal et al. (2017) Tuncel et al. (2019) Shlush et al. (2020) Jaganathan et al. (2018), Shimatani et al. (2017)

Inactivation of salicylic acid to 2,3-dihydroxybenzoic acid Wide-ranging resistance

de Toledo Thomazella et al. (2016)

Improved defence response

Prihatna et al. (2018)

Disables the assembly of virus Resistance to canker in citrus Immunity to viruses

Tashkandi et al. (2018) Jia et al. (2016) Chandrasekaran et al. (2016) Fang and Tyler (2016a, b)

Large virus resistance

(continued)

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Table 8.2 (continued) Plant species Camelina sativa Wheat

Target genes FAD2 ZIP4-B2

MLO GW2 a-Gliadin genes TaMLOA1, TaMLOB1 and TaMLOD1 EDR1 TaGW2 TaMLO

Maize

TaEDR1 TaLpx-1 TIFY1b ERF922 eIF4G SBEIIa ARGOS8

Soybean

Potato

CLE Wx1 TMSS ALS ARG0S8 Qsd1 GASR7 ALS F3H1, F3H2, FNSII1 CM3, CM16 PSY1

Resulting traits Reduction in polyunsaturated fats Enhanced recombination between homologous chromosomes Protection against powdery mildew Bigger and denser grains Grains with decreased gluten levels Powdery mildew resistance

Powdery mildew resistance Increased seed size Wide-ranging and tough resistance Resistance response Enhanced resistance response Enhanced resistance to cold temperatures Increased resistance against rice blast Improved resistance against rice tungro spherical virus Enhanced amylase content Reduced vulnerability to drought Maximised Kernel number High amylopectin content Thermosensitive male sterile Herbicide resistance Drought stress tolerance Extended periods of dormancy Increased particle size Herbicide resistance Soybean mosaic virus resistance and elevated isoflavone content Lessening of allergen concentration Inter-homologous somatic recombination

References Jiang and Doudna (2017) Martín et al. (2021)

Wang et al. (2014a, b) Wang et al. (2018a, b, c) Sánchez-León et al. (2018) Nieves-Cordones et al. (2017), Wang et al. (2014a, b), Shan et al. (2014), Kim et al. (2018), Wang et al. (2018a, b, c) Wang et al. (2014a, b)

Nalam et al. (2015) Huang et al. (2017) Wang et al. (2016) Macovei et al. (2018) Li et al. (2021) Shi et al. (2017) Liu et al. (2021a, b) Shi et al. (2017), Svitashev et al. (2016)

Abe et al. (2018) Zhang et al. (2016) Cai et al. (2015) Zhang et al. (2020)

Camerlengo et al. (2022) Hayut et al. (2017) (continued)

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Table 8.2 (continued) Plant species

Target genes ALS

Resulting traits Herbicide resistance

Wx1 Coilin gene Sweet potato Barley

ASN2 GBSS

High amylopectin content Impairment of host and virus interaction Decreased levels of free asparagine Low amylose content

Rapeseed

SBEII

High amylase content

SGR1, LCY-E, Blc, LCY-B1, LCY-B2 GAD2, GAD3 GABA-Ts, SSADH GBSS D-hordein

High levels of lycopene

ITPK FAD2 WRKY11, WRKY17 Barley

COMT-1

Arabidopsis

MP/CP, Rep/RepA, LIR Chromosome 1, chromosome 2 LIR, Rep/RepA

Switchgrass

eIF (iso)4E CP 4CL

Salvia

CPS1

Tobacco

NtAn1

A7, B7 and C3 sites Flax

EPSPS

High GABA content High GABA content Low amylose content Low prolamin and high glutenin content Low levels of phytic acid Enhanced oleic acid content Better resistance Increased concentration of bioethanol in mutant biomass Interferes with replication of virus Reciprocal translocation Interferes with replication of virus Interference of viral translation Disables the assembly of virus Generation of bioenergy as a lignocellulosic feedstock Tailoring secondary metabolite profiles Increased seed lipid accumulation for biodiesel production Interferes with replication of virus Overcoming herbicide-resistant weeds

References Choudhury et al. (2016) Makhotenko et al. (2019) Raffan et al. (2021) Wang et al. (2019a, b, c) Wang et al. (2019a, b, c) Li et al. (2018) Nonaka et al. (2017) Li et al. (2018) Zhong et al. (2019) Yang et al. (2020) Sashidhar et al. (2020) Okuzaki et al. (2018) Sun et al. (2018), Tashkandi et al. (2018) Lee et al. (2021) Kis et al. (2019) Beying et al. (2020) Baltes et al. (2015) Pyott et al. (2016) Liu et al. (2018) Park et al. (2017) Li et al. (2017a, b) Tian et al. (2021)

Ji et al. (2015) Sauer et al. (2016) (continued)

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Table 8.2 (continued) Plant species Cassava

Grapefruit

Target genes EPSPS AC2, AC3 Novel cap-binding proteins nCBP-1 and nCBP-2 CsLOB1 CsLOB1 promoter WRKY52 VvMlo7

Citrus

LOB1 WRKY22

Apple Cotton Cacao Banana

DIPM-1, DIPM-2, DIPM-4 GhMYB25 NPR3 ORF1, ORF2, ORF3

Resulting traits Overcoming herbicide-resistant weeds Virus resistance Interferes with translation and movement of the virus

References Hummel et al. (2018)

Resistance to canker in citrus Resistance to canker in citrus Transcriptional reprogramming to regulate disease resistance Broad-spectrum and durable resistance Susceptibility factor against Xcc Induces pathogen-triggered immunity Reduces host susceptibility

Jia et al. (2016) Jia et al. (2016) Wang et al. (2018a, b, c) Pessina et al. (2016)

Enhanced resistance to wilt Enhanced disease resistance Knockout of dsDNA

Li et al. (2017a, b) Fister et al. (2018) Tripathi et al. (2019)

Mehta et al. (2019) Gomez et al. (2019)

Peng et al. (2017), Jia et al. (2016) Wang et al. (2019a, b, c) Malnoy et al. (2016)

non-coding RNA transactivating crRNA (tracrRNA) forms a complex with crRNA. The formation of this complex is facilitated by the REC lobe. CRISPR/Cas basically consists of two main parts: a short identical repeat of 20–40 ladders and a non-identical spacer region. This spacer region was thought to be unique until Mojica et al. (2005) discovered similarity between the spacer regions of CRISPRs and sequences of bacteriophages, archaeal viruses and plasmids. CRISPR/Cas consists of a small guide RNA (crRNA) and has the ability to recognise a short spacer DNA (invading DNA) into the CRISP locus. The CRISP locus consists of an array of spacer sequences, and this CRISP array is preceded by an AT-rich ladder sequence called crisprRNA or crRNA and also sequences encoding trans-activating crRNA (tracrRNA). This region is flanked by a series of genes coding for CRISPRassociated (Cas) endonucleases, which makes Cas protein. The transcription of the CRISPR locus produces crRNA partially complementary to tracrRNA, leading to the formation of an RNA duplex. This duplex is recognised by RNase III and cleaves the double-stranded RNA to form crRNA–tracrRNA complexes. The crRNA–tracrRNA complexes drive the Cas protein to the target sequence, whereby it induces a double-strand break (DSB) in the presence of a short conserved protospacer-adjacent motif (PAM) downstream of the target DNA. Stages in CRISPR/Cas mechanism: Typically, CRISPR/Cas action mechanism involves three stages:

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Adaptation or Spacer Acquisition

During adaptation, a short DNA is excised from invading DNA and inserted into the CRISPR array. In some CRISPR/Cas systems, adaptation occurs when a sequence motif flanking the PAM region is recognised and a protospacer is inserted in the CRISP locus (Mosterd et al. 2021). The insertion of invading DNA into the CRISPR array enables the host to memorise the invader’s genetic material and display the adaptive immune system (Barrangou et al. 2007). Cas1 and Cas2 of type II CRISPR/ Cas system of E coli form a complex to integrate new spacers, and the catalytic activity of Cas2 helps in spacer acquisition. New spacers are usually placed at the leader-repeat boundary of the CRISPR array (Fig. 8.1c).

8.6.2

crRNA Processing

During crRNA processing, CRISPR array is transcribed into precursor crRNA (pre-crRNA), which is processed into mature crRNAs. The processing of crRNAs

Fig. 8.1 An overview of CRISPR/Cas technology: (a) Basic principle of CRISPR/Cas showing generation of double-strand break (DSB) followed by two types of repair mechanisms, i.e., non-homologous end joining (NHEJ) and homologous direct repair (HDR). (b) (i) Cas protein showing different domains represented by different colours in a map. (ii) 3D crystal structure of Cas protein (PBD:4CMP). (iii) Graphical representation of Cas protein comprising six components (RECI, RECII, RuvC, HNH, BRIDGE HELIX and PAM interacting domain). (c) Pictorial representation showing three steps in CRISPR/Cas system. Adaptation phase where Cas protein recognises and excises the foreign DNA/RNA and inserts into CRISPR array adjacent to the leader sequence at the CRISPR locus. crRNA processing phase where CRISPR array containing the foreign DNA is transcribed and processed into multiple crRNA interphase stage where mature crRNAs guide to interfere and cleave the foreign DNA

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in type II CRISPR/Cas systems involves Cas9 endonucleases and differs from other CRISPR/Cas types and subtypes. In type II systems, tracrRNA forms a duplex with pre-crRNA, which is stabilised by Cas9. When the host RNase III recognises the duplex, it processes into an intermediate crRNA, which matures into a small guide RNA (Deltcheva et al. 2011) (Fig. 8.1c).

8.6.3

Interference

In this stage, mature crRNAs are used as guides to interfere and to cleave the foreign DNA/RNA. The crRNA guide region hybridises to target DNA by R-loop formation or base-pairing between crRNA guide region and foreign RNA followed by cleavage and degradation of the target (Fig. 8.1c). Several reports on CRISPR/Cas-mediated targeted mutagenesis for improving agronomic traits including disease resistance, higher yield and superior quality have been documented in the past decades (Table 8.2). Much attention has been given to genes and regulators involved in the defence mechanism for plant disease resistance. In an attempt to develop rice lines resistant to rice blast caused by Magnaporthe oryzae, Wang et al. (2016)) developed CRISPR/Cas9 SSN (C-ERF922) targeting the transcription factor OsERF922 gene in rice. Compared to the wild types, the edited lines showed reduced blast lesion symptoms following pathogen infection at seedling as well as tillering stages without compromising the agronomic traits. Similarly, CRISPR/Cas9 disruption of OsSEC3A by Chen et al. in 2018 revealed enhanced defence response and resistance to the fungal pathogen Magnaporthe oryzae. Their study revealed OsSEC3A-mediated disease resistance by interacting with the rice SNAP25-type SNARE protein OsSNAP32 through the C-terminus and to phosphatidylinositol-3-phosphate [PI(3)P] through N-terminus both of which are involved in plant disease resistance, respectively. Similarly, several reports have been documented to protect the crops from the black disease (Fister et al. 2018; Xie and Yang 2013; Wenderoth et al. 2017). Genome editing of MLO in wheat (Wang et al. 2014a, b), tomato (S. lycopersicum) (Nekrasov et al. 2017) and grapevine (Vitis vinifera) (Wan et al. 2020) has brought forth a milestone in developing varieties resistant against powdery mildew. The MLO-S gene has been reported to specifically inhibit the penetration of black fungus by cell wall thickening (Pessina et al. 2016; Prabhukarthikeyan et al. 2020) and can become a potential target to develop mildew-resistant crops. Additionally, Pirrello et al. (2022)) showed that targeted knockdown of downy mildew resistance 6 (DMR6) in grapevine also conferred resistance to downy mildew disease in grapevine. Also, as the universality is observed in the mechanism of action of MLO and DMR6 against powdery mildew, the question emerges if multiplex editing of MLO and DMR6 genes could be a target in the development of powdery mildew in different crop plants. Peng et al. (2017) demonstrated a strategy for generating canker-resistant citrus cultivars through CRISPR/Cas9-mediated promoter editing of CsLOB1 by constructing five pCas9/CsLOB1sgRNA constructs to modify the CsLOB1 promoter in Wanjincheng orange. Direct delivery of CRISPR/Cas9 RNPs to the

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protoplast of golden delicious cultivar of apple targeting DIPM-1, DIPM-2, and DIPM-4 susceptible gene showed increased resistance to fire blight disease, and the MLO-7 susceptible gene showed increased resistance to powdery mildew in chardonnay cultivar of grapes (Malnoy et al. 2016). Using three different Cas9:sgRNA ratios, i.e., 3:1, 1:1, and 1:3, for protoplast PEG-mediated transformation in both apple and grape cultivars, Malnoy et al. (2016) also determined the highest mutation frequency obtained from 3:1, 1:1; 3:1 and 1:1 for DIPM-1, DIPM-2, and DIPM-4, respectively, in the apple cultivar and 3:1 ratio for MLO-7 in the grape cultivar. Multiplex targeting using CRISPR/Cas showing broad-spectrum effect has also shown promising results. Chandrasekaran et al. (2016) employed CRISPR/Cas9 system to develop virus resistance in cucumber (Cucumis sativus L.) by targeting eukaryotic translation initiation factor 4E proteins (eIF4Es), which are redundant genes to the host but confer resistance to viruses resulting in the development against multiple RNA viruses, viz., Cucumber vein yellowing virus (Ipomovirus), Zucchini yellow mosaic virus, and papaya ring spot mosaic virus-W. Using CRISPR/Cas9mediated genome editing, Olivia et al. (2019) introduced mutations in SWEET11, SWEET13, and SWEET14 promoters that serves as an effector binding element (EBE) for transcription-activator-like effectors (TALEs) secreted by Xanthomonas oryzae pv. oryzae (Xoo) to induce bacterial blight of rice. Multiplex targeting using CRISPR/Cas9 has created the ability to simultaneously edit all EBEs present in any single rice line, thus generating robust, broad-spectrum resistance to Xoo pathogen in rice. Therefore, targeted knockout of susceptibility genes and/or negative regulators through genome editing provides a powerful strategy for disease resistance breeding. Besides disease resistance, CRISPR/Cas system has been effectively employed for the identification of beneficial PGPM that can be successfully used as biofertiliser. Yi et al. (2018) employed the CRISPR/Cas9 system to perform gene knockout and chromosomal insertion in Bacillus subtilis HS3 and Bacillus mycoides EC18, potent plant growth-promoting bacteria to decipher the mechanisms of Bacillus-plant interactions. Their results revealed that while surfactin and fengycin family lipopeptides were responsible to impart antifungal activity to B. subtilis HS3, siderophore and bacillibactin were involved in the plant growth-promoting activity and root colonisation of B. mycoides. Its application has also been well documented in crop domestication and quality enhancement of important cereal crops such as maize, rice and wheat programmes. In maize, Liu et al. (2021b) engineered CLE genes in the CLAVATA-WUSCHEL pathway for enhancing grain yield-related traits using CRISPR/Cas9 strategy. CRISPR/Cas approach has been successfully used for improving several traits in rice such as enhanced grain weight, size and number (Zeng et al. 2020), enhanced accumulation of carotenoids (Banakar et al. 2020), production of high oleic and low linoleic acid (Abe et al. 2018), increased amylase content (Sun et al. 2017) and low cadmium rice (Tang et al. 2017). Using CRISPR/Cas9-based multiplexed gene editing (MGE) approach, Wang et al. (2018a, b, c) generated heritable mutations in hexaploid wheat in the GW2, Lpx-1 and MLO genes. Out of these, GW2 mutant showed increased seed size and grain weight. In another study conducted by Wang et al. (2019a, b), CRISPR/

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Cas9-mediated gene editing of TaGW7 in wheat that encodes a TONNEAU1recruiting motif (TRM) protein affecting grain shape in weight showed mutation in the B and D genome of TaGW7 exhibiting increased width and weight but reduced length. Another important area of CRISPR/Cas-mediated strategy is metabolite engineering of secondary metabolites to obtain higher production of bioactive secondary metabolites. CRISPR/Cas-mediated carotenoid biofortification has been achieved in rice with higher levels of carotene (Dong et al. 2021), tomato with fivefold higher lycopene content (Li et al. 2018) and banana with sixfold higher levels of β-carotene (Kaur et al. 2020). CRISPR/Cas-mediated enhanced GABA level has been achieved in tomato (Li et al. 2018).

8.7

Future Perspective: A Vast Expanse of Uncharted Science with Limitless Possibilities

As the world population is expected to rise from its estimated 7 billion plus numbers at present to almost 9 billion by 2050, there is a global threat to future food availability. This has led to the teetering of agricultural sustainability on the precipice of calamity due to rising climate variation, exploding population and a yet-tobe-arrested decline in soil health for crop cultivation. The study of beneficial and pathogenic microbes in relation to plant health and crop yield and stability needs to be conducted in the context of external challenges posed by global warming and climate crisis. Biotic and abiotic stresses impose major restraints on crop yield, food quality and global food security. Under stress, plants change in myriad ways, including physiologically, biochemically and molecularly. Since the use of inorganic fertilisers and pesticides in agriculture causes discernible degradation of soil fertility coupled with polluting of the environment, it is essential that ecologically sustainable means for agriculture production are explored. In this context, the application of plant growth-promoting microbes (PGPMs) and mycorrhizal fungi enhances plant growth, offering an economically fascinating and ecologically sound method for protecting plants against stressful conditions (Kumar and Verma 2018). At the same time, understanding the emergence of novel genetic variants that confer pathogenicity and virulence to the plant would open up the dynamics of interaction affecting and being affected by external factors and help in designing strategies to control plant diseases. In recent years, omics technology has brought about a paradigm shift in PM-interaction biology. The concept of one pathogen-one trait dogma is a thing of the past, and the new era of interaction of an orchestra of microbes within the plant’s biotic environment, to impart benefits or deteriorate its health status, has taken the lead. This transition from the concept of system biology to pathobiome would give a greater understanding of the mechanisms and the environmental conditions, including climate effects that control plant–microorganism assembly and activities and help us engineer microbial communities to optimise crop performance, particularly with microorganisms that are engineered using synthetic biology approaches. Moreover, with the ever-increasing advances in genome editing technologies, capturing

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the immune-receptor gene panels from the wild relatives of crops and understanding its surrounding microbiota would pose as an effective strategy to develop diseaseresistant lines. Further, harnessing pan-genome data from different recognition spectra and environmental condition of the variants and its rhizosoil would be an effective strategy to develop climate-resilient and disease-free crops. Thus, while unfettered development has led us to the imminent food crises, the very same development has also granted us the opportunity to modify characteristics of life forms such as plants and microbes beyond the cellular level. It is incumbent upon us to seize the opportunity presented by the advent and rapid development of the new genome editing techniques to accrue as much understanding of the multifarious processes involved while also devising ingenious and sustainable methodologies to achieve the overarching objective of global food and nutritional security.

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Biomedical Application of Advanced Microbial Approaches: Nutraceuticals, Biomedicine, and Vaccine Development Neha Namdeo, Ragini Arora, Harit Jha, Neha Namdeo, and Ragini Arora

Abstract

The products obtained from natural sources that provide physiological, medicinal, and nutritional values are termed as nutraceuticals. Medical foods, phytochemicals, designer foods, and functional foods are some of the terms by which nutraceuticals are known. It includes herbal products, dietary supplements, vitamins, and genetically engineered foods. It improves health issues and prevents fatal diseases. Various nutraceuticals are derived from distinct microbial sources. Fungi, bacteria, actinomycetes, microalgae-cyanobacteria, and diatoms are some key producers of novel bioactive compounds producing nutraceuticals. Growing demand for natural products is a major drive for microbial production of substances like vitamins, enzymes, organic acids, proteins, and antibiotics. These nutraceutical compounds are produced by several microorganisms like Lactobacillus species, Methylophilus methylotrophus, Bacillus subtilis, Aspergillus fumigates, and Saccharomyces cerevisiae. Nutraceutical compounds promote health as well as prevent diseases such as depression, arthritis, osteoporosis, oxidative stress, inflammation, diabetes, cancer, and cardiovascular diseases. The growth of the nutraceutical market has been greatly promoted through growing demands and interests to maintain human health. A wide range of bioactive metabolites are involved in food and pharmaceutical industries to synthesize novel drugs, which exhibit multiple drug-resistant properties against pathogens. Thus, our biodiversity and their metabolites might be utilized in order to discover novel nutraceutical compounds for biomedical research. Keywords

Nutraceutical · Biomedical research · Phytochemicals · Drugs · Therapeutics N. Namdeo · R. Arora · H. Jha (✉) · N. Namdeo · R. Arora Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_9

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Abbreviations 2-FL AMR AOS aP ARDS BCG BYG COPD CVD DTP FOS FTF GI GOS Hib HIV HMOs IBD LAB LC MenB USD UTIs

9.1

2-Fucosyllactose Antimicrobial resistance Algal oligosaccharides Acellular pertussis Acute respiratory distress syndrome Bacille Calmette-Guérin Brewer’s yeast glucan Chronic obstructive pulmonary disease Cardiovascular disease Diphtheria tetanus pertussis Fructans oligosaccharides Fructosyltransferase Gastrointestinal Galacto-oligosaccharides Haemophilus influenzae type b Human immunodeficiency virus Human milk oligosaccharides Inflammatory bowel disease Lactic acid bacteria Lung cancer Meningitis serogroup B United States dollar Urinary tract infections

Introduction

Nutraceutical, a term coined by Stephen L. DeFelice in 1989 as a blend of “nutrition” and “pharmacy”, was described as “any item that is a diet or a food ingredient that has medicinal or health advantages, including the treatment and prevention of illness”. In contrast to functional foods and medications, nutraceuticals now have a stronger emphasis on functionally and structurally varied bioactive compounds that have long-run physiological or therapeutic benefits in addition to solely nutritional or direct pharmacological effects (Wang et al. 2015). The majority of nutraceuticals come in a variety of forms, including foods, nutritional supplements, and prepared foods. They are vital for human existence and have strong physiological activities and bioactivities. Widespread nutritional deficits can lead to diseases that endanger people’s lives and health. An important group of compounds known as nutraceuticals has the potential to have long-term physiological effects, such as the prevention of illnesses linked to ageing, anxiety, asthma, osteoarthritis, digestive disorders, cardiovascular disorders,

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diabetes, and cancer (Yuan and Alper 2019). Focusing on human health has become more important as living standards have increased. Numerous research has tried to identify substances, or nutraceuticals, that are beneficial to ageing. These substances are powerful and active components in medicines and are crucial to the prevention of particular illnesses. The demand for interest in nutritional supplements to maintain human wellness has significantly fuelled the market’s expansion (Wang et al. 2015). The productivity of traditional nutraceutical industries, however, can hardly keep up with the growing nutraceutical market. The low concentration and purity of nutraceuticals, the high cost and availability of raw materials, and the strict quality control of suppliers all place restrictions on direct extraction techniques. Even though one alternative method is chemical synthesis, it can only be used to produce basic biochemicals and is impractical for producing intricate biochemicals, especially for those that have poor chemical composition (De Luca et al. 2012). Microorganisms have shown considerable advantages over conventional industrial processes in the biosynthesis of these compounds (Liu et al. 2015). Since germs are typically imperceptible to our senses, understanding microbiology and illness requires imagination. Microbiological research has achieved great gains in the detection, treatment, and prevention of infections. These developments have boosted human health and doubled life expectancy. Production of nutraceuticals is better suited for microbial synthesis. The substrates for microbiological synthesis are widely available, repeatable, and inexpensive to obtain. Microbes may exploit a variety of industrial waste and agricultural leftovers, and these materials have a great deal of promise for high nutraceutical output. Bioprocesses are also thought to be ecologically beneficial, and the end goods they produce are generally safe. However, when used in the pharmaceutical and food sectors, among others, strains need to be thoroughly described and chosen to fulfil safety criteria (Liu et al. 2015). The global need for value-added nutraceuticals for the prevention and treatment of human ailments has resulted in a multibillion-dollar business for nutraceuticals. However, the widespread usage of nutraceuticals is constrained by supply issues and the challenges associated with extracting ingredients of natural origin like fungi, plants, or animals. As a more ecologically friendly option, metabolic engineering using microbial production platforms has improved the synthesis of value-added nutraceuticals from simple carbon sources. Crosstalk between the body’s immune system and the microbiome throughout life is the foundation of their harmonious connection (Ciabattini et al. 2019). The development of efficient prevention techniques is required due to the ongoing spread of deadly pathogens and the decreasing availability of antimicrobial treatments. The prevention and treatment of microbial infections are one of the most difficult issues facing public health in the twenty-first century. A confluence of factors, including ageing populations at high risk of infection, increasing antimicrobial resistance (AMR), rising global morbidity and mortality due to bacteria and healthcareassociated infections, and a dearth of novel antibiotic classes, signal the beginning of a crisis in bacterial diseases. Vaccines have the potential to be extremely successful instruments in the fight against antibiotic resistance (AMR). Recent developments in vaccine technology

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have made it feasible to build vaccinations against targets that were previously difficult to attack. Understanding the vaccinations that are being developed at the moment and those that may be made accessible as tools to help manage AMR in the future is necessary (Klugman and Black 2018). Vaccines were first designed to shield kids from potentially fatal diseases. Early invention and manufacturing of potent toxoid vaccines against tetanus and diphtheria, cholera, whooping cough, tuberculosis, and typhoid were made possible by very simple processes. Acellular pertussis (aP) vaccines, Lyme disease vaccines, vaccines against simple polysaccharides and capsular polysaccharides covalently bound to a carrier protein, and later vaccines constructed with immunoprotective subunits (conjugate vaccines targeting Haemophilus influenzae type b [Hib], Neisseria meningitides, Streptococcus pneumonia, and most recently Salmonella enterica serovar Typhi) were developed. Recently, subtractive antibody screening and/or in silico antigen identification have been employed to create N. vaccinations for meningitis serogroup B. With a market value of 30 billion USD in 2016 and an expected 45 billion USD in 2022, the growth rate for vaccines is promising, given that more than 250 vaccines are being developed by US-based firms or international companies that are running drug trials in the USA. About 25 of the vaccines used to combat infectious illnesses can be linked to bacterial sickness. Streptococcus pneumonia, Neisseria meningitidis, and Haemophilus influenzae vaccines are under development for use against bacterial illnesses. Since national ministries of public health frequently distribute vaccinations, vaccine development and production costs must be cheap to allow for a worldwide reach of vaccines, particularly in emerging countries.

9.2

Commercially Available Nutraceuticals, Biomedicines, and Vaccines

9.2.1

Nutraceuticals

Nutraceuticals are significant natural bioactive substances that promote human health and provide therapeutic advantages (Wang et al. 2015). Clinical medications, cosmetics, healthcare items, and food additives are all examples of nutraceuticals for human health (Table 9.1). Numerous acute and chronic illnesses are prevented and treated by using nutraceuticals in conjunction with diet (Liu et al. 2015).

9.2.1.1 Inulin A prebiotic, or a substance that promotes the development of healthy gut flora, is inulin. Gut health and immunity are supported by helpful bacteria, which also lower illness risk. A fructan is an oligosaccharide that includes inulin. The gastrointestinal (GI) tract of people and animals is home to a broad variety of naturally occurring Lactobacillus species, which are thought to have a range of positive impacts on human health. Numerous probiotic functions of Lactobacillus gasseri have been

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Table 9.1 Value-added nutraceuticals produced using microorganisms Nutraceutical Inulin Galactooligosaccharides (GOS) 2-Fucosyllactose (2-FL) Brewer’s yeast glucan (BYG) Xanthan

Producing strains Lactobacillus gasseri Kluyveromyces lactis Escherichia coli

Saccharomyces cerevisiae Xanthomonas campestris

Functions Dietary supplement, diabetes, fat replacer Cosmetic, beverages, confectionary products, pharmaceutical, poultry Modulation of intestinal microbiota, anti-adhesive effect against pathogens, development of immune system Emulsion-stabilizing agent, waterholding agent, fat replacer A stabilizer and viscosifier in syrups, sauces, and bakery products, improves volume, texture, and shelf life of baked foods

Reference Leemhuis et al. (2010) Rodriguezcolinas et al. (2011) Lee et al. (2012)

linked to it, such as a decrease in the activity of faecal mutagenic enzymes (Pedrosa et al. 1995), adherence to intestinal tissues, and stimulation of macrophages (Kirjavainen et al. 1998, 1999; Tejada-Simon and Pestka 1999; Itoh et al. 1995; Conway et al. 1987). Numerous lactobacilli have been shown to have probiotic properties because they produce prebiotic fructans from sucrose using the enzyme fructosyltransferase (FTF) (Armuzzi et al. 2001). The fructose component of its substrate sucrose is polymerized by FTF enzymes into fructans having either inulin or levan structures, with b(2–1) and b(2–6) links, respectively. One of the possible reasons that provide certain lactobacilli with a probiotic role(s) is the fructansucrase enzymes’ ability to synthesize from sucrose. The inulosucrase enzyme InuGB of L. gasseri strain DSM 20604 produced both inulin oligo- and polysaccharides. Strain DSM 20604 generated a wide spectrum of inulin-type FOS spanning from DP2 to DP13 (Leemhuis et al. 2010).

9.2.1.2 Galacto-Oligosaccharides (GOS) The enzymes known as β-galactosidases (also known as β-D-galactoside galactohydrolases, EC 3.2.1.23) catalyse the hydrolysis of the galactosyl moiety from the non-reducing termini of oligosaccharides. Researchers and dairy product producers are interested in these enzymes because they can eliminate lactose from milk. β-Galactosidase catalyses transgalactosylation reactions in which lactose (along with the released glucose and galactose) serves as a galactosyl acceptor, resulting in a series of di, tri-, and tetrasaccharides (and eventually of higher polymerization degree) known as galacto-oligosaccharides (GOS). GOS make up the majority of the sales of dairy product (Martinez-Villaluenga et al. 2008). The ability of Kluyveromyces lactis to hydrolyse is too high. The production of GOS using K. lactis β-galactosidase in batch and continuous bioreactors is quite high. Several research on the immobilization of this enzyme with other carriers has also

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been documented. Lactis β-galactosidase has been reported (Rodriguez-colinas et al. 2011).

9.2.1.3 2-Fucosyllactose (2-FL) The most important component for the establishment of the intestinal microbiota in breastfed newborns is believed to be human milk oligosaccharides (HMOs). Additionally, it has been stated that HMOs are crucial in keeping infections and poisons from adhering to epithelial surfaces. HMOs with the fucosyl group include lacto-N-difucohexaose, lacto-Nfucopentaose, and 2-fucosyllactose. Infants are shielded against infection by toxin binding and enteric pathogens by fucosylated oligosaccharides, which function as growth-promoting substances for certain Bifidobacteria and soluble analogues of receptors for pathogenic bacteria (Morrow et al. 2004). Particularly, it has been found that 1,2-linked fucosylated oligosaccharides have anti-pathogen properties against Campylobacter jejuni and Enterotoxigenic E. noroviruses, E. coli, and Helicobacter pylori (Maqalhães and Reis 2010). Low levels of 2-FL in irritated mothers’ milk have been linked to a greater prevalence of diarrhoea in breastfed children. As a result, 2-FL is a potential oligosaccharide for nutraceutical and medicinal applications. 2-FL can be produced by α-1,2-fucosyltransferase (FucT2) enzymatic fucosylation of lactose, which needs guanosine 50-diphosphate (GDP)-L-fucose as a donor of L-fucose (Albermann et al. 2001). GDP-L-fucose is known to be synthesized by Escherichia coli since it is employed in the production of colanic acid, a primary component of the cell wall. Thus, 2-FL may be generated in metabolically modified E. coli by manipulating the GDP-L-fucose biosynthesis pathway and amplification of the fucosyltransferase gene. A range of uses for 2-FL as a nutraceutical additive may benefit from effective microbial 2-FL production (Lee et al. 2012). 9.2.1.4 Brewer’s Yeast Glucan (BYG) BYG is an abbreviation for marketed “brewer’s yeast glucan”, which might possibly include substances other than carbohydrates derived from S. cerevisiae. BYG is effective in enhancing the qualities of foods as a water-holding agent and thickener, or as a fat substitute that gives food a rich mouthfeel. It also strengthens gels in solutions (Reed and Nagodawithana 1991). Solid gels of BYG are generated after heating and chilling liquids with concentrations of more than 5–10%. The glycan also possesses emulsifying qualities and is said to improve the sensory properties’ aspects of meals to which it is introduced. After alkali extraction from homogenized cell walls, Thammakiti et al. (2004) studied the generation of a b-glucan from used brewer’s yeast that has a b-(1,3)glucose backbone chain, a little branch of b-(1,6)-glucose (approximately 3%) and an extra 4.5–6.5% of protein. The b-glucan in question might be used as an emulsion stabilizer in food.

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A similar substance known as baker’s yeast glycan, which is mostly used as a stabilizer/emulsifier in sauces and desserts, is made of D-glucose and D-mannose in a 3:2 ratio. The same yeast has been investigated and used to make medicinal glucans.

9.2.1.5 Xanthan Due to its viscosifying and stabilizing qualities, xanthan is perhaps the most frequent bacterial polysaccharide utilized as a food ingredient. Xanthan gum is produced by the Gram-negative plant pathogen Xanthomonas campestris as a mechanism of adhesion to plant surfaces. A trisaccharide side chain is attached to every other glucose molecule at C-3 in xanthan, which has a linear (1,4)-connected b-d-glucose backbone. It also has terminal mannose units connected to glucuronic acid residues (1,4) and (1,2). Xanthan has been used in a variety of products as a viscosifier and stabilizer in syrups, sauces, dressings, baked goods, soft cheese, restructured meat, and other things because of its strong freeze-thaw stability, good freeze-thaw stability, and remarkable suspending characteristics (Casas et al. 2000). In bakery products, xanthan gum is used to increase volume and texture, freezethaw stability of chilled doughs, water binding throughout baking, and shelf life of baked goods; replace egg white in low-calorie cakes; increase flavour release; and decrease syneresis in creams and fruit fillings, and more.

9.2.2

Biomedicine

The term “biomedicine” used to describe Western professional medicine stresses the fact that it is primarily a biological discipline. Gaines and Hahn (1982) gave what had previously been called “scientific medicine”, “Western medicine”, “allopathic medicine”, “cosmopolitan medicine”, and simply “medicine” the label “biomedicine” for these reasons. Indications of considerable potential in both pharmaceutical and nutraceutical applications come from the wide immunostimulating, anticancer, antibacterial, antioxidant, hypocholesterolaemic, and hypoglycaemic effects of polysaccharides (Table 9.2). Engineered E. coli can manufacture medicinally significant polysaccharides like heparosan and chondroitin. E. coli, with titres of 1.88 g/L and 2.4 g/L, respectively, is considered to be high (Wang et al. 2015).

9.2.2.1 Anticancer An important worldwide public health issue is cancer. In most parts of the world, cancer prevalence and death have been gradually increasing over the past century. Numerous epidemiological studies have shown that probiotics can help prevent cancer. The digestive tract benefits greatly from the presence of lactic acid bacteria and associated probioactive cellular components; additionally, they discharge a number of enzymes into the intestinal lumen, which may have additive effects (LAB) on digestion and lessen intestinal malabsorption symptoms. LAB-infused fermented dairy products may have anti-tumour properties. The reduction of

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Table 9.2 Commercially available biomedicines along with their producing microbial strains Biomedical application Anticancer

Producing strains Lactic acid bacteria

Infectious diarrhoea

Lactobacillus rhamnosus GG, L. reuteri, L. casei Shirota, and B. animalis Bb12

Allergy

Bacillus lactis and Lactobacillus rhamnosus GG

Inflammatory bowel diseases (IBDs)

Bifidobacteria, Lactococcus lactis LA 103, L. plantarum 299 V, Escherichia, and Enterococci

Urinary tract infection (UTI)

Lactobacillus sp.

Mechanism of action Lower enzymes involved in the production of carcinogens, mutagens, or tumour-promoting substances; limit mutagenic activity; and suppress tumours Boost the immune response, actively suppress receptorsite signals controlling secretory and motility defences, and produce compounds that directly inactivate virus particles Enhance mucosal barrier efficacy, which helps to reduce allergic reaction Restore the abnormal native microflora’s characteristics, strengthen the several lines of intestinal defence, and alter the ratio of proinflammatory (IL-12) to anti-inflammatory (IL-10) cytokines produced in the body Re-establish the equilibrium of the vaginal flora, act as a protective agent against pathogenic colonization, and synthesize the microbicidal chemical H2O2

Reference Kumar et al. (2010)

Kechagia et al. (2012)

Kechagia et al. (2012) Ouyang (2006), Mogra (2014)

Grin et al. (2013), Gil et al. (2013)

tumours, inhibition of mutagenesis activity, a decline in many enzymes involved in the production of carcinogens, neurotoxic or tumour-promoting chemicals, and epidemiology linking dietary patterns to cancer are all credited with these effects. In addition to altering cell-mediated immune responses, enhancing cytokine pathways, stimulating the reticulo-endothelial system, and controlling interleukins and tumour necrosis factors, lactic acid bacteria appear to have potent adjuvant effects. Intestinal bacteria may bind mutagens, inhibit the growth of bacteria that turn procarcinogens into carcinogens, reduce the amount of carcinogenic substances in the intestine, inhibit the deconjugation of bile acids, lower the enzymes

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b-glucuronidase and b-glucosidase, or simply boost the host’s immune system to prevent tumour growth (Kumar et al. 2010).

9.2.2.2 Infectious Diarrhoea The most well-known health advantages of probiotic bacteria are undoubtedly the cure and prevention of acute diarrhoea. The major cause of infant deaths worldwide and of acute neonatal diarrhoea is rotavirus. Probiotic addition of infant foods has been used to prevent rotaviral infections as well as treat existing sickness. Probiotics such as L. acidophilus have been established in well-controlled clinical research to be beneficial. L. rhamnosus GG, L. reuteri, B. animalis Bb12, and L. casei Shirota have been shown to reduce the occurrence of acute rotavirus diarrhoea, with the most data pointing to B. animalis Bb12 and L. rhamnosus GG (Shah 2007). The approaches proposed include the production of compounds that directly inactivate virus particles, as well as the competitive blockage of receptor site signals that govern secretory and motility defences. There is evidence that some food and non-food probiotic strains, in addition to rotavirus infection, can decrease the development and adherence of a number of diarrhoeal diseases (Kechagia et al. 2012). 9.2.2.3 Allergy Recent research shows that early bacterial exposure may have a defence role against allergies, as well as probiotics offer a safe substitute to the microbiological stimulation that young children’s growing immune systems require. They also improve mucosal barrier function, which is considered to aid in reducing allergic responses. Children and newborns with allergies and those without demonstrate quantitative and qualitative variations, with the former displaying colonization by a more adultlike kind of microflora, suggesting the function of gut microbiota in allergy. Food allergies and atopic dermatitis appear to be particularly affected by these probiotic effects (Marteau et al. 2002). The effectiveness of a small number of strains in the prevention and treatment of newborn allergy has been examined. B. lactis and L. rhamnosus GG were found to be useful in lowering the severity of atopic eczema in a recent study of breastfed babies. Furthermore, when administered prenatally to selected mothers who had at least one first-degree relative with dermatitis, asthma, or allergic rhinitis, L. rhamnosus GG was shown to be helpful in preventing the development of atopic dermatitis in high-risk babies (Kechagia et al. 2012). 9.2.2.4 Inflammatory Bowel Disease (IBD) IBD is a chronic condition characterized by intermittent stomach discomfort, altered bowel movements (diarrhoea and/or constipation), and additional gastrointestinal symptoms such as bloating and flatulence in the absence of structural intestine abnormalities (Weichselbaum 2009). IBD patients have abnormal enteric microflora, which includes a considerable rise in pathogenic and potentially hazardous enterobacteria and a significant decrease in bifidobacteria and lactobacillus. The pathophysiology of IBD may include

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abnormal microbiota. According to preliminary research, oral probiotic therapy may reduce intestinal inflammation in clinical trials and instances of animal colitis. Lactobacilli, Bifidobacteria, and few non-pathogenic bacteria like Enterococci and Escherichia are frequently found in probiotic mixtures. Probiotics taken orally can normalize the features of abnormal native microflora and strengthen several lines of intestinal resistance. However, different probiotic strains may have different immunological responses and distinctive properties while stabilizing in the inflammatory mucosa of patients or healthy people. In this regard, it may be feasible in the future to comprehensively explore the properties of various probiotic strains in order to choose and evaluate the best probiotic strains and relevant components for the therapeutic intervention of IBD. Ouyang (2006) and Drouault-Holowacz et al. (2008) discovered an increase in patients receiving a probiotic mix (L. acidophilus LA 102, B. longum LA 101, St. thermophilus LA 104, Lactococcus lactis LA 103) with time among those receiving a probiotic mix (L. acidophilus LA 102, B. longum LA 101, Lactococcus lactis LA 103, St. thermophilus LA 104) with satisfactory relief (Camilleri 2006). According to O’Sullivan and O’Morain (2000), L. rhamnosus GG had no impact on IBS symptoms, although L. plantarum 299 V showed a significant positive impact (Mogra 2014).

9.2.2.5 Urinary Tract Infections Urinary tract infections (UTIs), the most frequent illness in premenopausal adult women, are acute bacterial diseases caused by pathogens colonizing the vagina and ascending into the urinary system. They cause significant morbidity and cost the nation’s healthcare systems billions of dollars each year. At the moment, antimicrobials like trimethoprazine are the long-term, low-dose regimens clinically prescribed as prevention for these women. For years, Lactobacillus probiotics have specifically been investigated as a potential prophylactic for UTI. Several native lactobacilli species are found in the vaginal flora of healthy women and are thought to defend against pathogenic colonization (Cadieux et al. 2009). Probiotic Lactobacillus strains give a range of advantages, including the ability to (1) restore vaginal flora balance following antibiotic therapy for an initial UTI, (2) support the maintenance of a normal vaginal pH of 4.5, and (3) produce the microbicidal chemical H2O2 (Grin et al. 2013). L. crispatus was tested in a doubleblind, placebo-controlled experiment by Stapleton et al. Probiotic crispatus intravaginal suppository was used for premenopausal women to avoid recurrent UTI. In comparison to 27% of women who received a placebo, 15% of women taking probiotics experienced recurrent UTI (relative risk, 0.5; 95% CI 0.2, 1.2) and high levels of L. vaginal colonization. Only in the group that received probiotics was crispatus linked to a substantial decrease in recurrent UTI during the follow-up (Gil et al. 2013).

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Vaccine Development

One of the largest issues in modern public health is the prevention and treatment of bacterial infections. The worry that we will run out of effective therapies for infectious illnesses is only one aspect of the problem with AMR. AMR also jeopardizes various contemporary medical operations, such as surgery, organ transplants, and treatments for cancer, HIV, liver illness, renal disease, physical trauma, and liver and kidney disease. Several microbiological sources are used in the production of vaccines. These are living, attenuated microorganisms, such as the common cold virus, derived from dead, attenuated pathogens, or recombinant or neutralized genetic material, toxins, or surface proteins of the microbe. Some more recent vaccinations function by utilizing the antigens’ blueprints rather than the actual antigens (Table 9.3). For microbiology, vaccine manufacturing is important. The potential of developing novel vaccinations to stave against emerging illnesses has grown as a result of recent microbiological discoveries (Poolman 2020).

9.2.3.1 Tuberculosis Mycobacterium tuberculosis is an intracellular bacterium, and its lipoidal surface shape limits immune assault possibilities. It is spread by the inhalation of M. tuberculosis airborne droplets containing tuberculosis. In the majority of cases, M. tuberculosis causes latent infection, which seldom leads to active illness. Although tuberculosis can damage any organ, the most prevalent public health manifestation is an active lung infection with mycobacterial dissemination. Tuberculous meningitis or disseminated illness can develop in babies. Multidrug resistance of M. tuberculosis is a fast-growing issue (Poolman 2020). After infection, the formation of organized structures known as granulomas is supported by macrophage and monocyte aggregation, as well as accumulation of Table 9.3 Microbial strains producing some commercially available vaccines Disease Tuberculosis

Producing strains Mycobacterium tuberculosis

Vaccine Bacille Calmette-Guérin (BCG) vaccines

Diphtheria

Corynebacterium diphtheriae Clostridium tetani

Diphtheria-tetanus-pertussis (DTP) vaccine Diphtheria-tetanus-pertussis (DTP) vaccine Diphtheria-tetanus-pertussis (DTP) vaccine Hib vaccine

Tetanus Pertussis Pneumonia, meningitis Meningococcal meningitis

Bordetella pertussis H. influenzae type b (Hib) Neisseria meningitidis

Polysaccharide vaccines, conjugate meningococcal vaccines

Reference Poolman (2020), Miranda et al. (2012) Stratton et al. (2011) Przedpelski et al. (2020) Gupta et al. (1991) Mulholland et al. (1997) Christodoulides and Heckels (2017)

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chemokines that promote cell adhesion and recruitment of T cell. This typically results in a prolonged latent phase of the infection (Miranda et al. 2012). The live attenuated mycobacterial strains used in BCG immunizations are ancestors of attenuated BCG. In addition to its shown efficacy in treating babies with widespread sickness and tuberculous meningitis. BCG appears to enhance Mycobacterium tuberculosis transmission from alveolar macrophages to tissue- or lung-recruited macrophages and neutrophils by using antigen-specific CD4 T cells. It is anticipated that this transfer to phagocytes, as well as their activation and differentiation, occurs via yet-unidentified pathways that would boost the ability to limit bacterial reproduction. As a result of the trained innate immunity that BCG develops, innate effector cells are better able to respond to non-specific stimuli (Poolman 2020).

9.2.3.2 Diphtheria Vaccine The diphtheria vaccination is typically given to children in the majority of nations. It is brought on by toxic Corynebacterium diphtheriae and, sporadically, toxic C. ulcerans in tropical environments. C. ulcerans droplets and close physical contact are the main methods of transmission for the respiratory tract’s diphtheria; C. pseudotuberculosis, C. ulcerans, and zoonotic disease can potentially spread. Strong bacterial toxins occasionally induce upper respiratory tract obstructions or damage to the heart and other organs; however, clinical signs are often modest. The disease is very uncommon in nations with good vaccination rates against diphtheria. In populated areas with limited immunization programmes and lax sanitary standards, the incidence rises. Populations with poor rates of immunization against diphtheria, tetanus, and pertussis are more susceptible to exposure (DTP). National guidelines advise using booster tetanus and diphtheria vaccinations or suitably prepared combination DTP vaccines for main or booster immunization. Those who are younger than 7 years should take combinations with less diphtheria toxoid in them (Stratton et al. 2011). 9.2.3.3 Tetanus Tetanus vaccination is commonly offered to children in most countries. According to national standards, the vaccination should be administered to travellers who lack vaccinations. It is caused by the bacteria Clostridium tetani. Its spores can infect necrotic, anaerobic tissue and grow into toxin-producing, vegetative bacteria. Strong microbial neurotoxins generated by proliferative C. tetani may cause both localized and broad muscular convulsions and stiffness. Tetanus is often fatal if left untreated. The risk is associated with actions that increase the likelihood of unclean or contaminated injuries. This danger may not generally rise while travelling. Travellers should be inoculated against diphtheria-tetanus, or DTP, according to government guidelines. Tetanus-containing combinations with less diphtheria toxoid should be given to children under the age of 7 (Przedpelski et al. 2020).

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9.2.3.4 Pertussis In most countries, children are routinely vaccinated against pertussis. Unvaccinated travellers should be immunized in accordance with national norms. It is caused by the bacteria Bordetella pertussis. Bordetella bacteria exclusively colonize ciliated cells of the respiratory mucosa, causing an early respiratory infection with violent, spasmodic coughing fits. Pertussis in early childhood can be unusual and occasionally fatal. Disease signs become less striking as one gets older, especially in adults. Pertussis incidence is affected by DTP vaccine coverage; countries with poor DTP vaccine coverage tend to have higher rates of the illness, whereas those with high coverage tend to have lower rates. Newborns who are unvaccinated and travelling to countries with low DTP immunization rates are particularly at risk. For both initial immunization and booster shots, whole-cell pertussis vaccines should be given in fixed combination with diphtheria (D) and tetanus (T) vaccinations. The schedule ought to follow federal regulations. Diphtheria toxoid-free combinations should be administered to children under the age of 7 (Gupta et al. 1991). 9.2.3.5 Haemophilus influenzae Type b Many nations regularly immunize children against Haemophilus influenzae type b (Hib). Unvaccinated travellers under the age of 5 should be given a vaccine, according to official guidelines. The bacterium H. influenzae type b (Hib) causes it. Hib influenzae type b (Hib) is spread by respiratory droplets. Hib is a leading cause of meningitis, pneumonia, epiglottitis, septicaemia, infection, as well as other potentially deadly diseases in youngsters aged 3–5 years. It is frequent in countries with inadequate Hib immunization coverage. In a situation with poor Hib vaccine coverage, the risk is likely to increase. All licenced Hib vaccinations contain conjugated Hib vaccines. They differ in terms of the carrier protein, the chemical conjugation method, the size of the polysaccharide, and the adjuvant. The Hib vaccine is available in a number of various formulations, such as liquid Hib conjugate (monovalent), liquid Hib conjugate mixed with DTP and/or hepatitis B vaccination, meningococcal antigens coupled with Hib conjugate, Hib conjugate lyophilized with saline diluent (monovalent), Hib conjugate lyophilized for use with liquid DTP, or DTP combined with additional antigens, such as in baby dosage regimens that include three primary doses without a booster, two primary doses plus a booster, or three primary doses with a booster, with the first dose administered as soon as practically possible after 6 weeks of age. Over the age of 5, healthy children do not require the Hib immunization (Mulholland et al. 1997). 9.2.3.6 Meningococcal Disease Its development is caused by Neisseria meningitides bacteria, frequently serogroups A, B, C, W, X, and Y. The two primary mechanisms of transmission are direct individual contact and fluids of infected symptomatic or asymptomatic meningococcal carriers. With the greatest attack rates in newborns between the ages of 3 and 12 months, endemic illness mostly affects children and adolescents.

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Intense headache, nausea, fever, vomiting, stiff neck, and additional neurological abnormalities appear out of nowhere in meningococcal meningitis. There are frequent long-term neurological effects, and 5–10% of patients die from the illness. The symptoms of meningococcal septicaemia include haemorrhagic skin rash, hypovolaemic shock, and a high mortality rate. The vaccinations that are now available are as follows: 1. On the worldwide market, meningococcal polysaccharide vaccines are offered as bivalent (A and C), trivalent (A, C, and W), or tetravalent vaccinations (A, C, W, and Y). Following a single dose, these vaccines provide excellent serogroupspecific protection for 2–4 years in adults and children older than 2 years. Conjugate meningococcal vaccinations are increasingly often used in place of meningococcal polysaccharide vaccines. 2. Meningococcal conjugate vaccinations are accessible in monovalent form vaccines against serogroups A and C, two-valent vaccines against serogroups A and C or C and Y, and four-valent vaccines against serogroups A, C, W, and Y. Conjugate vaccines are extremely immunogenic (>90%) and serogroup specific. Additionally, vaccinations against Hib and Neisseria meningitides serogroup C (Hib MenC) as well as serogroup C and Y-tetanus toxoid conjugate (Hib MenCY) are sold (Christodoulides and Heckels 2017).

9.3

Microbial Diversity: Nutraceuticals

A great range of aquatic plants and critters can be found in the dynamic marine environment. Since there is so much biodiversity in marine water, it serves as a rich supply of distinctive bioactive chemicals in both quantity and variety. The majority of bioactive substances have a variety of biological functions that are discovered to function as nutraceuticals for living creatures. Nutraceuticals from the water are referred to as marine nutraceuticals. The benefits of marine nutraceuticals for human health have already been proven, and their usage in improving the health of animals has also been demonstrated to be effective. More than 20,000 marine bioactive compounds have been isolated to date, but their application in the management of aquatic animal health is still in its infancy. Advances in aquatic animal health research had paved the way for the search for new novel compounds, which also include marine nutraceuticals (Table 9.4) (Ande et al. 2017). Scientists found the marine environment as a treasure trove for discovering unusual chemical substances. However, there are a lot of limitations when it comes to natural products derived from aquatic sources, especially the availability of sufficient amounts of metabolites (Montaser and Luesch 2011). Marine microorganisms have drawn increasing attention as a substitute source of marine natural products since the identification of the constraints related to physiologically active metabolites obtained from marine animals and plants. Exceptionally varied marine microorganisms create substances with distinct structural characteristics, and pharmacological activities, viz. antibacterial, antiviral, antituberculosis, antimalarial,

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Table 9.4 Nutraceuticals derived from marine microorganisms and functional foods at present commercially available in the market (Dewapriya and Kim 2014) Product Astaxanthin

Microorganisms Spirulina platensis

Omega-3 oil

Schizochytrium sp.

Multivitamin

Spirulina platensis

Beta-carotene

Dunaliella salina

Assorted algae pastes (functional food for aquaculture) Phytoplankton powder

Tetraselmis, Nannochloropsis, Isochrysis Marine phytoplankton

Concentrated marine phytoplankton

A blend of marine phytoplankton

Brand name BioAstin Hawaiian Algae Omega Spirulina Pacifica® Nature’s Life Mixed Carotenoids Instant Algae

Sea Trinity UMAC CORE

Company Nutrex Hawaii Nordic Naturals, Inc. Nutrex Hawaii NutraMarks, Inc. Brine Shrimp Direct, Inc. Pure Healing Foods UMAClife

anthelmintic, antiprotozoal, antiplatelet, anti-diabetic, anti-inflammatory, and anticancer activities, are only a few of the pharmacological qualities these compounds have (Imhoff et al. 2011). Literature demonstrates the potential pharmaceutical applications of aquatic microbial metabolites. Numerous compounds now in marine drug development pipeline, according to Waters et al. (2010), are almost certainly made by microbes. Additionally, some marine microorganisms’ food-grade metabolites with potential medicinal capabilities have been discovered, and there is a chance to commercialize those active components as contemporary nutraceuticals and useful foods (Dewapriya and Kim 2014).

9.4

Therapeutic Applications of Nutraceuticals

9.4.1

Role of Nutraceuticals Against Myocarditis and Lung Diseases

Young adults are most commonly affected by myocarditis, an inflammatory condition of the myocardium. Viral infections, medications, autoimmune diseases, and inflammatory conditions are among the common causes of the disease. Nearly 50% of myocarditis cases are caused by a post-viral immune response in the presence of a known or unknown infection. Clinical manifestations can range from asymptomatic courses to baby and young patient’s sudden death. The phytochemicals act as strong therapeutic adjuncts in the treatment of myocarditis. The clinical and preclinical studies have demonstrated the protective effects of natural products in myocarditis treatment and prevention (Enayati et al. 2022).

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Lung disorders such as chronic obstructive pulmonary disease (COPD); infections such as acute respiratory distress syndrome (ARDS), influenza, asthma, and pneumonia; as well as lung cancer (LC) are common causes of death. In addition, probiotics, antiplatelet, antiviral, anti-inflammation, and antioxidants linked to lung disorders have proved biologically useful therapies against cancer (Rahman et al. 2022).

9.4.2

Benefits of Nutraceuticals for Health

Nutraceuticals have many therapeutic values as compared to any other agents. Consuming nutrients from plants, nuts, whole grains, cereals, and seafood is essential for maintaining good health and preventing disease (Rajasekaran et al. 2008; Khalaf et al. 2019). Lutein, folic acid, and cod liver oil capsules are popular and accepted nutraceuticals. The most widely consumed functional foods and drinks are omega-3-enriched poultry and dairy products, calcium-enriched juices, and non-alcoholic beverages. The majority of dietary supplements offer a range of therapeutic advantages. The following disorders are said to be physiologically benefited by or protected against by nutraceuticals: cardiovascular disease (CVD), obesity, Parkinson’s disease, cancer, diabetes, chronic inflammatory disorders, and Alzheimer’s disease (Fig. 9.1) (Khalaf et al. 2021).

9.4.3

Algal Polysaccharides in Nutraceutical Applications

Depending on their polysaccharide and pigment composition, macroalgae are differentiated into three groups: brown algae (Phaeophyta), red algae (Rhodophyta), and green algae (Chlorophyta) (Keith et al. 2014). When it comes to their bioactive potential and uses in food, green algae have received the least attention (Surget et al. 2017). Ulva and Enteromorpha genera green algae are made of the ulvan sulphated polysaccharide. Iduronic, glucuronic, and sulphated rhamnose are only a few of the uncommon sugars found in the polydisperse hetero-polysaccharide known as ulvan. Ulvan uses are constrained by their high molecular weight, which ranges from 189 to 8200 kDa, although it is said to have anti-influenza, anti-proliferative, hepatotoxic, and antioxidant properties (Kidgell et al. 2019; Jagtap and Manohar 2021). The possible uses for algal polysaccharides from different macroalgae include dietary fibres, hydrocolloids, anticancer and anticoagulant compounds, drug delivery, and tissue engineering (Tanna and Mishra 2019). The production of functional foods and beverages is constrained due to their high molecular weight, poor biocompatibility, and water solubility qualities. These polysaccharides are hydrolysed to produce low-molecular-weight, biocompatible, and soluble oligosaccharides, which have more extensive uses in the medical field than their parent polysaccharide (Cheong et al. 2018). It is possible to hydrolyse macroalgal complex polysaccharides either through chemical conversion into algal oligosaccharides (AOS) or by enzymatic activities.

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Lung disease Alzheimer’s disease

Cardiovascular disease

Cancer

Prevention from different diseases using nutraceuticals

Inflammatory disorders

Parkinson’s disease

Obesity

Fig. 9.1 Prevention from different diseases using nutraceuticals

Chemical hydrolysis involves concentrated acid used to cure polysaccharides and heated for several hours at a high temperature to obtain the ultimate product (Lee and Lee 2016). This results in the formation of undesired harmful by-products like furfural and monomers. Elevated concentrations of reducing sugars impair the purity of oligosaccharides. The use of AOS in functional food and nutraceutical applications is constrained by these unwanted by-products (Wang et al. 2017).

9.4.4

Use of Nutraceuticals in Dairy Products

Lactic acid bacteria (LAB) have become more popular as probiotics in recent years. The primary characteristic of these bacteria in addition to acid and bile resistance is the ability to produce antibacterial substances to bind to and colonize human intestinal mucosa while fighting off pathogenic and cariogenic bacteria. Additionally, probiotics help to colonize the intestine mucosa by the formation of antimicrobial compounds because these compounds increase their competitive advantage over the natural gastrointestinal microbiota. Capsular polysaccharides have been shown to promote bacterial growth through attachment to membrane surface (Khalaf et al. 2021).

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Probiotics have several advantages, including boosting immunity, acting as a supplementary vaccination, attaching to human gut cells, enhancing vitamin K and B production, fortifying the digestive system’s barrier against damage from radiation therapy, and avoiding diarrhoea brought on by rotavirus. Additionally, it works well to cure constipation, reduces blood pressure and cholesterol levels, prevents inflammatory bowel problems, and has anticancer effects (Andersson et al. 2001). Certain lactobacilli strains exhibit antioxidant activity, which may reduce the danger of free radical formation, according to research (Wang et al. 2017). Probiotics also control inflammation and hypersensitivity responses. The control of cytokine function may be the reason for this. They lessen the likelihood of childhood eczema and prevent the recurrence of inflammatory bowel disease in adulthood. They also alleviate milk allergies. By boosting phagocytosis, increasing the proportion of natural killer cells to T lymphocytes, and increasing the number of plasma cells that generate IgA, probiotics enhance immune function (Bodera and Chcialowski 2009). The consumption of fermented milk made with various LAB strains can result in temperate blood pressure because fermentation produces the ACE inhibitors, such as peptides, which block the formation of angiotensin during the process (Rai et al. 2017).

9.5

Biomedicine: Approaches

An interdisciplinary topic that has just emerged and has a significant impact on pharmacology, agriculture, industry, and the environment combining nanotechnology with nanomedicine was made possible by biotechnology. It has produced innovative advancements in the treatment of numerous illnesses and disorders, medication delivery techniques, and detection and diagnosis of various diseases. However, there are yet few applications for microbially produced nanoparticles in health and medicine. Nanomaterials have been thoroughly investigated for industrial uses (Fariq et al. 2017). The field of nanomedicine is currently developing in the age of sophisticated nanotechnology and nanorobotics. By using nanoscale structures for illness diagnosis, treatment, and prevention, nanomedicine helps to improve human health (Patil et al. 2008). The characteristics of nanoparticles are very different from those of macro- and micro-size materials that are useful in medicine and human health. The formation of nanoparticles through biological processes is relatively safe, economical, and sustainable (Ahmed and Ikram 2016). To create novel therapeutics for the treatment of disease and the repair of tissue, novel drug delivery systems and quick and sensitive diagnostic tools are essential (Karunaratne 2007). Microbes have been discovered to be tiny nano-factories, and the microbial production of nanoparticles (Table 9.5) has combined nanotechnology, microbiology, and biotechnology to create a new discipline called nano-biotechnology. For bioleaching and biomineralization, metal-microbe interactions have been extensively employed, but nano-biotechnology is still in its infancy. Due to its powerful advantages, it might have intriguing uses in nanomedicine. Plants and bacteria have been used all across the world to synthesize nanoparticles biologically (Rai and Duran 2011). Microbes are typically chosen for the synthesis of nanoparticles (NPs) due to their

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Table 9.5 Different nanoparticles biosynthesized from microbial sources Microorganism Streptomyces sp. Geobacillus sp. Stereum hirsutum Salmonella typhimurium Cladosporium cladosporioides Lactobacillus crispatus

Type of nanoparticle Silver Gold Copper/copper oxide Copper Silver Titanium dioxide

References Zonooz and Salouti (2011) Correa-Llantén et al. (2013) Cuevas et al. (2015) Ghorbani et al. (2015) Balaji et al. (2009) Abdulsattar (2014)

high metabolic and multiplication rate and tolerance to a wide range of environmental conditions. Through their enzymatic processes, microbes react with metal ions to convert them into their elemental form (Li et al. 2011).

9.5.1

Applications of Biomedicine

There are some intriguing potential uses for magnetic nanoparticles in biomedicine. Nanoparticles have dimension-matching small cells and/or biomolecules. It improves the ability of nanoparticles to interact with or tag these macromolecules. The unique physical characteristics of nanoparticles with magnetic properties open up a wide range of additional potential uses of nanoparticles in biomedicine (Pankhurst et al. 2003). Numerous areas of medicinal and biological study have effectively used magnetic separation. It has been found to be a very responsive method for extracting rare tumour cells from blood, and it works particularly well for separating sparse populations of target cells. For instance, this has improved the ability to detect malarial parasites in blood samples by using the parasite’s magnetic characteristics or by labelling the red blood cells with an immunospecific magnetic fluid (Seesod et al. 1997). Bacterial polymers have drawn a lot of interest throughout the years since they are so widely used in biological applications. Through applying metabolic engineering approaches, it is likely to better understand metabolic processes and design better production strategies while synthesizing custom polymer materials for use in biomedicine. This focuses on a variety of biocompatible polymeric materials with wound healing, antioxidant, anticancer, and antibacterial properties, including polysaccharides, polyesters, polyamides, and polyphosphates. Focus is also placed on the benefits of using different biomaterials to achieve controlled or prolonged drug release, tissue engineering applications in the biomedical field, and utilization of microbial polysaccharides as pharmaceuticals in the pharmaceutical industry. The most common biomedical uses of bacterial biopolymer materials including bandages for wound healing, drug delivery, tissue engineering, ortho-dental uses, and hydrogels are also studied (Jose et al. 2022).

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Environmental Medicine on a Cosmic Scale in Space Biomedicine

Space biomedicine is one of several modern biomedical improvement approaches that promote technology treatments to make people more “flexible” by going “beyond what is ‘normal’ or essential for life and well-being” to support human life outside its natural habitat. The thing that goes “beyond” space and time to improve human life, according to space biomedical practitioners, is not an improved biological being. Instead, it is a living milieu of interacting humans, machines, and natural phenomena (Olson 2010).

9.6

Vaccine Development: Approaches and Applications

The utmost effective strategies for preventing contagious infections during the past century have been vaccinations. In many parts of the world, measles and poliomyelitis have declined to very low levels, while smallpox has been completely eradicated. However, infectious diseases continue to be the predominant reason for death globally. In order to prevent diseases like AIDS or malaria for which there is presently no vaccine, as well as to increase the efficiency and safety of current vaccinations, it is still of utmost importance to create new vaccines. The approaches that have been employed thus far to discover potential vaccine candidates will undoubtedly need to be significantly changed in order to achieve such lofty targets in the near future. The most recent advances in molecular biology and immunology could greatly boost contemporary vaccination (Leclerc 2003). The technology used to build up vaccines has continued to be untouched for several years, despite the fact that immunization has significantly decreased the mortality and morbidity caused by infectious illnesses. In fact, it follows an experimental investigation approach and produces effective vaccines up to this point with little knowledge of the immunological processes behind the protection they provided. Recently, the three major types of vaccine production technologies were entire dead microorganisms, live attenuated microbes, and molecule or macromolecule antigen-based vaccines. However, aforementioned methods showed lacunae against dynamic pathogens like Plasmodium falciparum or human immunodeficiency virus (HIV). Vaccination had been used as a preventive measure. There has not been much work put into creating therapeutic vaccinations that can treat existing infectious diseases or non-infectious disorders. The situation is changing quickly, and several efforts are being made to create vaccinations against long-term illnesses like AIDS or cancer, both for primary prevention (such as cancers caused by viruses like the papillomavirus) and for the treatment of existing tumours. And last, autoimmune and immunological conditions pose yet another fresh problem for vaccines. To activate specific memory or effector immune responses against the target antigens, such vaccines will need to be developed using novel, out-of-the-box approaches based on synthetic or engineered antigens, RNA, DNA, etc. It is now promising to develop vaccinations on the basis of coherent advancement, increasing efficacy and

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safety for the first time in the history of vaccination, and expanding of different approaches accessible for creating vaccines. In this chapter, we examined a few strategies that have the potential to significantly boost the effectiveness of vaccine discovery in the coming years (Leclerc 2003). The use of vaccines has significantly improved the management of infectious diseases. In addition to being potentially the least expensive method of preventing infectious diseases, widespread vaccination can eradicate diseases, as was the case with smallpox, under the right circumstances. Vaccines have not had much of an impact on human parasitic illnesses, despite their enormous potential. Furthermore, we still have a limited understanding of the immune regulatory mechanisms of loads of parasite contagions, which defines an efficient protective response, as well as to create more worthy immunological reminiscence (Tarleton 2005). Vaccines aim to overcome deficiencies in immunity to infection. The aim of vaccinations, whether prophylactic or therapeutic, can also be illness prevention rather than sterile immunity. The ability to respond more quickly and efficiently to infection after vaccination is established by the enhancement of immunological memory, which is essential for prophylactic vaccinations to be successful. In both situations, it is critical to remember that the majority of parasitic diseases require the development of vaccines and vaccination procedures that are more successful at eliciting the necessary immune responses than the current infection (Tarleton 2005).

9.7

Conclusion and Future Prospects

The market is currently looking for substitute items that are natural (organic) and offer dietary and health advantages. Seaweeds, marine microalgae, marine lipids, and other naturally occurring organic compounds with increased health-promoting properties are known as marine nutraceuticals. To satisfy everyone’s (fish farmers and consumers) need for the aquaculture industry and generally increase aquaculture production, marine nutraceuticals will likely play some role in the near future. Thus, future opportunities for marine nutraceuticals in the treatment of aquatic animal health will be enormous. Advancements in basic science, artificial natural science, and hereditary engineering are expanding the variety and quantity of accessible host microorganisms to satisfy the needs for the present and future nutraceutical supplements. To better comprehend the whole range of physical processes and consequences that collectively determine whether a given application will ultimately be successful, there is an extent for major involvements through the arithmetical modelling of multifaceted systems. The development of a successful vaccination for any parasite illness is still expected to take several years in the future, even though the tools available for discovering vaccines and our knowledge to trigger protective immune responses have both grown significantly in recent years. The possibilities for antiparasite vaccinations are nevertheless quite promising for a variety of reasons. The knowledge from genome sequencing programmes has served as the basis for the development of vaccines, and the necessary instruments already exist or are on the horizon.

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Clearly, this finding has set the stage for quick advancement in the creation of vaccinations to fend off communicable diseases. However, we are moving in a new era of vaccination, one in which our ability to trigger and control protective responses will be used in a variety of medical specialties. It is still hard to say how long it will take for such novel approaches to develop, but it is understandable that new vaccines will appear in the coming years to treat conditions like cancer, allergies, asthma, and autoimmune illnesses. Acknowledgements The authors acknowledge the Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G.), for this work.

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Microbial Technology for Neurological Disorders

10

Asmita Dasgupta

Abstract

The emerging understanding of the interaction of the gut microbiome with the central nervous system points towards the ability of the microbiota to modulate the brain neurochemistry, and these interactions can influence the behaviour towards health or disease. These concepts indicate that the gut microbiota first influences the enteric nervous system, which in turn can impact the neuroendocrine and the immune system to the extent of modulating fluid intelligence and cognition. The challenge is to identify the microbial flora and specific molecules that can augment cognitive performance, prevent autism spectrum of disorders, lower stress, prevent neuropsychiatric disorders, and stall cognitive decline and neurodegeneration. This chapter aims to integrate the available knowledge in this domain and identify the areas that will require the use of microbial technology in delivering these evolving solutions. Keywords

Gut-brain axis · Gut microbiome · Metabolite · Inflammation · Neurodevelopmental disorders · Neurodegenerative disorders · Neurovascular disorders · Probiotics · Prebiotics · Synbiotics · Postbiotics

Abbreviations 4-EPS AD

4-Ethyl phenyl sulphate Alzheimer’s disease

A. Dasgupta (✉) Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_10

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ADHD AMP ASD BBB BMEC BMEC Cas CRISPR DCs EAE ENS ET FFAR FMT FOS GABA GI GI GLP-1 GOS GPR HDAC IBD IBS IPA LCA LCFAs L-dopa LPS LTP MCTs MDD MGEs MIA MOS MS mUPR NASH PAMPs PD PYY RCT RGNs RR-MS SCFA

A. Dasgupta

Attention deficit hyperactivity disorder Antimicrobial peptide Autism spectrum disorders Blood-brain barrier Brain microendothelial cells Brain microvascular endothelial cell CRISPR-associated proteins Clustered regularly interspaced short palindromic repeats Dendritic cells Experimental autoimmune encephalomyelitis Enteric nervous system Enterotypes Free fatty acid receptor Faecal microbial transplantation Fructooligosaccharides γ-Amino butyric acid Gastrointestinal Gastrointestinal Peptide-1 Galactooligosaccharides G protein-coupled receptor Histone deacetylase Inflammatory bowel disorder Irritable bowel syndrome Indole 3-propionic acid Lithocholic acid Long-chain fatty acids Levodopa Lipopolysaccharide Long-term potentiation Monocarboxylate transporters Major depressive disorder Mobile genetic elements Maternal immune activation Mannan oligosaccharide Multiple sclerosis Mitochondrial unfolded protein response Non-alcoholic steatohepatitis Pathogen-associated molecular patterns Parkinson’s disease Peptide YY Randomized controlled trial RNA-guided nucleases Relapsing remitting-MS Short-chain fatty acids

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Th17 TLR2 TMA TMAO Tregs XOS

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T helper 17 Toll-like receptor 2 Trimethylamine Trimethylamine-N-oxide Regulatory T cells Xylooligosaccharide

Introduction

The brain is now well understood to be under the influence of molecules derived from the gut. Many of these molecules are metabolites released actively or passively by the microbes that reside in the gut and travel through circulation to reach the brain to influence behaviour. Reports as early as 1969 confirm neurological diseases arising after partial gastrectomy (Williams et al. 1969). In recent times, the field of gut brain axis had some seminal discoveries that delineate the relationship between gut microbiota and behaviour (Dinan and Cryan 2017a). The interaction of the gut microbiota and its metabolites with the brain of the host organism is two way and modulates both immunity and behaviour, making the impact both intricate and pervasive. The gut is populated by trillions of microorganisms that live in symbiotic association within the host and interact with each other and the host to form the microbiota. The microbiota includes bacteria, archaebacteria, fungi, and others that interact with each other and their host, and their cumulative genomes form the gut microbiome. The net mass of bacteria in the gut can be as much as one kilogram in an adult human, and these microbes produce a multitude of chemicals that are not limited to, but include, small peptides, vitamins, short-chain fatty acids (SCFAs), as well as most of the relevant neurotransmitters. The proportion of the cells of the host and that of the microbiota is almost 1:1 in case of humans, making the contribution of their cumulative genomes highly significant for the normal life of the human host (Sender et al. 2016). Alteration in this microbiota is now accepted to be a key player in the initiation, establishment, as well as maintenance of various pathological states of the brain. This becomes possible because of the involvement of metabolic, neural, endocrine, and immune pathways (Willyard 2021; Nunez et al. 2018). Studies have unearthed equal reasons of thinking of gut microbiota as an agent that could stimulate both disease and well-being. Healthy human microbiome must be an ecologically stable population that has a resilient community structure and performs a set of desirable functions supplementing the metabolic and trophic requirements contributing to human health and longevity (Bäckhed et al. 2012). This definition of healthy microbiome while being generous enough to allow for host variations depending on diet, stage of life, and lifestyle profiles requires that the microbiomes function to fulfil the dietarily unmet metabolic and trophic factors for the host contributing to its health and longevity. While this structure could somewhat change

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under the conditions of stress, it is inherently expected to be able to return to its normal on its own. Driven by this rationale, the Human Microbiome Project, funded by the National Institute of Health in 2008, used high-throughput multi-omics analyses to identify and study by sequencing >1300 reference strains from the healthy human body including the gut (Human Microbiome Project Consortium 2012a, b). Understanding this healthy microbiome is integral to the designing of age-wise dietary interventions and/or microbial modification through prebiotic or probiotic supplementation towards sustained healthy, extended, and productive lifespans. Moreover, the same can also be used to design dietary intervention and prebiotic and probiotic supplementation for stalling the progress or recovering from age-associated degenerative diseases.

10.2

The Healthy Human Gut Microbiome

The initial establishment of microbiota in the gut is by those that are acquired from the mother since both the amniotic fluid and the placenta have been found to be colonized by bacteria (Jiménez et al. 2008; Wassenaar and Panigrahi 2014). Thereafter, the infant’s gut gets colonized by microbes based on the mode of delivery, surrounding environment, diet, maternal microbiota, and antibiotic usage and develops uniquely for every infant. Subsequently, this matures to a more common adult microbiota in due course. The colonization of human gut in these first 3 years of life is predominantly by Enterobacteriaceae, Bifidobacteriaceae, Lachnospiraceae, Bacteroidaceae, Clostridiceae, Bacteroidaceae, and Ruminococcaceae. These species participate in the enteric metabolism in the first 6 months of life after birth that involves simple carbohydrate degradation, amino acid transport, and vitamin synthesis and thereafter for the next two and half years contribute to complex carbohydrate degradation to give SCFAs, methane production, amino acid, and vitamin biosynthesis (Dinan and Cryan 2017b; Derrien et al. 2019). The profile gets modified with age as the pattern of diet changes (David et al. 2014). In older children of about 7–12 years, Bifidobacterium and Faecalibacterium spp. are predominant, whereas in adults, Bacteroides spp., particularly Bacteroides vulgatus and Bacteroides xylanisolvens, dominate the diversity (Hollister et al. 2015). Bifidobacterium levels may progressively decline with approach to adulthood though certain species continue to persist (Derrien et al. 2019). For an adult human, 90% of the gut microbiota are Bacteroidetes and Firmicutes (Qin et al. 2010; Kim and Jazwinski 2018). An overview of age-wise changing of typical human gut microbiota is given in Table 10.1. The adult microbiota profile however changes with progression of age, and the diversity that is gained through the adult life is lost with the change in diet associated to age-related loss of teeth, salivary function, digestion, and intestinal transit time. This loss in microbiota diversity can be correlated to altered interaction between the microbiota and the host, leading to age-related dysbiosis and frailty. In particular, the rise in Bacteroidetes and Clostridiales subpopulation and a loss of butyrate-making

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Table 10.1 Overview of the age-wise changing of typical healthy human gut microbiome Age group 0–3 years

7–12 years

Adults, 19 + years

Elderly, 65+ years

Predominant genus Enterobacteriaceae, Bifidobacteriaceae, Lachnospiraceae, Bacteroidaceae, Clostridiceae, Bacteroidaceae, Ruminococcaceae Bifidobacterium, Faecalibacterium spp., Bacteroides spp.

Bacteroides, Bifidobacterium, Bacteroidetes, Eubacteria, Fusobacteria, Streptococci, Firmicutes spp., Clostridia, Veillonella, Coliform bacteria, Lactobacilli, Proteus, Staphylococcus, Pseudomonas, Yeasts, Protozoa Bacteroidetes spp., Clostridiales spp.

Predominant species

Reference Derrien et al. (2019)

Prevotella copri, Bifidobacterium pseudocatenulatum complex, Bacteroides fragilis, Bacteroides ovatus, Bacteroides clarus, Bacteroides caccae, Bacteroides xylanisolvens, Bacteroides stercoris, Bacteroides dorei/ vulgatus, Prevotella disiens, Bacteroides finegoldii, Bacteroides coprophilus, Bacteroides thetaiotaomicron, Prevotella timonensis, Bacteroides uniformis, Bacteroides coprocola Bacteroides vulgatus, Bacteroides xylanisolvens, Ruminococcus albus, Blautia hydrogenotrophica, Eubacterium hallii, Bifidobacterium animalis, Methanobrevibacter smithii, Clostridium hylemonae, Clostridium butyricum

Hollister et al. (2015), Zhong et al. (2019)

Qin et al. (2010), Hollister et al. (2015), Cresci and Bawden (2015), Zhong et al. (2019), Kim and Jazwinski (2018)

O'Toole and Jeffery (2015), Cresci and Bawden (2015)

populations as Bifidobacteria, Bacteroides, and Clostridium cluster IV are linked to age-associated frailty (O'Toole and Jeffery 2015; Cresci and Bawden 2015). Phylogenetic study for core microbiota across all age groups from young (22–48) to semi-supercentenarian (105–109) however found Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae families to be common in all ages, the

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last two of which belonging to Firmicutes. Thus, the common core function in microbiomes across all ages belongs to the members of abundant taxa. Healthy ageing may involve modulation of composition within the core microbiome function to tailor these functions to increasing age. Hence, the three families reduce in abundance in the centenarians and semi-supercentenarians, and in parallel healthassociated species become more abundant (Biagi et al. 2016; Kim and Jazwinski 2018). Since gut microbiota population is connected to its function, the loss of microbiota or a change in its profile leads to complete loss of or decrease in function resulting in reduction of cardiometabolic rate and increase in inflammatory processes leading to age-accelerated loss of health. Thus, a minimum diversity in gut microbiome could serve as an indicator of healthy vs. unhealthy ageing.

10.2.1 Enterotypes of Gut Microbial Community The gut microbiome diversity shows significant variations and composition patterns among populations. To understand the patterns within the diversity and use this for therapeutic purpose, it is essential to have a classification of enterotypes (ETs) among human populations. Hence, gut microbiomes have been classified to enterotypes that are described as “densely populated areas in a multidimensional space of community composition”. These classifications are independent of geography, cultural background, age, and gender. The enterotypes are a co-occurring network of microorganisms converging towards one driver taxon, which serves as an indicator that correlates best to that enterotype. Thus, the best indicator for enterotype 1 (ET I/ET-B) is Bacteroides; enterotype 2 (ET II/E-P) is determined by Prevotella, a genus whose abundance reciprocally correlates with Bacteroides; while enterotype 3 (ET-III/ET-F) is best represented by Firmicutes most commonly by Ruminococcus (Arumugam et al. 2011; Costea et al. 2018). The next-level associations can be either the strong positive or the strong negative correlations. These are again unique for each enterotype as summarized in Table 10.2. Classifying microbiota to enterotypes recognizes the taxonomic diversity within the sample and across the samples. The taxonomic diversity within the samples is called alpha diversity, while the taxonomic similarity between the samples is called beta diversity. Recognizing these two different diversities is critical because the microbiota though has a defined community structure has both flexibility and resilience to slightly alter depending on the changed conditions within the host. Antibiotic treatment in particular adversely affects the gut microflora, and a deeper look into the interdependence among the various species of the microbiota led to the concept that there are some species that are foundational to the rebuilding of the complete microflora diversity to its previous diversity status prior to the antibiotic treatment. Such foundational species whose members have a large impact on the community stability and its recovery after perturbation of the whole microbiota are called keystone species (Martino et al. 2022; Chng et al. 2020; Gibbons 2020). Among the three enterotypes, the ET-F has maximum diversity while the ET-B has the least diversity (Arumugam et al. 2011; Costea et al. 2018).

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Table 10.2 Summary of co-occurring taxa for the three enterotypesa Enterotype (ET) Predominant genus Positive correlates

Diet association Disease susceptibility

a

1/I/ET-B Bacteroides

2/II/ET-P Prevotella

Parabacteroides, Lactobacillus, Clostridiales, Alkaliphilus, Slackia

Akkermansia, Ruminococcaceae, Rhodospirillum, Desulfovibrio, Rhodospirillum, Escherichia/Shigella, Holdemania, Staphylococcus, Peptostreptococcaceae, Leuconostoc, Helicobacter, Veillonella, Eggerthella Fibre-rich diet

Fibres and fructans, proteins, and animal fats C-reactive protein, insulin resistance, low-grade inflammation

Crohn’s disease risk allele, colitis

3/III/ET-F Firmicutes (Ruminococcus) Akkermansia, Gordonibacter, Ruminococcaceae, Marvinbryantia, Symbiobacterium, Dialister, Staphylococcus

Non-alcoholic steatohepatitis, ROS

Based on the three enterotypes described by Arumugam et al. (2011) and Costea et al. (2018)

Investigations on the functional role of each enterotype in their host systems have been performed using the gene ontology and functional classification of the microbiome based on their differential taxonomic composition. Such analysis has shown that individuals with fibre-rich or non-Western diet have ET-P predominant with Prevotella. This also correlated well to the hydrolases present in these organisms, which are more specialized in plant fibre degradation and have lower capacities of lipolysis or proteolysis. On the contrary, ET-B or Bacteroides with carbohydrate-active and proteolytic enzymes are enriched in individuals whose diets are enriched in animal proteins and saturated fats (Costea et al. 2018). There have been some significant associations of enterotypes with susceptibility to certain disease phenotypes. ET-B has been significantly associated to non-alcoholic steatohepatitis (NASH), coeliac disease, colorectal cancer, and immune senescence. ET-B individuals have constant low-grade inflammation accompanied with increased lymphocyte counts and C-reactive protein (Zhu et al. 2013; Costea et al. 2018). ET-P on the other hand has been linked to rheumatoid arthritis, long-term antibiotic usage, and type 2 diabetes (Scher et al. 2013; Larsen et al. 2010; Costea et al. 2018). Lastly, ET-F has been linked only with an increased risk for atherosclerosis. ET-F individuals have high microbiota diversity, the highest of all three enterotypes. They also have decreased host inflammatory status (Karlsson et al. 2012). Thus, enterotype can be a susceptibility factor for certain disease conditions. It could also serve as a biomarker for a certain stage of such disease conditions.

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Other than this, the microbiota participates in xenobiotic metabolism. The pharmacokinetics and pharmacodynamics of drug metabolism can be influenced by the enterotype. Thus, the enterotype may influence the treatment response. A prior knowledge of the same can help in making enterotype-driven correct choice among the treatment options. Additionally, many diseases will have different attributes based on the enterotype, and a better understanding of the same will enable treating the patients with the same disease according to their differential enterotypes. The gut microbiota can modify therapeutic drugs by binding to them and reducing their bioavailability as in case of Helicobacter pylori reducing the bioavailability of levodopa by direct binding. Microbiota-derived metabolites and xenobiotics also compete for the active site of the host enzymes. One example is tyrosine being metabolized to p-cresol by Clostridium difficile, which in turn interferes with acetaminophen O-sulphonation by the human liver enzyme SULT1A1, a hepatic cytosolic sulfotransferase that enables excretion of this paracetamol (Costea et al. 2018; Carmody and Turnbaugh 2014). Hence, prior knowledge of the microbiotadrug interaction and gut microbiota profile of the patient enables informed drug prescription and ensures greater success in disease therapy.

10.2.2 Gut Microbiota-Host Interaction The gut microbiota can be influenced by age, diet, hormones, treatment with antimicrobial agents, pharmaceuticals as proton pumps, xenobiotics, status of health or disease, and medications (Franzosa et al. 2015; Durack and Lynch 2019). The gut environment determines compositional and functional effectiveness of the microbiota, which interacts with the host through pathogen-associated molecular patterns (PAMPs) and metabolites to modulate the host immune homeostasis (Durack and Lynch 2019). Throughout the lifetime, the microbiota and the host communicate and modify each other. Such communication chiefly happens in four distinct ways (Fig. 10.1). The gut epithelia of the host could release cationic antimicrobial peptides (AMPs), which are lethal for the unwanted microbes, while the symbiotic microbiota is resistant to these peptides (Fig. 10.1a). The source of this resistance of symbiotic microbiota could be an endogenous phosphatase as identified in the gut Bacteroides. This enzyme dephosphorylates the lipopolysaccharide core lipid A that is di-phosphorylated in gram-negative species to reduce its negative charge. The phosphatase mutants are not able to steadily colonize the murine gut since they remain negatively charged and get eliminated through the cationic AMPs (Raetz and Whitfield 2002; Cullen et al. 2015). In another mode of communication, the microbial communities can release antibiotics which kill certain other microbes that are sensitive to them (Fig. 10.1b). These sensitive microbes could be communities that are pathogenic to the host since the host has co-evolved with its own commensal gut microbiota, which in turn has been competing for its ecological space within the host by competing out these other pathogenic microbial communities. In cases where the host microbiota weakens due to the use of external antibiotics by the host, the opportunistic pathogenic bacteria could gain an advantage

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Fig. 10.1 Gut microbiota communication with the host leading to context-dependent beneficial as well as pathological responses. (a) The gut epithelia of the host could release cationic antimicrobial peptides, which are lethal for the unwanted microbes, while the symbiotic microbiota is resistant to these peptides. (b) The microbial communities can release antibiotics which kill certain other microbes that are sensitive to them and are pathogenic to the host since the host has co-evolved with its own commensal gut microbiota, which in turn has been competing for its ecological space within the host by competing out these other pathogenic microbial communities. (c) The gut microbes release certain small molecules as short-chain fatty acids (SCFAs) that pass the gut epithelium to enter host circulation and modify the host biology. (d) The host molecules get modified by the resident microbiota to make antibiotics that are lethal to certain other microbes. Hepatic (e) oxidation and (f) sulphonation processes convert the microbiota-generated metabolites as trimethylamine (TMA) and 4-ethyl phenol (4-EP) to neurotoxic molecules trimethylamine oxide (TMAO) and 4-ethyl phenyl sulphate (4-EPS), respectively, that travel through systemic circulation to the brain where they cause Alzheimer’s disease (AD) and autism spectrum disorder (ASD) phenotypes, respectively

causing further manifestation of pathogenicity. For example, increase in Enterobacteriaceae species has a strong association with inflammation related to Crohn’s disease (Sokol et al. 2008; Mar et al. 2016). In yet another mode of crosstalk, the gut microbes release certain small molecules as SCFA that pass the gut mucosal epithelium and enter the circulation to modify the host biology, by

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binding to G protein-coupled receptor (GPR) 41/free fatty acid receptor (FFAR)3 and GPR 43/FFAR2 to downregulate inflammatory response through regulatory T cells (Tregs) (Fig. 10.1c) (Dalile et al. 2019; Maslowski et al. 2009; Smith et al. 2013; Furusawa et al. 2013). Finally, the host molecules get modified by the resident microbiota to make antibiotics that are lethal to certain other microbes. One good example of this is the secondary bile acid, lithocholic acid (LCA) that binds to the vitamin D receptor (Fig. 10.1d). This interaction is stronger than the binding of vitamin D to the vitamin D receptor and suggests a role of this receptor in detecting bacterial metabolism of primary bile acids (Makishima et al. 2002). It is also likely that other orphan receptors assigned with non-physiological ligands essentially respond to metabolites of microbiota origin.

10.2.3 Gut Microbiota Interactions with Central Nervous System: Role in Cognition The structure of the gut microbiome is associated to fluid intellect in healthy young adults. The “Ruminococcaceae and Coriobacteriaceae dominant community”, that consists of 14 genera, are positively correlated to cognitive performance with higher fluid intelligence scores, suggesting that gut microbiota interventions may improve cognition (Oluwagbemigun et al. 2022). Earlier, positive correlations have been drawn between Bacteroidetes in the gut during late infancy, with subsequent neurodevelopment in human males but not females (Tamana et al. 2021). The foundation of this connection between microbiota and cognitive development lies in the fact that most of the neurotransmitters and their precursors can be synthesized by diverse gut bacteria. Table 10.3 gives a summary of the range of neurotransmitters, or their precursors released by certain genus of gut microbiota. This can be connected to the shared evolutionary history of the microbiome with host where microbiota and host cells communicate with each other with these neurotransmitters they share, and with this they also gain the ability to mutually monitor each other. The hypothesis is that the neurochemical communications have evolved because of horizontal gene transfer (Iyer et al. 2004). Monoamines released by the gut microflora influence the behaviour of germ-free mice (Nishino et al. 2013). Subsequently, other neurotransmitters, neuroactive growth factors, and neuroprotective metabolites have also been found to be released by the gut microbiota. The neurotransmitters synthesized by the microbiota get released either actively or passively after the breakdown of the microbiota. These microbiotaderived neurotransmitters then cross the intestinal mucosa and reach the enteric nervous system (ENS) and the brain through the bloodstream to modulate physiology, mood, and behaviour (Fernandez-Real et al. 2015). Human gut microbiota species of Lactobacillus and Bifidobacterium may produce over 20 mg/ml of γ-amino butyric acid (GABA) in vitro with the availability of appropriate substrate. This can be used at a therapeutic level by altering the gut microbiome through dietary synbiotics, which comes under the prospect of microbial technology.

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Table 10.3 Neuroactives released by predominant genus of gut microbiota Microbiota genus Bifidobacterium infantis, Bifidobacterium bifidum; Bifidobacterium spp.

Neuroactives Tryptophan, acetylcholine, GABA; conjugated linoleic acid

Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus spp.

GABA, serotonin, dopamine, acetylcholine, histamine, brain-derived neurotrophic factor, indole, indole-3acetate; conjugated linoleic acid, 10-hydroxy-cis-12octadecenoic acid Serotonin, dopamine, histamine

Streptococcus

Enterococcus Escherichia coli K-12

Serotonin, histamine Serotonin, norepinephrine, dopamine, indole

Bacillus longum, Bacillus fragilis Campylobacter jejuni

Norepinephrine, dopamine, acetylcholine, indole Glutamate

Saccharomyces spp.

Norepinephrine

Candida spp.

Serotonin

Clostridium sporogenes, Clostridium bartlettii

Indole 3-propionic acid, indole

References Desbonnet et al. (2008), Barrett et al. (2012), Kawashima et al. (2007), Shamsipour et al. (2021) Komatsuzaki et al. (2008), Thomas et al. (2012), Landete et al. (2008), Kawashima et al. (2007), Ranuh et al. (2019), Shamsipour et al. (2021)

Landete et al. (2008), Yano et al. (2015), Legan et al. (2022) Landete et al. (2008) Shishov et al. (2009), Yano et al. (2015), Legan et al. (2022) Kawashima et al. (2007) Van der Stel et al. (2015), Queiroz et al. (2022), Baj et al. (2019) Lyte (2013 2014), Wall et al. (2014) Lyte (2013), Lyte (2014), Wall et al. (2014) Jellet et al. (1980)

10.2.4 Gut Dysbiosis: Inflammation and Stress Modulation The commensal host for the gut microbiota benefits from the various endogenous biomolecules produced in its digestive tract. Among these microbiota, synthesized biomolecules are vitamins, essential amino acids, fermentation products as SCFAs, long-chain fatty acids (LCFAs) and their conjugates, hormones, and even growth factors. Of these, the SCFAs, LCFAs, and their conjugates are the other major by-products of gut microbiota activity on resistant starch and fibres. Most of the complex carbohydrates remain undigested in the human upper gastrointestinal (GI) tract. Nutrition from these components of diet is retrieved through fermentation by the gut microbiota as Bacteroides, Bifidobacterium, Propionibacterium, Eubacterium, Lactobacillus, Clostridium, Roseburia, and Prevotella in the proximal large intestine. Fermentation releases the SCFA by-products in colon as acetic, propionic, and n-butyric acids in a molar ratio of 60:20:20 (Cummings et al. 1987). The SCFAs have immediate local effect on the intestinal epithelia, its permeability, as well as

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integrity. The SCFAs bind to their receptors GPR43/FFAR2 and GPR41/FFAR3 (Brown et al. 2003). FFARs show a wide array of effects depending on the expressing cell type. In enteroendocrine cells, they stimulate the secretion of glucagon-like peptide-1(GLP-1) and peptide YY (PYY) (Cherbut et al. 1998); in pancreatic beta cells, they stimulate insulin secretion (Puddu et al. 2014); while in adipocytes, they induce leptin production through GPR41/FFAR3 to regulate host adiposity and inhibit lipolysis, promote adipogenesis, and suppress plasma free fatty acids through GPR43/FFAR2 activation (Xiong et al. 2004; Hong et al. 2005; Samuel et al. 2008; Ge et al. 2008). Hence, SCFA can have a variety of effects on the host including metabolism, transport, regulation of carbohydrate and lipid metabolism by liver, epithelial cell growth, differentiation, and acting as energy resource for the muscles, kidney, heart, and brain (Topping and Clifton 2001). The SCFAs released by microbiota in the proximal large intestine permeate through the blood-brain barrier (BBB) and are taken up by brain microvascular endothelial cells (BMECs), astrocytes, and to some extent neurons, all of which express at least one or more of the monocarboxylate transporters (MCTs), with the rate of uptake of butyrate being the highest followed by propionate and acetate through arterial infusion (Dalile et al. 2019; Vijay and Morris 2014; Oldendorf 1973). These SCFAs are a significant energy resource for the developing brain, and they also affect the BBB integrity (Maurer et al. 2004; Rafiki et al. 2003). FFAR2 binding of SCFAs is the molecular connection between diets and microbiota-mediated metabolism of fermentable dietary fibres by the commensal microflora such as Bifidobacterium and Bacteroides species. SCFA binding to FFAR2 leads to downregulation of release of inflammatory cytokines as TNFα by the immune cells causing reduction in inflammatory responses (Maslowski et al. 2009). Apart from these, there are a few gut metabolites that have been identified to either improve host functions enhancing longevity or attenuate neurodegenerative processes. Colanic acid is one such commensal bacterium-derived exopolysaccharide that encourages mitochondrial fission. Under stress conditions, it also increases the mitochondrial unfolded protein response (mUPR). Colanic acid is reported to promote C. elegans longevity (Kim and Jazwinski 2018; Han et al. 2017). Another gut microbiota metabolite of particular interest is indole-3-propionic acid (IPA), a product of tryptophan catabolism that is produced particularly by the bacterium Clostridium sporogenes (Jellet et al. 1980). IPA is one of the gut microbiota metabolites that has been identified to be a candidate molecule for Alzheimer’s disease therapy due to its high antioxidant and neuroprotective properties (Bendheim et al. 2002; Hwang et al. 2009; Chyan et al. 1999).

10.3

Gut Microbiota in Immunity, Disease, and Therapy

Since healthy microbiota is a defined set of community which changes slightly with age and diet, alterations in the gut microbiota community more than its accepted range of diversity alters the immune response. Gut microbiota maintains both the

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innate and the adaptive immune response at homeostasis. Immune dysregulation is the foundation of many GI tract diseases as irritable bowel syndrome (IBS), Crohn’s disease, ulcerative colitis, metabolic syndrome, and obesity (Durack and Lynch 2019). The intestinal mucosal epithelial barrier integrity is maintained by the normal gut microflora as Bifidobacterium adolescentis and segmented filamentous bacteria, protecting the host against pathogens. T helper 17 (Th17) cells (Geva-Zatorsky et al. 2017) and CD4 + Foxp3 + regulatory T cells (Tregs) get modulated by the SCFAs produced by the gut Clostridium species, particularly those of clusters IV, XIVa, and XVIII (Atarashi et al. 2011; Atarashi et al. 2013; Geva-Zatorsky et al. 2017). Additionally, surface polysaccharide A of Bacteroides fragilis binds to the Tolllike receptor 2 (TLR2) in dendritic cells (DCs). DCs encourage the release of IL-10, the anti-inflammatory cytokine, by the Treg cells to stimulate immune tolerance (Dasgupta et al. 2014; Geva-Zatorsky et al. 2017). Thus, both innate and adaptive immunity are modulated by the gut microflora, their cellular constituents, and metabolites, and these modulations play an important role in tilting the balance between the healthy and the disease state of the host. Particularly, the increase in Enterobacteriaceae species has a strong association with inflammation related to Crohn’s disease, a type of inflammatory bowel disorder (IBD) (Sokol et al. 2008; Mar et al. 2016). Lactobacillus murinus enrichment can provide protection against gut dysbiosis through expansion of Treg cells. This demonstrates the capacity of Lactobacillus spp. in modulating intestinal inflammation of IBD type. Thus, restoration of depleted microbiota functions in diseased patients is possible by microbiome manipulation through supplementation with specific restorative strains (Tang et al. 2015). Clostridioides difficile causes both diarrhoea and colitis and is also associated with diarrhoea associated with antibiotic administration. Metabolites from the resident gut bacteria as Clostridium scindens as secondary bile acids obtained after dihydroxylation of bile acid 7α mediate inherent resistance against colonization by the pathogenic Clostridioides difficile. The hydroxylated bile acids or secondary bile acids thus work as antibiotics and are an inbuilt preventive measure building resilience against C. difficile colonization (Sorg and Sonenshein 2008; Buffie et al. 2015). Hence, prophylactic microbial supplements combined with antibiotic capsules are nowadays prescribed for avoiding antibiotic-associated diarrhoea and colitis.

10.3.1 Gut Microbiota, Blood-Brain Barrier, and Neurological Disorders The fact that the gut microflora can make many of the neurotransmitters and some of the neurotrophic factors (detailed in Table 10.3) shows that any substantial variations in this microbiota are going to adversely affect the CNS and consequently modulate cognition and behaviour (Dinan and Cryan 2017b). While host genetics has always been the key driver for most neurological disorders, the gut microbiota is also a significant contributor to the development of many neuropathologies (Buffington et al. 2021). Alteration of the gut microflora profile in gut dysbiosis leads to

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increased permeability of both the gut mucosa and the BBB (Tang et al. 2020). Blood-brain barrier (BBB) is the semipermeable specialized microvascular barrier consisting of the brain microendothelial cells (BMECs), perivascular feet of the astrocytes, pericytes, and basement membrane, which together separates the CNS from the peripheral circulation and maintains the immune-privileged status of the CNS (Obermeier et al. 2013; Daneman and Prat 2015). Loss of normal gut microflora in humans due to dysbiosis as well as studies in murine models show increased BBB permeability through altered tight junction expression that leads to behavioural changes (Braniste et al. 2014; Spadoni et al. 2017). The gut microflora is also known to impact brain morphology. Paediatric Crohn’s disease is known to influence the brain structure (Mrakotsky et al. 2016). Crohn’s disease patients show thinning of the grey matter in their brain, and the same also correlates with psychological distress (Bao et al. 2015). There are in vitro evidences of endotoxin-activated microglia inducing cell death in BMECs (Kacimi et al. 2011). Neurological disorders of neurodegenerative, neurodevelopmental, neuropsychiatric, and neurovascular types as Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), major depressive disorder (MDD), and atherosclerosis have all been connected to gut inflammation, immune response, and a compromised neurovasculature at various stages of life (Durack and Lynch 2019). In the following sections, the involvement of microbiota in a few of the key neurological disorders is being discussed.

10.3.2 Autism Spectrum Disorders Autism spectrum disorder (ASD) is a diverse group of disorders characterized mainly by difficulties in social interactions and communication in combination with different patterns of activity and behaviour. Typically, there are difficulties in transitioning from one activity to the next, lack of attention to details, and unusual reactions to sensations. Autism spectrum disorder has been the one disorder most connected to gut microbiota during development and infancy. The gut microbiota in ASD bears an altered fermentation profile relative to unaffected siblings. Selective increase in Collinsella, Corynebacterium, and Lactobacillus and the fungal pathobiont Candida is observed in these patients (Strati et al. 2017). Increased Firmicutes/Bacteroidetes ratio and a higher relative abundance of Clostridia species are reported in children with ASD (Liu et al. 2019; Srikantha and Mohajeri 2019). Overall, this leads to higher than usual concentrations of SCFA and ammonia that are considered neurotoxic. These unusual concentrations of SCFA and ammonia associated with ASD may promote some of the detrimental neurological phenotypes (Wang et al. 2012). ASD is also linked to maternal health factors as obesity, maternal immune activation, and maternal gut microbiota all of which affect the environment the foetus is exposed to throughout the critical period of neurodevelopment. Feeding high-fat diet (HFD) to pregnant mice leads to depletion of Lactobacillus reuteri from gut microbiota, and oxytocin from their hypothalamus induces behavioural

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alterations typical of ASD in their offspring. These behavioural deficits can be reversed by supplementing the affected pregnant mice with L. reuteri or cohousing them with mice that are fed regular diet, and interestingly both treatments promote oxytocin levels (Buffington et al. 2016). Gut microbiota enrichment from murine maternal immune activation (MIA) model of ASD to germ-free mice shows that the MIA mice can impart some of the behavioural deficits typical of ASD to the germfree mice (Hsiao et al. 2013). These unusual behavioural patterns were subsequently connected to 4-ethyl phenyl sulphate (4-EPS). 4-EPS is a hepatic sulphonation product of 4-ethyl phenol (4-EP), the gut microbiota catabolite of aromatic amino acids (Fig. 10.1f), whose level is 49-fold more in the serum of MIA mice. EPS alone is enough to induce the ASD phenotype (Fischbach and Segre 2016). More recently, host genetics and microbiome have been shown to interdependently regulate maladaptive behaviours typical of ASD in mouse model. Additionally, only social deficits could be rescued with precise microbial therapy, or microbe-induced metabolite-based therapy, but hyperactivity could not be improved (Buffington et al. 2021). In an open-label study, microbiota transfer therapy was found to alter the gut ecosystem and recover both the gastrointestinal and autism symptoms (Kang et al. 2017). Thus, neurodevelopmental disorders may be partially improved through microbial therapy.

10.3.3 Attention Deficit Hyperactivity Disorder Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by maladaptive behaviour involving impaired attention, hyperactivity, and difficulty controlling one’s impulses (Jiang et al. 2018). ADHD is associated with dopaminergic reward pathways, particularly including the deficits in reward processing involving the ventral striatum. The risk factors include both genetic and environmental (Aarts et al. 2017; Thapar and Cooper 2016). Among the other risk factors are early exposure to antibiotics and caesarean section delivery at birth (Slykerman et al. 2017; Curran et al. 2015). Analysis of differences in microbiota composition between treatment-naïve juvenile ADHD patients and healthy controls showed significant decrease in Dialister, a succinate-utilizing bacterium whose abundance positively correlates with dietary carbohydrates. Presence of Dialister in ADHD patients also correlated to lower levels of IL-6. This indicates that imbalanced diet, antibiotic treatment, or inflammation could be causative factors contributing to ADHD. Apart from this, Faecalibacterium negatively correlates with the severity of ADHD symptoms (Jiang et al. 2018). A nominal rise in the Bifidobacterium genus is also observed in ADHD. This rise in the Bifidobacterium genus is linked significantly to enhanced expression of cyclohexadienyl dehydratase in ADHD. Cyclohexadienyl dehydratase is involved in the synthesis of phenylalanine, the dopamine precursor, and thus correlates to abnormal dopaminergic signalling in ADHD (Aarts et al. 2017). Mice colonized with ADHD microbiota show reduced structural integrity of both white and grey matter regions of internal capsule and hippocampus and are more anxious in the open-field test. They also have

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reduced connectivity between right motor and right visual cortices (Tengeler et al. 2020). Thus, altered microbial composition leads to altered brain structure, function, and behaviour. Overall, these experiments point to the fact that altered microbiota could be one of the contributing factors to ADHD and such patients could benefit from microbiota reconstitution through probiotic and prebiotic administration.

10.3.4 Alzheimer’s Disease Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive dementia combined with impaired cognition involving accumulation of cerebral amyloid beta plaques and hyperphosphorylated neurofibrillary tangles. AD can be familial when caused by genetic defects in molecular pathways in beta-amyloid processing, but these form only a minor fraction of the disease cases, most of which are sporadic, and such cases usually happen in older individuals typically above 65 years in age. The key driving factor associated with sporadic Alzheimer’s disease is ageing, and ageing-associated gut dysbiosis is now understood to be one of the driving factors. Recent interest in gut microbiota axis has connected gut dysbiosis with increased permeability of the intestinal mucosal barrier and enhanced inflammation. The breach of the intestinal mucosal barrier with the increased vascular permeability across the BBB leads to neuroinflammation. The change in the gut microbiota profile in gut dysbiosis results in change in neurotransmitter and trophic benefits associated with the normal gut microbiota (see Table 10.1). Gut dysbiosis is also known to contribute to obesity and type 2 diabetes mellitus, both of which are also risk factors for the pathogenesis of AD. The key species significantly reduced due to high-fat diet (HFD) are Bifidobacterium spp., Eubacterium rectale, Clostridium coccoides group, and Bacteroides-related group (Jiang et al. 2017). Metabolic disorders also lead to increased vascular permeability, neurovascular damage, and amyloid aggregation and deposition, which are also characteristics of AD (Stanciu et al. 2020; Sweeney et al. 2018). Yet another link between the gut microbiota, diet, and AD is the level of trimethylamine-N-oxide (TMAO), a molecule that is produced exclusively by the gut microbiota action on both free and phospholipid-conjugated dietary choline and carnitine. TMAO is elevated in CSF of individuals with AD. Gut bacteria harbouring one of several related catabolic gene clusters extrude trimethylamine (TMA) from the dietary precursor. This molecule reaches liver through circulation where it is hydroxylated to TMAO, and this further reaches the brain through systemic circulation (Fig. 10.1e) (Martı’nez-del Campo et al. 2015). The elevated CSF TMAO is associated with neurodegeneration and biomarkers of AD pathology like phosphorylated tau and phosphorylated tau/Aβ42 (Vogt et al. 2018). Additionally, the altered gut microflora can add up to the lipopolysaccharide (LPS) and amyloid burden of the host inducing the proinflammatory cytokines and inflammasomes aggravating the pathogenic processes leading to AD (Kohler et al. 2016). Together, gut microbiota and plasma amyloid-β are projected as potential index for identifying pre-clinical AD (Sheng et al. 2022). HFD leads to two- to

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threefold increase in plasma LPS causing metabolic endotoxemia, leading to low-grade chronic inflammation, insulin resistance, and type 2 diabetes (Kohler et al. 2016; Ley et al. 2005; Turnbaugh et al. 2006). Plasma levels of LPS in AD patients are three-fold more than healthy controls, further indicating that gut dysbiosis is a risk factor in AD. Furthermore, the number of gut Bifidobacterium spp., which maintain the gut barrier integrity and prevent bacterial/endotoxin translocation, negatively correlates with plasma LPS concentration (Jiang et al. 2017). Gut microflora from AD patients intensified AD pathologies in germ-free transgenic mice model of AD. The symptoms could be associated with the polyunsaturated fatty acid metabolites, their oxidative enzymes, asparagine-endopeptidase pathway activation, and microglia activation and inflammation (Chen et al. 2022). Epigenetic regulation of systemic functions by the gut metabolite SCFAs, in particular butyrate-mediated inhibition of histone deacetylase (HDAC) activity, promotes gene expression. Such HDAC inhibition is associated with the promotion of long-term potentiation (LTP) and enhanced learning. Thus, SCFAs are learning and memory modulators. Other than this, the HDAC inhibitor sodium butyrate also stimulates neurogenesis in experimental models of stroke and mouse model of AD (Kim et al. 2009; Zhang et al. 2017; Silva et al. 2020). Hence, while age-associated gut dysbiosis could be a major risk factor in exacerbating neuroinflammation associated with AD, supplementation with prebiotics and probiotics could be a mode of therapy in this debilitating neurodegenerative disease.

10.3.5 Multiple Sclerosis Multiple sclerosis (MS) is an autoimmune neurodegenerative disease involving degeneration of myelin sheath in the CNS. Among the risk factors are environmental factors, obesity, low-grade chronic inflammation, viral infections, and deficiency of vitamins D and A. Insufficient vitamins D and A exacerbate the inflammatory pathways. MS pathology also involves activated microglia and astrocyte pathogenicity. Microbiota of the MS patients is high on Archaea while being low or depleted in Firmicutes and Bacteroidetes phyla. MS patients also show increased levels of Blautia, Haemophilus, Pseudomonas, and Dorea genera, while Prevotella, Bacteroides, Parabacteroides, Haemophilus, Sutterella, Adlercreutzia, Coprobacillus, Lactobacillus, Clostridium, Anaerostipes, and Faecalibacterium genera levels are much lower (Chen et al. 2016; Buscarinu et al. 2017). Patients with relapsing remitting-MS (RR-MS) have a microbiota that has higher amounts of Pedobacteria, Flavobacterium, Pseudomonas, Mycoplana, Acinetobacter, Eggerthella, Dorea, Blautia, Streptococcus, and Akkermansia, compared with healthy controls. Restoration of the gut microbiota reduces inflammatory events in RR-MS patients (Schepici et al. 2019). Gut dysbiosis leads to intestinal inflammation and leakage of intestinal mucosal barrier and BBB, encouraging neuroinflammation and infiltrations of cells with impaired Treg function into the CNS (Dendrou et al. 2015; Riccio and Rossano 2018). Food components have the capacity to induce proinflammatory or anti-

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inflammatory pathways and encourage gut dysbiosis or healthy microbiota. Bifidobacterium and Lactobacillus are able to diminish the severity of inflammatory indicators on allergic and autoimmune responses in rodents (Ezendam et al. 2008). Nutrients including nondigestible dietary fibres encourage gut eubiosis by encouraging healthy gut microbiota that encourage the anti-inflammatory pathways to bring back homeostasis (Dendrou et al. 2015; Riccio and Rossano 2018; Adamczyk-Sowa et al. 2017). The Bacteroides and Clostridia species can suppress inflammation by induction of FoxP3+ Tregs (Chu et al. 2018). Another emerging component in gut microflora involvement in MS is the modulation of serotonin production in gut by the resident microbiota (Correale et al. 2022). This further reinforces that microbiota restoration to eubiosis can be an important component of therapy against MS, and either dietary intervention with prebiotics, probiotics, and postbiotics or transplantation with defined microbial communities can all be a part of restorative therapeutic strategies of multiple sclerosis.

10.3.6 Cerebrovascular Diseases Cerebrovascular diseases are diseases caused by impaired cerebral circulation and may present themselves as ischaemic and haemorrhagic stroke, atherosclerotic plaques in the carotid arteries, and arteriovenous malformation in the brain. The gut microflora affects the gut mucosal barrier permeability, and the same in turn also affects the BBB permeability. Gut microbiota can promote atherosclerosis through trimethylamine-N-oxide (TMAO). Platelets exposed to TMAO become hyperreactive and release Ca2+ from intracellular stores, leading to possible thrombosis. Hence, blocking the bacterial trimethylamine lyase using small-molecule inhibitors can prevent atherosclerosis in apoE-null mice (Zhu et al. 2016; Wang et al. 2015). Despite the plasma TMAO being associated with cognitive damage, following ischaemic stroke, patients with higher choline and betaine levels have lower risk of cognitive impairment (Zhong et al. 2021). Hence, dietary intake of choline through animal-derived food needs to be moderated in high-risk group and the same can be supplemented with probiotic support to populate the gut with microbial populations that do not have the catabolic gene clusters to metabolize dietary choline and carnitine.

10.3.7 Chronic Stress and Depression Chronic stress leads to despair behaviour and is a result of elevated adrenal derived cortisol levels due to over-activation of the hypothalamus-pituitary-adrenal axis. Such elevated cortisol can alter the gut microbiota leading to gut dysbiosis (Farzi et al. 2018; Foster and McVey Neufeld 2013). The GI tract is extensively innervated by the CNS through the vagus nerve as well as the ENS (Furness 2012; Breer et al. 2012). In response to restraint stress, germ-free mice show increased levels of ACTH and corticosterone. This effect is fully reversible by reconstitution with

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Bifidobacterium infantis (Sudo et al. 2004). Chronically stressed mice show depletion of Lactobacillus reuteri in gut microbiota, leading to changes in serum metabolites of the tryptophan kynurenine catabolic pathway, thus removing the availability of tryptophan for serotonin biosynthesis. Supplementation with L. reuteri can reverse these changes (Marin et al. 2017). Additionally, the connection of the gut microbiota-circHIPK2-astrocyte axis is associated with depression, and gut microflora from NLRP3 KO mice can reverse such astrocyte dysfunction (Zhang et al. 2019). The microbiota-derived SCFA butyrate also exerts antidepressant-like effect through inhibition of histone deacetylase (Schroeder et al. 2007). Moreover, stress also induces hyperphagia and obesity that can be linked to lack of satiety which involves the gut-brain neuroendocrine signalling. Microbiotaderived propionate and butyrate can induce intestinal gluconeogenesis possibly by activating the neuropeptide detecting μ-opioid receptor in the portal vein nerves (Duraffourd et al. 2012; De Vadder et al. 2014). This indicates microbiota involvement in both physiological stress response in the host and maintenance of metabolic homeostasis.

10.4

Microbial Technology in Neurological Disorders

Accumulating evidence linking neurological disorders of development, degeneration, psychiatric, as well as vascular types to gut microbiota has led to investigations on interventions in gut microbiota as a therapy against neurological disorders for both proof-of-concept and mechanistic studies in animal models of many diseases. Some of these microbiota intervention studies have also extended to randomized control trials and early-phase clinical trials. Reengineering of microbiome composition, functional genes, and metabolic output can be accomplished by administration of defined live microbial strains. However, microbiota heterogeneity across populations makes it complex to prescribe such generalized administration to reengineer microbiome towards therapeutic ends. Hence, personalized precision microbiota administration may become more effective in naturalizing disturbed gut microbiomes towards effective disease prevention or management (Aggarwal et al. 2023). Algorithms for tailoring nutritional advice to personalized glycaemic responses taking into account modifying gut microbiota have been worked out for at-risk groups with type 2 diabetes and cardiovascular disease (Zeevi et al. 2015). Gut microbiota can also influence the effectiveness of checkpoint blockade drugs in treating advanced melanoma. Live Bifidobacterium supplementation alters dendritic cell activity and leads to better prognosis by improving the tumour-specific T cell function (Fischbach and Segre 2016). Before going into a discussion of therapeutic strategies with microbiota supplementation in various neurological diseases, through probiotics, prebiotics, synbiotics, and postbiotics, it will be apt to discuss the effect of antibiotics on gut microbiota, the keystone species in restoration of gut microbiota, probiotics, and prebiotics. It also needs to be mentioned here that faecal microbial transplantation (FMT) is another mode of precision and personalized microbial therapy that alters

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gut microbiota and has been used in both experimental animal models and randomized control trial. A detailed discussion of FMT and possibilities of its use in treating neurological disorders is outside the scope of this chapter. Readers are however encouraged to refer to the latest reviews (Sorbara and Pamer 2022; Antushevich 2020; Vendrik et al. 2020) to understand the opportunities in FMT towards precision therapy. Hence, we move on to directly discuss the role of antibiotics in gut microbiota dysbiosis and its consequences.

10.4.1 Antibiotics, Gut Microbiota, and Neuroinflammation With the progress being made on the gut microbiota research and increased understanding of the need to maintain a healthy gut microflora, the general opinion of antibiotics being an overall beneficial class of drugs has drastically changed. Antibiotics can lead to loss of taxonomic and functional diversity, reduce the resident beneficial gut microbiota, and dilute off its metabolic products that downregulate inflammatory responses to maintain a healthy mucosal barrier of the intestine. Hence, it is now well accepted that antibiotics can be potentially harmful and can be responsible for a weakened immune system because of an impaired gut microbiota. This leads to reduced resistance to colonization by invading pathogens, increasing the possibilities of antimicrobial resistance (Lange et al. 2016). Reduction in beneficial resident microbiota can encourage opportunistic infection by species like Clostridium difficile causing IBDs and colitis, further leading to leaky gut and inflammation of the intestine, subsequently leading to metabolic disorders. Persistent low-grade inflammation in the intestinal mucosal barrier may in turn lead to a weakened BBB encouraging neuroinflammation, activating microglia, causing astrocyte pathology and neurodegeneration that leads to debilitating degenerative diseases impairing normal healthy life. Factors as the class of antibiotics, its dose, and route of administration all have a role to play in this antibiotic-induced gut dysbiosis. Another notable aspect in this is exposure to antibiotics on the developing infant gut microbiota. The developing immunoglobulin M (IGM) profile in the infant gut is highly dynamic and can be disrupted by antibiotic exposure. This can lead to disruption of the gut microbial architecture in the infants and increased possibility of host diseases of both the communicable and the non-communicable type in the subsequent adult life (Gibson et al. 2015). However, antibiotics can also be a dependable therapy for non-communicable diseases as hepatic encephalopathy or inflammatory bowel syndrome (IBS). Moreover, antibiotics can act positively providing a eubiotic effect on gut microbiota, increasing the abundance of beneficial species (Ianiro et al. 2016). Long-term treatment of APPSWE/PS1ΔE9 mouse model of AD with a combinatorial broadspectrum antibiotic regime led to prolonged changes in gut microflora composition and diversity, with concomitant reduction in Aβ plaque formation. This was accompanied with elevated levels of soluble Aβ and attenuated both plaquelocalized glial reactivity and altered microglial morphology, indicating that the gut microflora community diversity can impact Aβ amyloidosis (Minter et al. 2016).

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Hence, there lies a potential of identifying and developing antibiotics towards major non-communicable disorders. This new generation of antibiotics are expected to bring out eubiotic effects and thus play a therapeutic role beyond the cure of communicable diseases.

10.4.2 Probiotics in Therapy of Neurological Disorders The accepted definition of probiotics according to 2001 consensus of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) of the United Nations (FAO/WHO) was “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. However, a clearer definition was on call under the updated context when probiotics are being considered therapeutically useful and are hence being marketed as a product. The International Scientific Association for Probiotics and Prebiotics (ISAPP) has therefore spelt out its updated consensus definition of probiotics in its 2013 meeting. Henceforth, probiotics without a defined health claim include “a member(s) of a safe species, which is supported by sufficient evidence of a general beneficial effect in humans OR a safe microbe(s) with a property (e.g., a structure, activity or end product) for which there is sufficient evidence for a general beneficial effect in humans with proof of viability at the appropriate level used in supporting human studies”. Probiotics with claims of specific health benefits include “only defined probiotic strain(s) with proof of delivery of viable strain(s) at efficacious dose at end of shelf-life”. Fermented food with undefined microbial content is not probiotics as per the ISAPP (Hill et al. 2014). Common probiotic strains for human consumption belong to Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, and Enterococcus and gram-positive bacteria of Bacillus genus and some yeast strains belonging to Saccharomyces genus. Some of these are commonly used in prescription drugs as a probiotic combinatorial therapy or probiotic-enriched food supplements to protect the gut microbiota (Markowiak and Śliżewska 2017; Simon 2005). A recent double-blind randomized controlled trial (RCT) tried to establish the cognitive and emotional benchmarks of administering a commercial probiotic product with nine bacterial strains, namely, Lactobacillus casei W56, Lactobacillus acidophilus W22, Lactobacillus paracasei W20, Bifidobacterium lactis W51, Lactobacillus salivarius W24, Lactococcus lactis W19, Bifidobacterium lactis W52, Lactobacillus plantarum W62, and Bifidobacterium bifidum W23, for 4 weeks. On assessment of brain function, behaviour, and gut microbial composition, the study identified subtle shifts in microbiota profile along with changes in emotional memory, emotional decision-making tasks, and brain activation patterns (Bagga et al. 2018). Probiotic interventions are also associated with normalizing the heightened cognitive reactivity and transient changes in sad mood, which are also the hallmarks of susceptibility to depression and are hence the targets for interventions. A tripleblind placebo-controlled pre- and post-assessment RCT was conducted with healthy volunteers to look into the effect of probiotics into cognitive reactivity to sad mood. The participants received a 4-week multi-species probiotic food supplement

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containing Bifidobacterium bifidum W23, Bifidobacterium lactis W52, Lactobacillus acidophilus W37, Lactobacillus brevis W63, Lactobacillus casei W56, Lactobacillus salivarius W24, and Lactococcus lactis (W19 and W58). Significantly reduced overall cognitive responsiveness to sad mood was observed in the participants receiving the probiotic supplement indicating that probiotics may help lessen negative thoughts associated with sad mood (Steenbergen et al. 2015). Earlier, when a single organism Bifidobacterium longum 1714 had been used as a translational psychobiotic in healthy volunteers to modulate stress, there were improvement in a few parameters of memory performance and neurocognition with a concomitant change in brain activity (Allen et al. 2016; Queiroz et al. 2022). Probiotics have been used in pre-clinical and clinical studies to modulate gut microbiota as a therapy against neurological disorders. Many of these studies are at varying stages of progress towards turning into acceptable clinical applications with some being in their early pre-clinical proof-of-concept stages with animal models to obtain mechanistic validation, while others are randomized controlled trials (RCTs) with patients of neurological disorders to validate the effects of probiotics if any in the actual patients. Some of the significant studies that use probiotics to modulate gut microbiota as a therapy against neurological disorders are listed in Table 10.4. Most probiotic microorganisms identified and put into clinical trials so far are natural gut microbiota isolates or beneficial isolates derived from fermented food. However, there remains a huge scope of using updated molecular approaches to identify and isolate newer microorganisms that are compatible to the host, their resident commensal microbiota, and produce significant therapeutic responses. Ideally, these newer microorganisms should affect a specific host immune pathway in a well-controlled, physiologically compatible manner. One example of identification of 17 strains of CD4(+) FOXP3(+) Tregs is by inducing strains of Clostridia spp. from the human indigenous microbiota using genome sequencing. These strains lacking major toxins and virulence factors can be a community that provides both PAMPs and TGF-β-rich environment to help induction of Treg cells. Experimental validation of the same was observed when oral administration of a combination of these 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea (Atarashi et al. 2011, 2013). Thus, identification and use of novel isolates may allow for tailored therapeutic management.

10.4.3 Prebiotics in Therapy of Neurological Disorders The consensus definition of prebiotics as per the ISAPP meeting of 2016 is “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Hence, prebiotics include non-carbohydrate substances. Also, prebiotics can be applied to body sites other than the GI tract. Diverse categories of substances other than food are also included under prebiotics if they have documented evidence of selective microbiota-mediated mechanisms towards effective benefits to health. The target host upper gastrointestinal enzymes should not degrade dietary prebiotics. Hence, prebiotics can now include complex carbohydrates,

Pediococcus acidilactici

Bacteroides fragilis

Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus fermentum Bifidobacterium longum NCC3001

Bifidobacterium longum NCC3001 R

5.

6.

7.

9.

8.

Bifidobacterium animalis

4.

Depression

Anxiety

Alzheimer’s disease

Multiple sclerosis Multiple sclerosis

Multiple sclerosis

Mouse model of chemically induced colitis RCT with patients with IBS

60 AD patients; double-blind, RCT

EAE

Mouse model of EAE

Rat model for EAE

Anxiolytic effect, involves activating CNS vagal pathways for ENS Reduced responses to negative emotional stimuli in amygdala and fronto-limbic regions Reduces depression scores

Impact on migratory patterns of Foxp3+ CD4 Tregs in CNS autoimmunity via regulation of CD39 ectonucleotidase Affects cognitive function and some metabolic statuses in AD patients

(continued)

Bercik et al. (2011) Pinto-Sanchez et al. (2017)

Akbari et al. (2016)

Takata et al. (2011) Wang et al. (2014)

Ezendam et al. (2008)

Lactobacillus rhamnosus GG

3.

In offspring of dams of mice fed with HFD Seventy-five normal infants at 6 months, RCT

Restores social behaviour, ameliorates ASD-like behaviour ADHD or AS was diagnosed in 6/35 (17.1%) children in the placebo and none in the probiotic group (P = 0.008) Shorter duration of clinical symptoms, positive effect on total body weight gain, possible involvement of Tregs Inducing IL-10-producing Tregs

Lactobacillus reuteri

2.

Reference Hsiao et al. (2013) Buffington et al. (2016) Partty et al. (2015)

Microbiota intervention Bifidobacterium fragilis

Sl. No. 1. Mechanism/results Restores integrity of intestinal barrier

Table 10.4 Gut microbiota modulation strategies against neurological disorders Model organism MIA mouse model

Microbial Technology for Neurological Disorders

Condition/ disorder Autistic-like behaviour Autistic-like behaviour ADHD and AS

10 321

Lactobacillus reuteri

Winclove’s Ecologic® Barrier

Yoghurt with Lactobacillus acidophilus 145, Bifidobacterium longum 913

Lactobacillus acidophilus 145, Bifidobacterium longum 913

Akkermansia muciniphila

Lactobacillus plantarum ZDY04

11.

12.

13.

14.

15.

16.

Atherosclerosis

Atherosclerosis

Atherosclerosis

Major depressive disorder Atherosclerosis

Stress and despair

Condition/ disorder Anxiety and depression

ApoE-/- 1.3% choline-fed mice

ApoE-/- mice

Clinical trial, 15 normocholesterolaemic and 14 hypercholesteraemic women Human, double-blind RCT, hypercholesterolemic patients

Patients, triple-blind RCT

Chronically stressed mice displaying despair behaviour

Model organism Adult male BALB/c mice

Decreased serum total cholesterol, LDL cholesterol, and HDL cholesterol levels. No effect on serum triglyceride or fasting blood glucose levels Preventing endotoxemia-induced inflammation Strain-specific lowering of TMAO

Increased serum HDL cholesterol and lead to the desired improvement of the LDL/HDL cholesterol ratio

Mechanism/results Reduced stress-induced corticosterone and anxiety- and depression-related behaviour. Differential GABA(B1b) and GABA(Aα2) expression in brain regions Vagus involvement Restores despair behaviour from chronic stress Suppression of tryptophan/kynurenine metabolism Reduction in cognitive reactivity

Qiu et al. (2018)

Rerksuppaphol and Rerksuppaphol (2015) Li et al. (2016)

Kiessling et al. (2002)

Chahwan et al. (2019)

Marin et al. (2017)

Reference Bravo et al. (2011)

Abbreviations: Alzheimer’s disease (AD), Asperger syndrome (AS), attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), experimental autoimmune encephalomyelitis (EAE), gamma amino butyric acid (GABA), high-density lipoprotein (HDL), high-fat diet (HFD), low-density lipoprotein (LDL), maternal immune activation (MIA), randomized controlled trial (RCT), trimethylamine oxide (TMAO)

Microbiota intervention Lactobacillus rhamnosus (JB-1)

Sl. No. 10.

Table 10.4 (continued) 322 A. Dasgupta

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polyphenols, polyunsaturated fatty acids, and conjugated fatty acids provided that documented evidence exists in such being beneficial to the host through microbiotamediated mechanisms. Human milk oligosaccharides, fructooligosaccharides (FOS), galactooligosaccharides (GOS), mannan oligosaccharide (MOS), xylooligosaccharide (XOS), and inulin are thus all accepted prebiotics (Gibson et al. 2017). Dietary fibres may be prebiotic in one host and not on the other, and the same may vary from site to site within the same host based on their selective utilization by the microbes to promote health (Delcour et al. 2016; Roberfroid et al. 2010). With respect to neurological disorders, prebiotics containing FOS and GOS are reported to have antidepressant and anxiolytic effects on chronic stress in mice (Burokas et al. 2017). FOS also ameliorates cognitive deficits and neurodegeneration in APP/PS1 transgenic mice through modulating gut microbiota (Sun et al. 2019; Zhang et al. 2020). FOS upregulates neprilysin to mediate protection against AD (Wu et al. 2020). GOS can also reverse cognitive and behavioural dysfunction in these mice and reduce symptoms of depression (Yang et al. 2021). More studies are underway to elucidate the beneficial effects of prebiotics on neurological disorders.

10.4.4 Synbiotics in Therapy of Neurological Disorders Synbiotics are functional foods with probiotic as well as prebiotic properties that are created to enable the probiotics to survive the upper GI tract. Hence, synbiotics are an appropriate blend of probiotics and prebiotics in a single product with superior effect, compared to the activity of either of them alone. Synbiotics improve survival of favourable microbes added to food or feed and stimulate proliferation of particular natural bacterial strains present in the GI tract (Markowiak and Śliżewska 2017). A synbiotic agent consisting of GOS and a multi-strain probiotic with Lactobacillus helveticus and Bifidobacterium longum improved the tryptophan signalling in MDD with concomitant reduction of the symptoms of depression (Kazemi et al. 2019). A few synbiotic-based therapies have also been through RCTs. In one such study, tolerability of Bifidobacterium infantis with a bovine colostrum product (BCP) as a source of prebiotic oligosaccharides was put through a double-blind RCT of 12 weeks in children with ASD to evaluate GI microbiome, and immune factors showed that the combination is well tolerated. Participants from both treatments saw reduced incidence of aberrant behaviours with concomitant reduction in IL-13 and TNF-α production (Sanctuary et al. 2019). Thus, synbiotics provide an effective route to therapy for both neuropsychiatric and neurodevelopmental disorders.

10.4.5 Postbiotics in Therapy of Neurological Disorders Postbiotics are metabolites, biogenics, soluble factors, or cell-free supernatants of bacterial fermentation obtained from live bacteria or released after bacterial cell lysis (Sorboni et al. 2022). Postbiotics thus can include short-chain fatty acids (SCFAs),

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endo- and exopolysaccharides, antibiotics, cell surface proteins, vitamins, plasmalogens, and organic acids (Sorboni et al. 2022; Aguilar-Toalá et al. 2018). Of these, SCFAs are known to downregulate inflammatory pathways and reduce stress through GPR 43/FFAR2 and GPR 41/FFAR3 (Sanna et al. 2019; van de Wouw et al. 2018; Maurer et al. 2004; Rafiki et al. 2003; Maslowski et al. 2009). However, SCFAs, particularly propionate, in excess have been shown to be a causative of ASD-like behaviours in experimental mice models (Shultz et al. 2008). Apart from this, coelomic acid and IPA are two metabolites that have been identified as gut microbiota-derived neuroprotective agents with the latter already being used in clinical trials against Alzheimer’s disease (Bendheim et al. 2002). Thus, microbiota-derived molecules including but not limited to metabolites form an extremely important resource for therapeutics against neurological disorders that are yet to be substantially explored and utilized with documented evidences of their therapeutic benefits.

10.5

Precision Microbiome Engineering and Challenges for Microbial Technology

Trillions of microorganisms inhabit the gut, and hundreds of genes from the microbiome may be interacting with the host either maintaining homeostasis and health or causing disease. The challenge is to identify the causal contribution of one single gene or a group of genes along with the microorganism to which it belongs and associate it with either health or disease state of the host. The added complexity in this is the fact that the disruptive or positively contributing gene may be belonging to some non-model gut microbe, making it difficult to target genetically. However, genetic manipulation of gut microbes is necessary for single-gene interrogation in a complex microbiome, making building of tools and experimental pipelines for such interrogations essential to enable mechanistic dissection of single-gene involvement out of the whole microbiome and their impact on host physiology. For example, gene transfer methods and gene manipulation tools for non-model gut Firmicutes/ Clostridia have been developed to enable functional analysis of a microbiota gene. This pipeline has been tested with the commensal gene baiH that mediates SCFA and host bile acid pool, gut microbiome composition, and colon inflammation in the host with a complex microbiome (Jin et al. 2022). The host circulation carries significant amounts of metabolites and breakdown products from gut microflora. Hence, understanding the role of each microbiotaderived molecule in the host biology is challenging. Selective depletion of these molecules may be a necessary step in delineating their contribution to host health or disease. Towards these ends, tools for making clean genetic deletions in Clostridium spp. have been developed for selective knockout. The method has been applied by its developers to knock out ten genes from the model commensal Clostridium sporogenes. The mice colonized by the C. sporogenes with deleted branched SCFA production showed that branched SCFAs have immunoglobulin A-modulatory activity (Guo et al. 2019; Ronda et al. 2019).

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As the link between the microbiota and human diseases is getting established and PAMPs are getting identified, the concept of precise tailored microbiome-based therapeutics is also emerging. It is now known that variation in microbiota among patients leads to differential outcome of treatment with prescription drugs. Overall, there is a huge scope for personalized tailored therapy for each patient based on their specific requirements and responses. This brings forward a scope for individualized microbial therapy or microbiota product-derived therapy. Whether using the elimination approach with antibiotics or supplementation approach with probiotics, understanding the disease-associated features in the first place using high-throughput sequencing and other omics-based studies of individual patient and their microbiota will have a significant role to play in such approach to precision microbiota-based therapy. Tailoring specific microbiota component and deriving their specific metabolites can be yet another approach towards precision therapy (Ryu et al. 2021). Microbiome engineering can be used to modify the composition of the gut microbiome and its derivatives and their function towards a therapeutic end against the specific neurological disorder (Fig. 10.2). Such engineering may involve delivering alternate genetic message through a carrier microorganism within a plasmid with other mobile genetic elements into the gut where the genetic element gets delivered to the microbiota through conjugation and then integrates into the gut microbiota genome. The genetic message can further be tailored to enable its selective or generalized expression in the microbiota. Otherwise, an engineered microbe by itself can be the therapy by synthesizing metabolites that can work as drugs being inhibitors to the harmful enzymes within the microbiota. In a third concept, the engineered microbe can be enabled to metabolically transform the

Fig. 10.2 Strategies of engineering microbiome composition and functionality by genetic manipulations or enzyme inhibitor

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harmful microbial or host metabolite to harmless or beneficial molecules. Though some of these strategies are being tried for other gut microbiota-associated diseases, applying any of these to neurological disorders requires better understanding the mechanisms of action of the microbiota and its specific components in neurological disease development (Aggarwal et al. 2023). One such case relevant to neurological disorders is presented by the human gut metabolism of levodopa (L-dopa) through an interspecies pathway. Tyrosine decarboxylase from Enterococcus faecalis converts L-dopa to dopamine, which is thereafter converted to tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta. This limits dopamine availability in the host despite being administered carbidopa, the inhibitor to the host aromatic amino acid decarboxylase in the gut (Rekdal et al. 2019). However, co-administering an inhibitor to the tyrosine decarboxylase from E. faecalis, (S)-a-fluoromethyltyrosine (AFMT) with L-dopa and carbidopa, prevents L-dopa decarboxylation and allows peak serum concentration of L-dopa in mice colonized with E. faecalis (Rekdal et al. 2019). This paves the way for personalized medicine for Parkinson’s disease patients whose gut microbiota includes E. faecalis, towards enabling drug bioavailability in such patients. Several elegant methodologies have been developed in the last 10 years to engineer bacteria or bacteriophages to create antimicrobials (Citorik et al. 2014), for programmable edits of target base in genomic DNA (Komor et al. 2016), recruitment of CRISPR-Cas systems by T7-like transposons, and RNA-guided DNA insertions with CRISPR-associated transposases (Peters et al. 2017; Strecker et al. 2019; Faure et al. 2019). Prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems in archaea and bacteria provide adaptive immunity against foreign genetic elements via guide RNA-dependent DNA or RNA nuclease activity to enable removing the mobile genetic elements (MGEs) from viruses, plasmids, and transposons (Strecker et al. 2019; Faure et al. 2019). This technology has been successfully used to create antimicrobials of the class RNA-guided nucleases (RGNs) whose range of activity can be designed to target specific DNA sequences. The advantage of such is that now RGNs targeting specific and customized undesirable genes for polymorphisms, antibiotic resistance, or virulence determinants can be selectively deleted once the RGNs are delivered to the host microbiome for horizontal gene transfer. Thus, RGNs form a selection pressure at the microbiome level, reducing the incidence of undesired genes while curtailing non-target effects and enabling programmable remodelling of microbiome (Citorik et al. 2014). However, applicability of RGNs towards therapeutic ends to cure neurological disorders needs further research. Sometimes, base editing is ideal for correcting genetic errors or creating essential point mutations. Base editing tools have been evolved using engineered fusions of CRISPR-Cas9 and a cytidine deaminase enzyme that can be programmed with a guide RNA, without causing dsDNA breaks to mediate the direct conversion of cytidine to uridine, thereby effecting a C → T (or G → A) substitution (Komor et al. 2016; Aggarwal et al. 2023). This can be used to correct mutations in gut microbes to reactivate beneficial enzymes or to inactivate the deleterious ones. Bacterial and

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archaeal genomes have many Tn7-like transposons with minimal CRISPR-Cas systems that use RNA-guided transposition (Peters et al. 2017; Faure et al. 2019). Sometimes, these Tn7-like transposons have co-opted nuclease-deficient CRISPRCas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. The DNA-targeting complex cascade and the transposition protein TniQ can enable programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli. This is an RNA-guided integrase that enables fully programmable genomic manipulations without causing double-strand breaks and thus does not require homology-directed repair (Klompe et al. 2019). Similarly, Tn7-like transposase subunits and V-K CRISPR effector (Cas12k) that catalyses RNA-guided DNA transposition from cyanobacteria Scytonema hofmanii (ShCAST) can integrate DNA into targeted sites in the E. coli genome (Strecker et al. 2019). Nevertheless, the challenge remains to identify appropriate cases and apply these technologies towards engineering disease-relevant microbiota to meet therapeutic ends of neurological disorders.

10.6

Conclusion

Advances in high-throughput sequencing and other omics-based technologies have enabled a deeper understanding of the microbiome-host interactions, more specifically for the human host. This has unravelled newer understanding of ways in which the commensal and symbiotic microflora within our gut influences the function of our CNS and in many cases has a mechanistic role to play in states of our health and disease. The understanding of this gut-brain axis has further elucidated the ways in which gut dysbiosis can cause low-grade inflammation, disrupt the gut mucosal barrier, and increase permeability of the BBB causing an inflow of undesirable endotoxins from systemic circulation into the central nervous system, triggering microglia activation and astroglia reactivity, and further initiating neurodevelopmental, neurodegenerative, neuropsychiatric, and neurovascular diseases. Hence, restoring the gut homeostasis to reduce inflammation will be integral to the therapy of most neurological disorders. As discussed, discriminating and precise antibiotics, engineered probiotics, prebiotics, and other microbiotaderived postbiotics are all exciting avenues of therapy against neurological disorders. Importantly, gut microbiota diversity significantly varies within and between population. This presents the need of precise and personalized microbiota engineering. However, in order to perform such precision engineering, specialized tools for gene manipulation are essential. Many such tools have evolved through the past 10 years. Now with tools as RNA-guided nucleases, precise sequence insertion tool for non-model gut microorganisms, base editing tools, RNA-guided integrases, and systems for conducting clean genetic deletions in non-model gut microorganism, it remains to be seen what applications of these wide array of tools can determine precise relationships of genetic components of microbiome-to-host interaction with respect to the neurological disorders. Once such proof-of-concept studies are

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performed, upscaling the same to a commercial product will be yet another challenge towards meeting the therapeutic needs of the wide array of neurological disorders. Acknowledgements Acknowledgements are due to all investigators who have invested themselves in gaining understanding of the complex host-microbiome interactions and have painstakingly developed tools for precision gene targeting and modifications that have far-reaching scope and implications. Works of many such investigators are cited here. However, those who could not be cited due to the limitations of space and time are also thanked for their contribution to this rapidly evolving field of study.

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Frontiers in Fungal Endophytes Associated with Medicinal Orchids

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Bishal Pun and S. R. Joshi

Abstract

Orchids are diverse group of plant species important for their medicinal value. Endophytic fungi are important component of micro-ecosystem in orchids. Population structure and distribution pattern of fungal endophytes are significantly affected by several components which include ecological environment and genetic constitution of the plant harbouring them. Recent research of medicinally important orchids has centred on identification and characterization of endophytic fungi that somehow influence the development and vigour of the host along with the production of important bioactive compounds. The explored group of fungal endophytes are scanty, which implies that the scope of discovering novel species or genetic variants and realizing their importance in host fitness and stress tolerance is crucial. Also, the room of mining bioactive compounds from these fungi associated with medicinal orchids is abundant. Optimization strategies for enhanced and sustainable production of bioactive compounds from fungal endophytes are also available to maximize their utility commercially. Thus, the study of these associations gives insights of the host-microbe interaction to understand the ideal production of new and important fungal metabolites for sustainable development of bioproducts. Keywords

Endophytic fungi · Bioprospection · Bioactive compounds · Production optimization

B. Pun · S. R. Joshi (✉) Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_11

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Introduction

Orchids are well known for their ornamental, food and medicinal value. Some orchid species are medicinally very important while other possess both aesthetic and medicinal values (Singh and Duggal 2009). In many parts of the world, orchids are used as herbal medicine to treat a number of diseases (Kasulo et al. 2009). Traditional Chinese medicine largely uses orchids to cure a number of diseases (Bulpitt 2005). Also, in the Indian system of medicine called Ayurveda, orchids are widely used as one of the ingredients owing to their therapeutic properties (Singh and Duggal 2009). Studies carried out on different genera of medicinal orchids have reported the presence of many important phytochemicals (Yang et al. 2006). Endophytes are microorganisms living in the internal tissue of plants without causing any apparent symptoms of disease (Stone et al. 2000). Over one million fungal endophytes are estimated to thrive in nature (Faeth and Fagan 2002). They are well known to form an important component of micro-ecosystems in orchids. The long-term coexistence of endophytic fungi and orchids, coupled with evolutionary process, has established important inter-relation to expand the adaptability of both the partners. These endophytes increase tolerance capacity of the host to different forms of stress conditions (Demain 2014). These colonizers also biosynthesize host secondary metabolites (Chutolo and Chalannavar 2018). Orchids’ fungal endophytes are reported to synthesize antimicrobial compounds that provide a defence system in the host plants to a broad range of pathogens (Alurappa et al. 2018). Polyphyletic groups of fungi are isolated as endophytes from medicinal orchids and explored for their biotechnological applications (Ma et al. 2015). It is reported that they have a pivotal role in the production status of bioactive compounds owing to mutual fungus-host interaction (Faeth and Fagan 2002). Understanding the population structure and functional attributes of endophytic fungi associated with medicinal orchids is important to obtain and make better use of the resources commercially through sustainable production strategies for the wellbeing of mankind.

11.2

Classification of Fungal Endophytes

Based on their ecological roles and diversity, fungal endophytes can be classified broadly as clavicipitaceous and non-clavicipitaceous fungal endophytes; former are predominant in grasses and latter are mostly common in vascular and non-vascular plant species (Rodriguez et al. 2009). On the basis of their mode of transmission in the host, they can be classified as vertically transmitted and horizontally transmitted endophytes. Vertically transmitted endophytes are true endophytes and are transmitted directly from the parent plants to their progenies (Saikkonen et al. 2002). On the other hand, horizontally transmitted endophytes are transferred from different plants in a particular population. This means of transmission is mostly exhibited by sporetransmitted endophytes which commonly show weak pathogenicity against insect herbivores (Higgins et al. 2007). Based on the preferential tissue localization of

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fungal endophytes in the host plant, they can be grouped as root and foliar endophytes. Many fungal endophytes have been reported to exhibit localized instead of systemic colonization. Fusarium sp., Piriformospora sp. and Glomus sp. are mostly found within the plant roots and are called root endophytes. Others such as Pochonia chlamydosporia and Beauveria bassiana invade the plant leaves, and stems are grouped as foliar endophytes (Behie et al. 2015).

11.3

Relationship of Fungal Endophytes and Orchids

Orchids are most unique of all plants as the survival and adaptation are supported by the fungal endophytes. The mode of nutrition in orchids includes direct and often obligate relationships with fungi. For germination of miniature seeds of orchids which lack endosperm and have insufficient energy supply, seeds come into symbiotic association with the fungal symbionts. The colonizing symbionts nourish the germinating seeds and get closely associated with the growth and adaptation of the orchids (Arditti and Predgeon 1997). In orchids, it has been studied that mechanism of transmission of endophytes to the host plant can be horizontal or vertical (Gostincar and Turk 2012). In vertical transmission, fungal endophytes come in contact with the seeds and subsequently associate with the host plant. It generally occurs in species-specific manner. On the other hand, horizontal transmission is barely species specific. It requires the aerial dispersal of exterior spores from the host plant to other new hosts (Stone, 2000). Fungal endophytes differ from fungal pathogens in the manner of colonization and penetration in orchid hosts. The endophytes colonizing the host from the stomata through anticlinal wall epidermal cells localize intercellular region in the shoots. Fungal pathogens penetrate directly from the plant cell wall and colonize extracellularly (Schulz and Boyle 2005). Fungal endophytes residing in the cortical or velamen tissues of roots in orchids are called orchidaceae root-associated fungal endophytes. These root cortical tissues are targeted primarily for studying symbionts in orchidaceae. Ascomycota, basidiomycota and mucoromycota members have been isolated and identified from roots of orchids (Novotna et al. 2018). Till date, several orchid species which are studied harbour diverse genus of fungal endophytes. Some common species documented from orchid shoots are Alternaria sp., Colletotrichum sp., Geopora sp. and Acremonium sp. (Ma et al. 2015).

11.4

Factors Influencing Diversity and Dynamics of Fungal Endophytes

Distribution pattern of fungal endophytes is closely linked with ecological environment and genetic background of the host. Ecological parameters like geographic location, vegetation, temperature, humidity and edaphic conditions considerably influence the population structure of endosymbionts. A particular set of environmental conditions decides the distribution pattern of host plants which eventually

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decides the colonizing species of fungal endophytes. As a result, composition of endophyte population displays certain extent of regional specificity. However, same host plant species from one region show very low degree of similarity of endophytic species with same host species from another region (Jiang et al. 2010). The presence of particular species of fungal endophytes is also limited to certain host species and certain host genotypes (D’Amico et al. 2008). The genotype of host plant plays a pivotal role which ultimately affects the population structure of fungal endophytes determining the fungus-host relationship. These endosymbionts demonstrate parasitic as well as mutualistic state, based on the gene expression and response in both partners. Therefore, association of both the partners is a flexible interaction, and difference in genome and gene expression status determines the outcome of the symbiosis (Moricca and Ragazzi 2008). Additionally, age and colonizing tissue of host plant govern the species composition and population structure of endophytic community (Sieber 2007). Specific endophytic species show specific distribution pattern in different tissues of host plant with different ages. Such definite allocation of endosymbionts is associated with their ability to make use of particular substrates produced by particular plant tissue (Rodrigues 1994). Restricted distribution explains the importance of population structure of endophytes in determining the beneficial properties conferred to the host plant. Also, fungal endophytes produce diverse class of host-associated secondary metabolites. Therefore, bioprospection of endophytic fungi associated with medicinal plants is an important approach to discover a plethora of bioactive compounds that can be exploited as important medicinal resources (Rodriguez et al. 2009).

11.5

Fungal Endophytes and Their Role in Medicinal Orchids

11.5.1 Promoting Growth and Fitness of Host Plant Phytohormones show a major role in the growth and development of plants. Endophytic symbionts have a profound influence on the synthesis of these plant hormones. They synthesize phytohormones, siderophores and nutrients assimilating enzymes that help in acquisition and growth promotion of plant (Malla et al. 2004; Sherameti et al. 2005; Sirrenberg et al. 2007; Khan et al. 2009). They promote the vigour of orchids by regulating the production of hormones such as auxin, cytokinin, gibberellin and ethylene. Several studies show their importance in the germination, growth and better acclimatization. Orchid-associated fungal endophytes such as Rhizoctonia zeae and Piriformospora indica are reported as beneficial partners and increase the survivability of the host plant (Aggarwal et al. 2012; Varma et al. 2013). Nutrients are essential for the plants. Endophytic fungal association also helps the host plant in obtaining minerals (Stockel et al. 2014). Mycoheterotrophic orchids completely rely on fungal endophytes for the supply of carbon. Endophyte Inocybe species colonizes the rhizome of a mycoheterotrophic orchid, Achlorophyllous

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Fig. 11.1 Role of endophytic fungi in medicinal orchids

epipogium, and supply a major quantity of carbon to the host (Roy et al. 2009) (Fig. 11.1). Orchids require endophytes for uptake of carbon for germination of seed due to the fact that their seeds lack the endosperm which serves as carbon source (Gennaro et al. 2003; Zimmer et al. 2007). Many researchers have reported the role of fungal association in carbon acquisition and utilization in orchids like Cephalanthera damasonium, Dendrobium officinale, Pseudorchis albida and Epipactis helleborine (Gebauer and Meyer 2003; Wang et al. 2013; Stockel et al. 2014).

11.5.2 Stress Tolerance of Host Plant Orchids experience biotic and abiotic stress but they commonly overcome the stress conditions. Stress tolerance develops in the plant not just from the plant itself but also from the fungal colonizers residing in the host tissue. Recent advances in fungal ecology have revealed that fungal colonization exhibited priming in the expression of a group of stress-related genes and promoting production of stress hormones in the host plant compared to plants with no cryptic fungi (Sherameti et al. 2008). Endophytic fungi may also elicit antioxidant enzyme biosynthesis by host plant to tolerate stressful conditions (Baltruschat et al. 2008). Several metabolomic studies have reported that endophytic genes associated with important metabolites increase stress tolerance of host plant by affecting its defensive pathways and physiological response (Arnao and Hernandez-Ruiz 2018).

11.5.3 Production of Bioactive Metabolites Endophytes can co-produce identical or much alike biological compounds as their host. There are various means through which these bioactive compounds are simultaneously produced in plants and their endophytes. In some cases, the biosynthesis of the same metabolite in both the partners develops a pathway separately (Bömke and Tudzynski 2009). The co-production of bioactive metabolites may also result from horizontal gene transfer between the host plants and their microbial

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counterparts (Taghavi et al. 2005). Some of the important bioactive compounds co-produced by these partners include azadirachtin (insecticide), podophyllotoxin (anticancer drug) and camptothecin (anticancer drug) (Kusari et al. 2012; Puri et al. 2006; Puri et al. 2005). Fungal endophytes are storehouse of numerous bioactive compounds. They are valuable source of many important therapeutic compounds. Therefore, it is important to device a fit strategy to cultivate them for industrial production. Growth conditions such as medium composition, temperature, pH and aeration must be optimum for better quantity and quality of the desired end product. Fermentation of endophytic fungi for large-scale production of bioactive compounds yields reproducible and unlimited supply of the desired product (Strobel et al. 2004). With the application of modern tools of biotechnology, improved strains can be developed to exploit them commercially. Fungal endophytes such as Pestalotiopsis sp. and Talaromyces sp. are applied as elicitors to enhance the growth, development and metabolite production in orchids (Zhu et al. 2018). It is known that they can produce bioactive compounds which are also produced by the host plant (Heinig et al. 2013). Fungal endophytes associated with medicinal orchids serve as a reservoir of therapeutic compounds. However, very few studies have been done on medicinal orchid-fungal endophytes to investigate their bioactive potential for pharmaceutical application.

11.5.4 Host Protection and Biocontrol of Disease To minimize the application of chemicals in agriculture, fungal endophytes are gaining attention as one of the subjects for research and application in plant pathology (Zabalgogeazcoa 2008). They can confer protection to the host plant from several phytopathogens. It can be through direct targeting the pathogen or by indirectly activating the host immune system (Mishra and Sarma 2018). Endophytes have the ability to produce antibacterial, antifungal and insecticidal compounds which strongly inhibit the plant pathogen. Cryptosporiopsis sp. and Colletotrichum sp. are examples of some endophytic fungi which are active against phytopathogens such as Phytophthora capsici and Gaeumannomyces graminis (Sudha et al. 2016). Endophytic fungus Pestalotiopsis sp. isolated from orchid Dendrobium officinale has antifungal properties against pathogenic strains of fungi (Baltruschat et al. 2008). Endophytes indirectly control plant pathogens by conferring aid to the host plant through secondary metabolites (Strobel and Daisy 2003). Additionally, orchids get many benefits from the mutualistic relationship with the fungal endophytes to control diseases caused by insects (Mishra and Sarma 2018). Endophytes are reported to protect the host from the insects by several strategies such as repelling insects, inducing weight loss, reduction in growth and development and increased pest death rate. The endophytic colonizers produce secondary metabolites such as alkaloids to act against insect pests (Patterson et al. 1992) (Table 11.1).

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Table 11.1 Role of endophytic fungi isolated from medicinal orchids Host plant Anoectochilus formosanus Anoectochilus setaceus Bulbophyllum neilgherrense Cymbidium sinense Dendrobium nobile Dendrobium officinale Dendrobium sp. Gastrodia elata Pleione yunnanensis Vanda testacea

11.6

Endophytic fungi Epulorhiza sp. Xylaria sp. Aspergillus flavus and Aspergillus niger Mycena orchdicola Cephalosporium sp., Gliocladium sp. and Mycena dendrobii Pestalotiopsis sp. Biscogniauxia petrensis Penicillium sp. Fusarium sp., Trichoderma sp. and Paecilomyces sp. Aspergillus ochraceus and Penicillium sp.

Role of endophyte Enhanced growth of host plant Antibacterial Antibacterial Secrete plant growth hormones Seedling growth

Reference Tang et al. (2008) Ratnaweera et al. (2014) Sudheep and Sridhar (2012) Zhang et al. (1999) Dan et al. (2012)

Antifungal and cytotoxic Cytotoxic Antimicrobial Seed germination

Wu et al. (2016)

Antifungal

Sudheep and Sridhar (2012)

Ma et al. (2020) Duan et al. (2016) Yang et al. (2008)

Molecular Interaction Between Endophytic Fungi with the Host Orchid

The ability of plants to select compatible fungi from among the diverse group of microbes thriving the surrounding has been an interesting area of research. Studies have shown that molecular coordination between the host plant and the residing fungi is important to establish the mutual relationship. Some studies suggest that flavonoid and phenolic compounds are key molecules responsible for the fine signalling involved in orchid-microbe interaction (Mandal et al. 2010). The association brings about a series of structural changes in the colonizing fungi from its germination of spores to hyphal differentiation (Bonfante and Perotto 1995). Recruitment of symbiotic fungi is commenced by the interaction between the chemicals of the root exudates (De Weert et al. 2002). Different compounds like flavonoids and coumarins (Huang et al. 2019; Stringlis et al. 2018) function as signalling molecules during the initial phase of colonization of orchid tissue by endophytic microbes. Also, phytohormones like strigolactones released from the host roots have been reported to perform as signalling molecule to selectively recruit the endophytic microbes (Rozpadek et al. 2018). Microorganisms adhere to the host plant and penetrate the surface by either active or passive pathways. Active process involves lipopolysaccharides (LPS) and exopolysaccharides (EPS), (Suarez-Moreno et al. 2010) and on the other hand, passive process is mediated by plant pores, cracks or cuts (Hardoim et al. 2008). After invasion to a specific host, an intricate crosstalks

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of signalling come into action. In the initial stage, exudation of fungal effectors and lectins takes place, and in the later stage, several hydrolytic enzymes are produced by the symbionts (Lahrmann and Zuccaro 2012). For successful establishment of this relationship, invading endophytes must avert or suppress the host immune response. Endophytes have developed several tactics to overcome the host defence system (Yu et al. 2019). A fungal endophyte Cladosporium fulvum produces two lectin-type chitin-binding effectors, Aver4 and Ecp6, to toughen its cell wall and protect it from hydrolysis by host chitinases (De Jonge et al. 2010). Also, endophytes secrete enzymes with antioxidant properties to combat reactive oxygen species (ROS) produced by plant defence response (Zeidler et al. 2004; Mengistu 2020). Silencing of the pathways mainly targeted by miRNAs for host defence is also a major strategy to combat host immune response (Plett and Martin 2018). Additionally, some secrete only low levels of hydrolytic enzymes to evade host defence response (Elbeltagy et al. 2000). Endophytes hijack the host hormone signalling pathways with the help of effectors to promote the symbiotic association. Rhizophagus irregularis produces the SP7 effector and associates with JA/ethylene inducible-ERF19 to prevent ERF19-activated defence-related genes to establish mutualism with the host (Kloppholz et al. 2011).

11.7

Omic Approaches to Understand Orchid-Endophyte Interactions

A thorough comprehension of orchid-endophyte association is essential to understand the full potential of these fungal colonizers which are reservoir of bioactive compounds. Modern high-throughput genomic analyses have transformed the exploration of microbiome studies. In-depth studies by whole genome analysis of endophytic fungi provide essential data to comprehend the endophytic lifestyle. This supports the identification of important genes associated with plant growth promotion (PGP) and other important metabolic mechanisms involved in different bioactivities (Dubey et al. 2020). Whole genome of several endophytic fungi has been sequenced which is available as model systems to improve the data analysis in omic studies. Comparative genomic sequencing also gives wider understanding of endophytic behaviour to explore their potential application. Diversity of unculturable and culturable endophytic community can be studied with the availability of next-generation sequencing technique (Akinsanya et al. 2015). Metagenomic approaches coupled with in silico analysis uncover the functional and phylogenetic relationship of the microbiome (Tian et al. 2015). Microarray tool is the modern genome-based technique which is employed for gene profiling and transcriptomic analysis (Felitti et al. 2006). This tool can carry out profiling of genes associated with the interacting host-endophyte partners (Barnett et al. 2004). Metaproteomics focuses on identification of proteins and metabolomics on spotting metabolites, and together, these approaches form a reliable tool to understand the metabolic interaction among the host and the endophyte. Some studies have been done with the help of different omic approaches to study endophyte diversity and

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host-endophyte interaction and discover novel bioactive compounds (Kumar et al. 2014; Andres et al. 2017).

11.8

Biosynthetic Gene Clusters of Secondary Metabolites

Natural compounds or natural product-derived compounds constitute about 75% of anti-infective agents (Newman and Cragg 2010). Understanding the biosynthesis of most of these bioactive compounds at the genetic level reveals the presence of single gene clusters in their production and regulation. Several works have aimed to isolate these biosynthetic gene clusters and exploit the technique of genetic engineering to feasibly produce biologically active secondary metabolites. One of the most important class of secondary metabolites are polyketides. Polyketides are synthesized by large multimodular enzyme complexes called polyketide synthases (PKS). This enzyme system comprises of three core domains which include acyltransferase (AT), acyl carrier proteins (ACPs) and ketosynthase (KS). AT domain specifically selects and activates the substrate, ACPs covalently link the growing intermediate acyl chain through phosphopantetheine arm and KS domain catalyses the elongation of the polyketide chain through C-C bond formation. Thioesterase (TE) domain aids in the termination of the elongating chain. PKS systems are of three types. Type I PKSs consist of multimodular, multifunctional enzymes in which each module comprises of a set of non-iterative domains that carry out the catalysis of one cycle of chain elongation (Bisang et al. 1999). Type II PKSs are monofunctional iterative enzymes (Hertweck et al. 2007). Type III PKSs consist of homodimeric, iterative enzymes with several ACP-independent modules (GitaBangera and Thomashow 1999). Similar to polyketides, oligopeptide antibiotics are synthesized by large enzyme complex called non-ribosomal peptide synthetases (NRPSs) with multiple modules. Each module comprises of adenylation domain with catalytic units for amino acid selection, peptidyl carrier protein for activation and condensation domain for bond formation of the growing peptide chain. NRPSs are classified into three types based on the catalytic potential. Linear (type A) NRPSs are multimodular enzymes organized in a sequential manner, and individual module adds single monomer to the elongating chain. Iterative (type B) NRPSs consist of modules that are utilized repeatedly in the building of a single multimeric peptide. Nonlinear (type C) NRPSs comprise a complex system with nonconventional organization forming unique structures and synthesizing nonlinear peptides (Mootz et al. 2002). Many researchers have indicated a strong correlation of biosynthetic gene clusters and bioactivity in bioprospection studies. Thus, genetic and bioactivity screening method provides a streamlined approach for drug mining.

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Bioactive Compounds from Orchid-Associated Fungal Endophytes

Studies on the phytochemical and pharmacological properties of extracts and metabolites of different genera of orchids have reported important drugs derived from orchids. Researchers have reported the isolation of phytochemicals such as alkaloids, terpenoids and flavonoids from different orchid species (Zhao et al. 2003). Owing to the fact that endophytic fungi are capable of producing bioactive compounds similar to their host compounds, exploration of medicinal orchidassociated endophytic fungi for its numerous applications in medicine has been validated by scientists. Researchers have studied the crude extracts of fungal mycelia of orchid endophytes and have noticed the presence of bioactive molecules accountable for the bioactive compounds in orchids (Zhu et al. 2018). Penicillium chrysogenum isolated from the orchid Cymbidium aloifolium produces hydroxamate-type siderophore which has strong antibacterial activity against major phytopathogens of groundnut and rice (Chowdappa et al. 2020). Fusarium oxysporum isolated from Dendrobium lindleyi produced several bioactive compounds such as indole acetic acid that are majorly engaged in host defence systems against phytopathogens, gibepyrone A with antimicrobial activity and 2-coumaranone which has antioxidant properties and pyrrolo(1,2-a)pyrazine-1,4dione and hexahydro-3-(2-methylpropyl) which have cytotoxic effects on cancer cell lines (Bungtongdee et al. 2019). In a study, the composition of the crude extract of fungal endophytes from Thai orchids was analysed employing phytochemical methods and GC-MS (gas chromatography-mass spectrometry). Investigation of the fungal extracts unfolded the presence of gibepyrone A and indole acetic acid that acts as plant elicitors and activates plant defence mechanisms against pathogens. The fungal extracts also revealed the presence of an antioxidant, 2-coumaranone. Therefore, endophytes isolated from the orchids are reservoir of novel drugs for pharmaceutical utility (Bungtongdee et al. 2019). There is very scanty exploration of orchid-associated fungal endophytes as storehouse of bioactive compounds. Several works reported on endophytic fungi from medicinal orchids is limited to experimental studies. Additional attention should be put into bioprospection and scale-up. This can be achieved through relentless advancement in the screening procedure of bioactive metabolites and their structure elucidation so as to understand their bioactivities and facilitate production of novel drugs.

11.10 Fermentation Methods for Secondary Metabolite Production Endophytic fungi have great potential to produce several bioactive molecules. Therefore, it is necessary to device a feasible and economical production procedure for their commercial application. These fungi can be grown using different culture techniques and growth conditions. It is important to design a desirable cultivation

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system with optimum temperature, pH, aeration and medium composition to achieve best mycelial development and metabolite production (Kumar and Kaushik 2013). Fermentation techniques such as submerged (liquid) fermentation and solid-state fermentation are chiefly employed for fungal mass cultivation. These cultivation techniques are economical and environment friendly. Submerged or liquid fermentation has gained tremendous utility for the production of biomedicinal products. Luxuriant mycelial growth in short period and active metabolite synthesis are the advantages of submerged batch fermentation. Temperature beyond or below the optimum level has reflective effect on fungal growth and is responsible for reduced metabolite production. Effect of pH on the mycelium development and metabolite concentration has been studied on endophytic fungi Penicillium microspore and shows varied trend with increase in pH of the growth medium. Steady pH is maintained by balancing the ratio of carbon and nitrogen supply (Yuniati et al. 2018). In solid-state fermentation, the key advantages comprise of decreased catabolite repression and higher metabolite production. This method needs simpler downstream processing steps and has been reported for production of secondary metabolites using endophytic fungi (Su et al. 2014). Conditions of the culture fermentation can alter the gene expression level and subsequently production rate of secondary metabolites (Iwashita 2002). Different culture methods like liquid culture, agar plate and shake flasks, applied for the growth of A. oryzae, show dissimilarity in growth and production pattern of proteins. Gene transcriptional profiles by DNA microarray studies revealed that liquid culture and agar plate demonstrated to some degree similar pattern; however, the shake flask profile was relatively dissimilar. Enzyme activities were considerably elevated in liquid culture and agar plate compared to shake flask culture (Imanaka et al. 2010).

11.11 Strategies for Improved Production of Secondary Metabolites Although the prospective of fungal endophytes as rich source of biomedicinal products is tremendous, improvement of yield and productivity is required in order to cater on an industrial scale. Approaches for enhanced and sustainable production of natural products (Fig. 11.2) are discussed below.

11.11.1 Strain Improvement Strain improvement technique basically involves induction of mutation and selection of the fungal strain with the desired characteristics. Mutations through different agents induce changes in the genetic level, resulting alteration in regulatory genes and increased secondary metabolite production (Adrio and Demain 2006). Protoplast fusion is another widely used recombination method in endophytic fungi for

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Endophytic Fungi

Screening Studies

Strain with desired trait

Strains with axenic instability

Stable yielding strain

● Co-culture with other host endophytes ● Epigenetic modifiers

Strain improvement ●Mutant selection ● Protoplast fusion

Bioprocess optimization and scale-up Fig. 11.2 A strategic approach for improved production of secondary metabolites from endophytic fungi

enhancement of productivity. This method overcomes the common problem of genetic separation in progenies and optimization of mutagenesis conditions (Kai et al. 2009). Most of the works done on mutagenesis of fungal endophytes are related to taxol-producing endophytes. Protoplast fusion technique was used for two strains of Nodulisporium sylviforme to develop strain HDF-68 with increased taxol production compared to the parent strains (Zhao et al. 2013a, 2013b).

11.11.2 Bioprocess Optimization Strategies for providing optimum culture conditions are essential for enhancing important and high-value fungal compounds through fermentation technology. Enhancement strategies like optimization of medium composition and culture conditions like pH, temperature and aeration have been adopted for endophytic fungi to sustain high yield and productivity of secondary metabolites for industrial scale-up. One of the latest approaches in microbial fermentation technology is ‘one strain many compounds’ (OSMAC) method in which one microbial strain can be subjected to varying fermentation parameters to trigger the production of several compounds (Bode et al. 2002). This procedure taps the potential of endophytic fungi to biosynthesize multiple compounds through variations in cultivation conditions to

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elicit the silent genes and produce multiple and sometimes even novel metabolites (Bode et al. 2002). The method of using elicitors is an effective strategy to induce the production of secondary metabolites. The main advantage of this approach is that it is cost effective. These elicitors are signalling molecules and work effectively in endophytic fungal system where host signalling molecules actually play a significant role in secondary metabolite production. Addition of ethanol in Colletotrichum gloeosporioides culture medium enhances camptothecin production (Zhao et al. 2013a, 2013b). Improved huperzine A production has also been documented by ethanol and methanol addition in Fusarium solani suspension culture (Venugopalan and Srivastava 2015). Apart from elicitors, it is also reported that inhibitors can be used to divert the metabolite biosynthesis towards the desired product. Application of sterol biosynthesis inhibitors to reroute the flux of geranyl-geranyl pyrophosphate towards taxol biosynthesis has been reported in taxol-producing endophytes with about 50-fold increase in the taxol yield (Li et al. 1998). Precursor feeding is another method of enhancing the yield of selective products by adding precursor molecules or intermediates of the biosynthetic pathways to the growth medium. However, addition of exogenous molecules into the culture medium requires the consideration of permissible range of concentration which otherwise will have toxic effect on the culture growth (Mulabagal and Tsay 2004).

11.11.3 Improvement of Strains with Axenic Instability Although strain improvement and bioprocess optimization have helped tremendously on scaling up of endophytic fungal bioproducts, but often endophytic fungi in axenic culture exhibit genomic instability. Also, the presence of cryptic biosynthetic genes in endophytic fungi limits the biosynthetic potential of fungal strain in axenic culture. It has been reported through whole genome sequencing analysis that microbial genome contains cryptic biosynthetic gene clusters (Hertweck 2009). Silencing of the biosynthetic genes often occurs in the microbial strain in the standard culture conditions which are different from the microenvironment in the host plant (Brakhage et al. 2008). It is one of the obstructions in bioprospecting the full potential of new endophytic fungal isolates). Co-cultivation is a system which mimics the natural habitat of the endophytes for novel product isolation and as a yield enhancement strategy. The inoculum of co-cultured species should be such that all species can co-exist in the presence of optimum culture conditions and different growth morphologies should accommodate in the cultivation system (Weathers et al. 2010). Co-culture technique maximizes the yield of the desired product by one of the fungal endophytes. It is due to the co-existence of the cultures that lead to production and accumulation of novel compounds which otherwise are not produced (Marmann et al. 2014). Targeting the silent biosynthetic gene clusters is a new strategy for discovery of novel metabolite either through co-culture technique or epigenetic modifier addition (Scherlach and Hertweck 2009). When fungal biosynthetic gene clusters are present

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Table 11.2 Strategies for improved product yield and novel products from endophytic fungi Product Taxol

Strain Fusarium maire K178

Tanshinone IIA

Emericella foeniculicola NU152 Nodulisporium sylviforme UV40–19

Taxol

Optimization parameter Exposure to UV + diethyl sulphate Exposure to UV + NaNO2

Effect Yield enhanced by 8.6-fold

Reference Xu et al. (2006)

Yield increased by 1.46-fold

Ma et al. (2011)

pH, temperature, agitation rate and fermentation period optimized Carbon source, temperature, pH, agitation rate and fermentation period optimized Yeast extract as elicitor

Yield increased by 1.15-fold

Zhao et al. (2011b, 2011a)

Yield increased by 28.58%

Zhao et al. (2013a, 2013b)

3.2-fold yield increased

Zhao et al. (2011b, 2011a) Zhao et al. (2013a, 2013b) Li et al. (1998)

Huperzine

Colletotrichum gloeosporioides ES026

Palmarumycin C13

Berkleasmium sp. Dzf12

Huperzine

Colletotrichum gloeosporioides ES026 Pestalotiopsis microspora Ne32 Entrophospora infrequens RJMEF001 Fusarium tricinctum Acremonium sp.

Ethanol as elicitor

Yield increased by 51.89%

Sodium benzoate fed as precursor

Increased yield

Tryptophan fed as precursor

Yield increased

Amna et al. (2012)

Co-cultured with Bacillus subtilis Co-cultured with Mycogone rosea

Leucostoma persoonii

Sodium butyrate (HDAC inhibitor) as epigenetic modifier

1.4-fold yield increase Novel lipoaminopeptide produced Novel cytosporones R produced and cytosporones B yield increased

Ola et al. (2013) Degenkolb et al. (2002) Beau et al. (2012)

Taxol

Camptothecin

Lateropyrone Acremostatins A, B and C Cytosporone B

in heterochromatin state, they are often regulated by epigenetic modifiers to modify their chromatin state. Histone acetylation/deacetylation and DNA methylation/ demethylation in the chromatin induce the chromatin remodelling and regulation of the gene expression (Pettit 2010). Application of epigenetic modifiers is an important strategy to modulate the expression of secondary metabolite genes and produce important bioactive compounds from endophytic fungi (Brakhage and Schroeckh 2011) (Table 11.2).

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11.12 Conclusion and Future Aspects Endophytic fungi have a pivotal role on the growth, fitness and ecology of orchids. Since the population structure and distribution pattern of endophytic fungi extensively depend on the ecological environment and genotype of orchids, information on the diverse endophytic fungi under specific environmental conditions can provide important insights into their existence and variation. Endophytic fungi assist orchids by enhancing their fitness and stress tolerance capacity and accumulating plethora of bioactive compounds originally produced by the medicinal orchids. Endophytes with plant growth-promoting and stress-tolerant capabilities find application in agriculture. The most promising utility is to employ the benefits of endophytic fungi that can produce bioactive compounds for sustainable and higher-quality natural medicine with large-scale application. Moreover, taking into account the different strategies of yield enhancement present that can be customized with endophytic fungi, the prospects of endophytic fungi study for valuable bioactive compound production appear highly promising. However, a great deal of work described on endophytic fungi from medicinal orchids is limited to experimental studies, and extra attention should be set into bioprospection, field trails and scale-up to procure valuable therapeutic compounds. Also, the intricate role-play of both the partners in molecular signalling and interaction is still not completely defined. Thorough knowledge in overall aspect of the host-endophyte interaction will provide a wider understanding of the association which can further improve the potential of the endophytes in playing a key role in drug discovery and clinical utility. Acknowledgement The authors acknowledge the financial assistance provided to the parent department through UGC-SAP [F.4-7/2016/DRS-1 (SAP-II)] and DST-FIST [SR/FST/LSI-666/ 2016(C)] programmes. Bishal Pun acknowledges the Department of Biotechnology (DBT), Ministry of Science and Technology and Government of India [DBT/2020/NEHU/1333] for the financial support.

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Nutraceuticals: Advancement in Microbial Production and Biomedical Prospects

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Dixita Chettri, Manswama Boro, Shahil Ansari, and Anil Kumar Verma

Abstract

Nutraceutical is an emerging concept of foods with nutritional and medicinal benefits that have formed a unique niche in the food market and represent a rapidly growing industry. They are credited with various health benefits, such as antiaging, anticancer, antidiabetes, and other therapeutic properties and include various foods from natural to biofortified plants. Nutraceuticals are classified according to their origin and composition and can be derived from various natural sources such as plants and microbes. The diverse group of microbes from different habitats is proving to be a rich source of nutraceuticals, and research interest in identifying novel organisms and metabolites with nutraceutical properties is growing. Further technological advances have enabled the production of nutraceuticals on a large scale by overcoming the major hurdles in the production and extraction of these metabolites from natural sources. Keywords

Biomedicine · Metabolites · Microbes · Nutraceuticals · Therapeutics

Abbreviation CVDs DNA FIM

Cardiovascular diseases Deoxyribonucleic acid Foundation for innovation in medicine

D. Chettri · M. Boro · S. Ansari · A. K. Verma (✉) Department of Microbiology, Sikkim University, Gangtok, Sikkim, India e-mail: [email protected]; [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_12

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GMOs GRAS MUFA PUFAs SCFAs UN

12.1

D. Chettri et al.

Genetically modified organisms Generally recognized as safe Monounsaturated fatty acid Polyunsaturated fatty acids Short-chain fatty acids United Nations

Introduction

Since the beginning of the industrial revolution, meeting the food and nutritional needs of the exponentially growing world’s population has been a major problem. Although food security is second only to poverty on the top 17 priority list of the Sustainable Development Goals of UN, there has been a steady increase in the global hunger index and malnutrition (Iriti et al. 2022). According to the World Food Program report, the number of people living under food insecurity has increased from 135 million to 345 million since 2019, with 49 million at risk of famine. Meeting nutritional needs is another challenge as reports of growth disorders and obesity in children under 5 years of age are increasing (Mensi and Udenigwe 2021). It is estimated that the current demand for agricultural products will increase by up to 60% by 2030. While the use of agrochemicals and modern technologies could increase overall food production, they have serious environmental impacts. In addition, these chemicals degrade natural soil quality with each application, so dosages must be increased. Increasing consumer awareness of the potentially harmful effects of crops produced with these agrochemicals has led to ethical concerns about their consumption. All of these reasons have led to changes in food and farming practices. At the level of farming systems, the use of bio-based products in the form of biofertilizers and biological pesticides is gaining new momentum as a sustainable approach (Nosheen et al. 2021). Sustainable agriculture seeks to solve the first problem associated with world hunger, which is food security while maintaining the integrity of food and the environment. This practice of sustainable agriculture is still lagging in competing with the conventional method and competing for cultivable land, energy, and water resources with other purposes (Tripathi et al. 2019). In addition, modern lifestyles and unhealthy eating habits have a great impact on public health, leading to diseases such as obesity, diabetes, cardiovascular disease, etc. Therefore, there are more and more alternative food sources with high nutritional value and medicinal effects, which can contribute to food security while improving the overall health of the population (Popa-Wagner et al. 2020). Among the most widely used functional foods are fermented foods, which have been consumed for decades in various traditions around the world; probiotics, which contain microbes with health-promoting properties; and prebiotics, which promote the growth and multiplication of probiotics in the host and thus have beneficial effects. These

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functional foods are included in the food matrix and boost host immunity but do not have therapeutic properties to treat disease. Another term in this context is nutraceuticals, which, unlike functional foods, are in capsule form and also serve as drugs to treat various diseases (Domínguez Díaz et al. 2020), although various functional foods also have nutraceutical properties (El Sohaimy 2012). In this chapter, nutraceuticals are discussed in terms of their definition, classification, use as foods and drugs, and advances in their production.

12.2

Nutraceuticals

Nutraceutical was coined by Stephen DeFelice, MD, a founder and chairman of the Foundation for Innovation in Medicine (FIM), Cranford, New Jersey, USA, which is a portmanteau of the two words “nutrient” and “pharmaceutical.” He defined nutraceuticals as “foods or parts of foods that have a health benefit and are used to inhibit or treat a disease” (Nwosu and Ubaoji 2020). Although there is no legal definition, a nutraceutical can be defined as a broad range of products that may be foods or food extracts that are nontoxic and assist in the prevention and treatment of disease. There are already over 470 nutraceutical and functional foods on the market with proven health benefits (Maurya et al. 2022). A prerequisite for a substance to be a nutraceutical is that it serves some form of disease prevention or treatment. In recent years, rapid changes in people’s lifestyles and living standards have had a direct impact on their health and physiology. This has led to an increasing incidence of health problems such as dementia, malnutrition, and other degenerative diseases. Many people are reluctant to use conventional drugs due to their high cost and side effects. Due to their assured safety and profound health benefits, many people prefer nutraceuticals to conventional drugs. Apart from lifestyle disorders, nutraceuticals have also been proven effective for a number of conditions such as inflammation, immunodeficiency, allergies, arthritis, malignancies, digestive disorders, depression, sleep disorders, hypertension and cholesterol level control (Venkatakrishnan et al. 2020). The term “nutraceuticals” is not well known worldwide and is usually replaced by the term “dietary supplements” to comply with regulations. However, there is a tiny difference between the two terms, as nutraceuticals should always help prevent or treat disease, not simply replacing a specific food. Currently, nutraceuticals are available as whole food or concentrated in the form of pills, capsules, powders, and tinctures either as a single product or in combination.

12.2.1 Classification Though nutraceuticals can be classified in numerous ways, such as their sources, chemical nature, or the targeted mode of action against the diseases, the simplest and commonly followed classification is that based on their source, that is, natural or

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traditional nutraceuticals and unnatural or nontraditional nutraceuticals (Chávarri 2020).

12.2.1.1 Traditional or Natural Nutraceutical The nutraceuticals that fall into this category are those that contain natural bioactive compounds and provide health benefits without any modification (Nwosu and Ubaoji 2020). They contain ingredients that provide more benefits than the normal diet. Examples include omega-3 fatty acids, lycopene, and saponin found in salmon, tomatoes, and soy, respectively. Traditional nutraceuticals can be classified further into chemical ingredients, nutraceutical enzymes, and probiotic microorganisms. Chemical Ingredients The chemical ingredients imparting the nutraceutical properties are further divided into nutrients, herbals, and phytochemicals (Ali et al. 2019). Nutrients

Nutrients are substances derived from plants or animals that can be used by humans for nutrition and growth. Nutrients can be used directly by the body as precursors or intermediates in various metabolic pathways and provide energy or stability to the body. They are not obtained chemically but are contained in the food itself in the form of amino acids, fats, minerals or vitamins, etc. (Nwosu and Ubaoji 2020). A number of common diseases such as cataracts, cardiovascular disease, osteoporosis, cancer, and diabetes can be curbed with the nutrients found in naturally occurring food such as whole grains, vegetables, fruits, and animal products such as meat, dairy, and poultry. Other deficiency diseases such as osteoporosis and anemia can be improved by plant-based minerals. Flaxseeds and omega-3 PUFAs (polyunsaturated fatty acids) from salmon have been shown to reduce cholesterol and inflammatory processes and maintain and improve brain function. Herbals

Herbal foods include all plant parts that have nutraceutical properties and thus contribute to the prevention or curing of various chronic and acute diseases. There are many unique bioactive compounds in these herbs that have excellent benefits. Bioactive compounds that fall into this category include flavonoids, terpenoids, saponins, and polyphenols (Corzo et al. 2020). Parsley (Petroselinum crispum), for example, used mainly in European and Middle Eastern cuisine, contains flavonoids (apiol, psoralen) and has carminative, diuretic, and antipyretic properties. Another example is willow bark (Salix nigra), which has analgesic, antiinflammatory, antipyretic, anti-astringent, and anti-arthritic effects due to its active component salicin. Peppermint (Mentha piperita) is often used to treat colds and flu due to its menthol content. The presence of tannin in Lavender (Lavandula angustifolia) confers its nutraceutical value for the treatment of colds, asthma, and cough along with depression, stress, and high blood pressure. Cranberries (Vaccinium erythrocarpum) contain proanthocyanidin, which may be helpful for cancer, urinary tract infections, and ulcers (Bobis et al. 2020).

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Phytochemicals

Phytochemicals are plant nutrients that have a specific biological activity that is helpful for human health. They can be enzymes or cofactors themselves or modify some other biological metabolisms. They mainly include carotenoids, phenolic acids, and flavonoids. Carotenoids such as β-carotene, zeaxanthin, and lycopene, found in foods such as berseem, oat corn, eggs, citrus fruits, tomatoes, etc., help neutralize free radicals, promote health, and fight prostate cancer. Phenolic acids are mainly found in citrus fruits, red wine, etc. as caffeic acid or hydroxycinnamic acid. They promote health as they are antioxidants and have anticancer properties (Caponio et al. 2022). Flavonoids include various compounds such as proanthocyanidin, anthocyanidin, and catechin. They can reduce cardiovascular disease, are antioxidants, and reduce cancer risk. They are found in foods and food products such as coca, tea, fruits, babool pods, mustard cake, etc. Other examples of phytochemicals are the seeds of Barbarea verna and broccoli, containing isothiocyanates (glucosinolates) and possessing antitumor activity (Chanda et al. 2019). Nutraceutical Enzymes Enzymes play a very important role in an organism and sustain life by enabling various metabolic processes. Enzymes can sometimes be obtained directly from plant, animal, or microbial sources to provide health benefits. Further, they can be used for the treatment of various diseases such as gastroesophageal reflux disease, constipation, diarrhea, ulcerative colitis, and other common gastrointestinal disorders (Jha et al. 2021). Nowadays, enzyme therapies are widely used to treat various rare diseases such as Gaucher disease, Pome disease, and Fabry disease. Nutraceutical enzymes could be a potential therapeutic intervention for a patient suffering from diabetes (Nwosu and Ubaoji 2020). Probiotic Microorganisms Probiotics are microorganisms that, when consumed in sufficient quantities, provide health benefits to humans. They do not cause any disease in the host organisms and stimulate their immune system via promotion of proper digestion and absorption of certain nutrients. Probiotics are usually available as powders, liquids, gels, pastes, or fermented foods fortified with probiotics (Gu et al. 2022). Probiotics work by competing with pathogenic bacteria and inhibiting their growth. They help in the production of vitamins, especially vitamin K, and in the absorption of some nutrients. They ensure proper functioning and enhances the overall immune system response to various disorders. Probiotics have various mechanisms of action: they compete with pathogenic bacteria for the adhesion site on the intestinal epithelium, compete with them for space and food, can produce various antimicrobial substances, and alleviate the postinfection condition caused by pathogenic bacteria. Probiotics contain a wide range of bacteria such as Lactobacillus sp. (L. plantarum, L. acidophilus, L. casei subspecies rhamnosus, L. delbrueckii subspecies bulgaricus, L. brevis), Bifidobacteria sp. (B. bifidum, B. infantis, B. adolescentis, B. longum, and B. breve), Streptococcus thermophilus, etc. (Rajasekaran 2017). One of the best

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known is Bacillus vulgaris, often found in Bulgarian yogurt, which is regularly consumed by local farmers. Elie Metchnikoff (Nobel Prize winner, Institut Pasteur, Paris) postulated that farmers who consume this yogurt live longer than farmers who do not because of the probiotics contained in the yogurt (Maurya et al. 2022). Other probiotics such as Lactobacillus bulgaricus could have an antimicrobial effect against various harmful bacteria such as E. coli, Salmonella, Clostridium, Shigella, and Helicobacter pylori by producing lactic acid in the intestine and degrading β-glucuronidase. Treatment of chronic gastritis (leading to gastric ulcers) caused by H. pylori involves expensive therapeutic measures with potential side effects with probiotic-based nutraceuticals providing a cost-effective alternative (Rajasekaran 2017).

12.2.1.2 Nonnatural or Nontraditional Nutraceuticals Nontraditional nutraceuticals are artificial foods developed by biotechnological means or conventional breeding techniques or foods fortified with nutrients. The bioactive compounds they contain are manipulated to create foods that promote health. Unnatural nutraceuticals are divided into two types. Enriched/Fortified Nutraceuticals Enriched nutraceuticals include foods with fortified nutritional values obtained through traditional plant breeding techniques. They also include foods to which important minor ingredients have been added to improve the overall nutrient content of the food (Lachance 2000). Some examples include mustard oil fortified with vitamin A, minerals or vitamins added to cereals, flour with added folic acid, and orange juice fortified with calcium. Cholecalciferol is used to prepare fortified milk which has been found to be effective to treat vitamin D deficiency (Jha et al. 2021), while the soy ferritin gene-fortified banana can be used for iron deficiency. Recombinant Nutraceuticals Recombinant nutraceuticals include foods produced using recombinant DNA technology methods. Recombinant nutraceuticals include genetic engineering to obtain the desired product such as an enzyme or other biologically active compounds or the use of fermentation to obtain such products. The main foods that fall into this category are high-energy food, for instance, bread, cheese, yogurt, fermented starch, alcohol, etc. (Ayushree et al. 2022). They also include plants, animals, or microbes whose genes have been modified to produce an active ingredient used to treat certain deficiency diseases. A classic example is golden rice, which contains genes for betacarotene and is used primarily to treat people with vitamin A deficiency. Another example is a recombinant E. coli K-12 strain that produces the enzyme chymosin and is derived from coagulated milk.

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12.2.2 Biomedical Application As their definition suggests, nutraceuticals have various medical benefits that help improve the overall health of human. Some of the major diseases where nutraceuticals have been found to have beneficial effects have been discussed below.

12.2.2.1 Cardiovascular Diseases (CVDs) Cardiovascular diseases that include coronary heart disease, deep vein thrombosis, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, and pulmonary embolism are the major causes of death worldwide. It has long been known that minimal consumption of fruits and vegetables is associated with high CVD mortality. It is believed that about 50% of cardiovascular diseases can be prevented by timely screening and healthy eating. Dietary supplements containing vitamins, antioxidants, omega-3 fatty acids, fiber, and minerals are recommended along with physical activity to treat CVD. Ginger is an excellent antioxidant that has anti-inflammatory effects and has long been recommended for the prevention of high blood pressure and heart palpitations. Polyunsaturated fatty acids or PUFAs (e.g., α-linolenic acid) and monounsaturated fatty acids or MUFA, found in vegetables, salmon, flaxseed, nuts, and others, have been shown to be helpful in preventing coronary heart disease (Rajasekaran 2017). Consumption of flavonoids has been shown to be helpful in treating cardiovascular disease. They are mainly found in onions, black grapes, cruciferous vegetables, grapefruits, apples, cherries, berries, and others. The anthocyanins and tannins (proanthocyanidin) contained in grapes regulate metabolism in the body and help reduce the harmful effects of arterial disease (Ayushree et al. 2022). Omega-3 fatty acids contained in fish lower lipid and bad cholesterol levels and are therefore used as a dietary supplement to treat cardiac arrhythmias. Essential fatty acids help regulate blood pressure and lower triglycerides and cholesterol. 12.2.2.2 Cancer Cancer treatments nowadays are usually associated with chemotherapy, radiation, and surgery. However, these treatments are harsh in nature and can have long-lasting side effects. Nutraceuticals can therefore be an excellent alternative to these cancerfighting measures, potentially helping to prevent cancer in the first place (Chettri et al. 2022). They are inexpensive and have few side effects. Cancer prevention is primarily related to a healthy diet and lifestyle. Herbs have secondary plant compounds that have anticarcinogenic and antimutagenic properties, which have been shown to be helpful in preventing prostate and breast cancer (Jha et al. 2021). The property of carotenoids is that they are a powerful antioxidant; lycopene minimizes oxidative stress and helps in the treatment of cancer. Lycopene is found primarily in foods such as tomatoes, watermelon, pink grapefruit, guava, and papaya. Flavonoids from citrus fruits with antioxidant effects are also useful for cancer prevention. Lutein, a compound found in chicken eggs, spinach, tomatoes, and oranges, is very useful in preventing colon cancer. Foods that contain ginseng have

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anti-inflammatory properties and therefore may be useful in suppressing cancer that results from inflammation (Ayushree et al. 2022). Other nutraceutical substances such as saponins and tannins also have antitumor and anticancer properties. The risk of colon and lung cancer can be reduced by eating cruciferous vegetables. They act by inhibiting enzymes that stimulate tumor development. Β-carotene and pectin work against prostate cancer because they can effectively reduce free radicals. Garlic contains sulfur components that stimulate the immune system while reducing atherosclerosis and platelet aggregation. The biotransformed products of glucosinolates have been shown to be helpful in the treatment of various cancers such as colon, lung, breast, and liver cancers.

12.2.2.3 Diabetes High abnormal blood glucose (diabetes mellitus) is a common disease worldwide and has caused great concern as the number of patients is rapidly increasing every year, possibly due to changes in lifestyle and diet. Diabetes has two different types: type 1, which affects about 5% of the population and is an autoimmune disease, and type 2, which affects about 95% of people and is associated with obesity (Ayushree et al. 2022). In addition to pharmaceutical therapeutics, nutraceuticals have a crucial role in the prevention and treatment of diabetes. Nutraceuticals such as herbs and other products have been shown to help alleviate the symptoms of diabetes in patients. Isoflavones are phytoestrogens that are used in the treatment of diabetes type 2 and have successfully shown a decrease in mortality rates. Isoflavones are mainly found in soy, so regular consumption helps against diabetes. In addition, omega-3 fatty acids from salmon have been shown to normalize blood sugar levels by improving insulin sensitivity and reducing glucose tolerance, thereby decreasing mortality and diabetes prevalence (Jain et al. 2022). Other herbs and foods such as cinnamon, garlic, onion, ginseng, mushrooms such as Pleurotus ostreatus, bitter melon, and Gymnema sylvestre have been shown to counteract diabetes mellitus. They may act through several mechanisms, including decreased glucose uptake from the small intestine while lowering its production in the liver, increased secretion of insulin, and increased glucose uptake in peripheral tissues. Psidium guajava (guava) leaf extracts have been shown to prevent protein glycation and thus protect against the complications of diabetes. Lipoic acid is a dithiol molecule that acts as a crucial cofactor for mitochondrial enzymes associated with bioenergetics and is also a universally potent antioxidant that is now being used in Germany to treat diabetic neuropathy. The addition of lipoic acid as an essential dietary supplement in diabetic patients can protect them from the complications of the disease. High-fiber cherries have an antidiabetic effect primarily because of their antioxidant properties and their content of compounds such as anthocyanins and quercetin. High-fiber foods alleviate diabetes-associated symptoms by increasing the binding of insulin receptors and decreasing the absorption of glucose from the blood, thereby maintaining sugar levels in the body (Rajasekaran 2017). Other herbs such as fenugreek contain 4-hydroxyisoleucine and galactomannan and also the high fiber content help to lower blood sugar levels by decreasing the rate of digestion and absorption of

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carbohydrates from the intestines. They also increase insulin production and slow down blood sugar levels.

12.2.2.4 Obesity Obesity is an excessive deposition of unwanted fats in the body due to the consumption of high-fat, high-energy foods. Obesity can lead to various complications such as angina, hyperlipidemia, congestive heart failure, respiratory diseases, hypertension, renal vein thrombosis, cancer, decreased fertility, osteoarthritis, etc. (Rajasekaran 2017). Obesity is also a consequence of the increasing sedentary lifestyle. Therefore, a proper low-calorie diet and regular exercise are helpful in preventing obesity and also alleviating its symptoms. Nutraceuticals such as linoleic acid, psyllium fiber, and capsaicin have shown promising effects against obesity. Other substances such as ephedrine, fenugreek, and vitamin C have shown potent effects against obesity. Herbs such as caffeine, chitosan, ma huang-guarana, green tea, and ephedrine help to reduce a person’s weight (Jain et al. 2022). They increase metabolism in the body, thereby reducing fat deposition. The presence of dietary fiber is important in reducing body weight, fat, and the total mass. Dietary fibers such as glucomannan (natural gel-forming fiber) decrease the bioavailability of nutrients. They reduce the food caloric density by physically diluting and trapping nutrients, which further lowers digestion and absorption (bioavailability) of nutrients such as glucose and lipids. The soluble fibers form viscous, hydrated masses that lower appetite and prolong the absorption time of nutrients in the small intestine and also downregulate appetite-stimulating hormones. The proteins in buckwheat seeds also act similarly to fiber. A diet enriched with omega-3 fatty acids has been shown to improve obesity by modulating expression of lipogenic gene and increasing metabolism. Phytochemicals such as curcumin (diferuloylmethane) are beneficial in alleviating obesity-related inflammation and metabolic complications and promoting weight loss. Foods containing oolong tea (catechins), soybeans, garlic (organosulfur compounds), green tea (organosulfur compounds), psyllium (soluble fiber), fortified margarine (plant sterol and stanol esters), etc. have been shown to be beneficial for people suffering from obesity. They act to reduce body fat through various mechanisms, such as inhibiting pancreatic lipase, increasing thermogenesis, stimulating fat metabolism, and limiting adipocyte differentiation (Rajasekaran 2017).

12.3

Microbes in Nutraceutical Production

Nutraceuticals are found in all living organisms, from plants and animals to microbes. However, they are naturally present in very small amounts in these organisms, and their extraction from plant and animal sources is extremely complicated. The microbes with simple growth requirements and short generation time can be easily used for the industrial production of nutraceuticals.

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12.3.1 Sources of Nutraceuticals Nutraceuticals produced by various bacteria, fungi, and microalgae have been used knowingly or unknowingly by people around the world since ancient times. Certain bacteria have traditionally been consumed as probiotics, while various macro fungi have been used as traditional medicines for centuries. With the advancement of science, scientists have reported the production of nutraceuticals by these organisms that have multiple health benefits.

12.3.1.1 Microalgae as a Source of Nutraceuticals Microalgae are photosynthetic eukaryotic microorganisms that live in various freshwater and saltwater habitats. The cultivation of microalgae has gained much interest in recent years as a source of nutraceuticals. Several high-value bioactive metabolites such as beta-carotene, carotenoids, polysaccharides, omega-3 fatty acids, vitamins, polyphenols, and proteins from microalgae can be used as nutritional additives in food and feed products that improve human and animal health (Udayan et al. 2022). Microalgae are also known to treat deficiencies and health problems in humans. Tetraselmis, Chaetoceros, Spirulina, Chlorella, Nannochloropsis, Crypthecodinium, Dunaliella, etc. are some of the commonly known microalgae that produce nutraceuticals and are a rich source of micro- and macronutrients (Udayan et al. 2022). Astaxanthin is a widely known nutraceutical that has antioxidant and anti-inflammatory activity and is commercially produced by the microalgae Haematococcus pluvialis (Bjørklund et al. 2022). Common microalgae such as cyanobacteria are also known to produce phycobiliproteins, which are used as nutraceuticals (Manirafasha et al. 2020). Although microalgae have gained popularity as a source of nutraceuticals, there are some of the major limitations faced by the industry, such as identification and irradiation of natural toxins, low biomass yield, and high cost of downstream processing (Udayan et al. 2022). 12.3.1.2 Bacteria as a Source of Nutraceuticals Various nonpathogenic bacteria are commonly consumed as living organisms in the diet due to their beneficial properties for human health. These bacteria are commonly referred to as probiotics. Probiotics produce peptides, amino acids, bacteriocins, vitamins, short-chain fatty acids (SCFAs), and various other metabolites (Kumar et al. 2022), which help to improve intestinal dysbiosis, maintain the immune system, regulate metabolic disorders and associated diseases such as diabetes and obesity, and improve nutrient absorption in the gut (Boro et al. 2022). Bacteria such as Bacillus subtilis and Lactobacillus in the form of probiotics are commonly consumed to improve human metabolism (Al-Obaidi et al. 2021). Some reports of bacteria-producing compounds with nutraceutical properties are listed in Table 12.1. Escherichia coli, Lactococcus, and many other bacteria are often genetically modified to produce polyphenolic compounds (e.g., apigenin, eriodictyol, catechin, naringenin, etc.), alkaloids (e.g., (S)-reticulin, strictosidine, and indolyl glucosinolate), terpenoids (e.g., lycopene, β-carotene, zeaxanthin, and astaxanthin),

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Table 12.1 Bacterial bioactive compounds with nutraceutical properties Bacteria Lactobacillus uvarum LUHS245 and L. casei LUHS210 LAB, Lactobacillus, and Bifidobacterium

Lactobacillus acidophilus LA-5

Food source Oral ingestion of tablets

Nutraceutical In vivo fourcomponent nutraceutical

Benefits to health Immunomodulation and probiotic properties in pigs

Reference Grigas et al. (2021)

Fermented food products like yogurt, cheese, kimchi, and beverages Milk permeate and cheese whey

Bioactive compounds and metabolites

Numerous health benefits including immunomodulation, improved metabolism, etc.

CuvasLimon et al. (2021)

Linoleic acid (CLA), exopolysaccharides (EPSs), and bacteriocins (BACs) Alkaloids (guanosine and inosine)

Health-promoting effects like improved digestion, antidiabetic, etc.

Amiri et al. (2021)

Antianxiety, anticancer, and antiinflammation

Liu et al. (2015)

Anti-inflammatory and immunomodulatory activity ACE inhibitory and immunomodulatory activity

Shakya et al. (2021)

Endophytic Bacillus cereus, Aranicola proteolyticus, Serratia liquefaciens, Bacillus thuringiensis, and Bacillus licheniformis Lactobacillus brevis 174A

Medicinal plant – Pinellia ternata

Fermented Paeoniae radix

Pyrogallol

Lactobacillus helveticus R0389 and Lacticaseibacillus rhamnosus R0011

Fermented milk products

Peptides

Adams et al. (2020)

prebiotics (e.g., 2-fucosyllactose and galactooligosaccharides), polysaccharides (e.g., insulin, heparosan, hyaluronan, chondroitin, etc.), and polyamino acids (e.g., poly-ɛ-L-lysine, cyanophycin, poly-γ-glutamic acid, etc.) used as nutraceuticals (Wang et al. 2016).

12.3.1.3 Fungi as a Source of Nutraceuticals Macro- and microfungi from different habitats are rich sources of nutraceuticals. Certain species of fungi have been popular in traditional medicine around the world since ancient times. It has also been scientifically proven that several species of mushrooms contain bioactive compounds with various health benefits for humans

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(Al-Obaidi et al. 2021). Certain edible mushrooms such as Agaricus bisporus (tuberous-leaf mushroom), Lentinus edodes (shiitake mushroom), Pleurotus spp. (oyster mushroom), etc. which belong to macrofungi are rich sources of proteins, vitamins, minerals, and various other bioactive compounds such as polysaccharides, fatty acids, amino acids, and phenolic compounds, which are essential for maintaining human health and regulating diseases (Bakratsas et al. 2021; Niego et al. 2021). Due to the presence of a large number of bioactive compounds, fungi are often considered by researchers as mini-pharmaceutical factories (Niego et al. 2021). Ganoderma lucidum is another medicinal mushroom that has been used for centuries in East Asia, especially in traditional Chinese medicine. G. lucidum is a rich source of antioxidants such as triterpenes, fatty acids, nucleotides, polysaccharides, proteins, glycoproteins, steroids, sterols, peptides, and trace elements, which show promising properties such as anti-neurodegenerative, antiangiogenic, antidiabetic, antioxidant, antiproliferative, hepatoprotective, antimicrobial, antiviral, and immunomodulatory properties (Niego et al. 2021; Seweryn et al. 2021; El Sheikha 2022). The filamentous fungus Aspergillus oryzae, which is popularly used to produce Asian fermented foods, is a rich source of agmatine, which is used to treat depression and anxiety (Akasaka and Fujiwara 2020). Several fungi from marine environments such as Penicillium sp., Ampelomyces sp., Cladosporium sp., Pseudallescheria, etc. have been described with nutraceutical properties (Gupta and Prakash 2019). This suggests that fungi from extreme conditions and environments have the potential to produce new nutraceuticals that can be introduced into modern medicine.

12.3.2 Advanced Approaches for Nutraceutical Production (Fig. 12.1) Growing public awareness of the medicinal benefits of nutraceuticals has led to an exponential increase in demand in the global market, with the market estimated to be worth billions of dollars. However, conventional methods of direct extraction from natural sources are not sufficient to meet this increasing demand. Various natural sources, including plants and microbes, have limitations in terms of their availability and productivity, as well as the cost and purity of the extracted nutraceuticals. Production of nutraceuticals in plants usually occurs under stressful conditions, while microbial strains can produce toxic by-products. Therefore, microbes used for nutraceutical production should have generally recognized as safe status (GRAS). Alternative artificial synthesis of nutraceuticals using chemicals is extremely difficult due to their structural and chemical complexity, as well as ethical and environmental concerns. Modern methods such as metabolic engineering, synthetic biology, etc. are gaining attention to overcome this hurdle using microbial cell factories for the synthesis of various nutraceuticals (Wang et al. 2016; Madhavan et al. 2022). Since gene manipulation is easier in microorganisms compared to higher organisms, they are a suitable candidate for metabolic engineering for improved production of nutraceuticals with easier extraction of purified products. Metabolic

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Fig. 12.1 Various approaches to nutraceutical production

engineering involves making changes in metabolic pathways or adding recombinant genes through genetic engineering to improve the overall quality and quantity of a product. Although the process of metabolic engineering is feasible in native cells, the lack of knowledge of their metabolic pathways, the complexity of the genetic modification process, and the slower growth rate usually hinder the success of the process. As an alternative, a heterologous host organism can be used that expresses the recombinant genes for gene expression and generation of the target product (Pickens et al. 2011). The most commonly used hosts are E. coli and S. cerevisiae, as these organisms meet the essential criteria for their use in metabolic engineering. First, these organisms have a simple and well-recognized metabolic pathway that identifies all the major regulatory networks and are therefore very manageable. In addition, all the critical steps and enzymes involved in the synthesis of the targeted products have been identified, so they can be produced on a large scale. In addition, these organisms are considered GRAS for food and pharmaceutical applications. Other commonly used GRAS strains are lactic acid bacteria, Bacillus subtilis, Propionibacteria, Yarrowia lipolytica, Pichia ciferrii, etc., which are used for the production of various nutraceuticals such as gamma-aminobutyric acid, carotenoids, glutathione, folate, vitamin B12, hyaluronic acid, etc. (Pontrelli et al. 2018). Metabolic engineering of these GRAS strains for nutraceutical production has several advantages, such as enhanced expression of the metabolic pathway along with alteration of the pathway for overproduction and exogenous release of nutraceuticals. In addition, the genes of novel nutraceuticals can be converted from a wild-type strain without GRAS status to a GRAS strain, which enables their commercialization. Moreover, the introduction of ways to utilize cheap carbon

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sources such as organic waste biomass for nutraceutical production makes the overall process extremely cost-effective and environmentally friendly (Liu et al. 2017). Newer systems biology technologies such as omics, de novo DNA synthesis, etc. are helping to further improve the efficiency of metabolic engineering along with computational biology for in vitro analysis of these strategies. The multi-omic approach helps in the identification of novel biologics and microbes with nutraceutical properties, such as transcriptomics, proteomics, and metabolomics, and opens a whole new perspective for the development of metabolic pathways to enhance the expression of nutraceuticals (Ramalingam et al. 2021). Metabolomics provides the metabolic profile, while transcriptomics and proteomics provide the mRNA and protein profile, which can help in identifying novel compounds with nutraceutical properties and the metabolic pathways involved. This information can then be used for in vitro design and screening of the genetic element with high gene expression and regulation. All this information can then be used together to give direction to the metabolic process by identifying limiting steps, synthesizing new enzymes or modifying existing proteins, increasing metabolic flux, and de novo DNA synthesizing to convert the theoretical yield into an actual one (Ma et al. 2020). Another strategy for producing nutraceuticals is to use a microbial consortium. Since nutraceuticals are complex molecules, a microbial consortium can be used to produce them, and the metabolic by-product of one strain can be used by another strain to produce the desired nutraceutical. The process can be very beneficial as metabolic engineering can be applied in parallel to all strains to improve the most favorable property that complements the other strains and achieves overall improved production (Yuan and Alper 2019). However, very few of these metabolically engineered products are available for commercial purposes. The ethical issues and risk assessment of these genetically modified organisms produced by metabolic engineering require them to be monitored by a regulatory agency for their commercial purposes. The risk of reduced stability of these GMOs, and possible adverse effects on human health and the environment are the major obstacles that need to be addressed.

12.4

Conclusion and Future Prospect

Nutraceuticals are an emerging topic in the field of food and medical technology. They offer an answer to the issue of food safety and nutrition, while providing a therapeutic option for the treatment of various diseases. Considering the modern lifestyle and the fact that food plays a key role in various diseases, nutraceuticals offer a great alternative that has been proven to alleviate the symptoms of some of the major diseases while protecting against numerous others. The medicinal values and mechanisms responsible for the therapeutic value of nutraceuticals need further research to better understand them. Since natural sources are insufficient to meet the growing demand for nutraceuticals, metabolic engineering using the various GRAS microbes is being used. Further development of the technology with

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biotechnological advances such as omics, co-cultures, and systems biology is underway and requires further research to maximize the production of even nutraceuticals with complex chemical structures. Acknowledgments The authors would like to thank the Department of Microbiology, Sikkim University, for providing the computational infrastructure and central library facilities for procuring references.

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Hyaluronic Acid Microbial Synthesis and Its Explicit Uses in the Development of Nutraceuticals, Biomedicine, and Vaccine Development

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Priya Shukla, Pradeep Srivastava, and Abha Mishra

Abstract

A medicinal biopolymer with high economic value, hyaluronic acid (HA), is increasingly synthesized through microbial fermentation. Research and development have focused on enhancing hyaluronic acid’s crucial quality characteristics, purity, and molecular weight. Due to its adaptability, hyaluronic acid has played a significant role in biomedical research and has been used in numerous sectors, including tissue engineering and cancer treatment, in various forms. Micelles, polymersomes, hydrogels, and other hyaluronic acid-based nanomaterials are essential for effective drug administration and cancer treatment. Modern hyaluronic acid encapsulation techniques can adequately prevent probiotics and nutraceuticals from the severe conditions of food processing. Hyaluronic acid can also create a wide range of vaccinations as an efficient and biocompatible adjuvant. Also, developing nasal vaccinations against infectious diseases is a promising application for hyaluronic acid-coated micelles. This chapter examines the benefits and drawbacks of microbial hyaluronic acid generation and its diverse applicability in various industries. Keywords

Hyaluronic acid · Fermentation · Biomedicine · Nutraceuticals · Vaccine adjuvant

P. Shukla · P. Srivastava · A. Mishra (✉) School of Biochemical Engineering, Indian Institute of Technology, Varanasi, India e-mail: [email protected]; [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_13

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Abbreviations ATP BCG CDM DC ECM EDC/NHS GAG HA KH MW OA PDA PEG UDP

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Adenosine triphosphate Bacillus Calmette-Guerin Chemically defined medium Dendritic cell Extracellular matrix Ethyl(dimethyl aminopropyl)carbodiimide/N-hydroxysuccinimide Glycosaminoglycans Hyaluronic acid Sodium hyaluronate Molecular weight Osteoarthritis Polydopamine Polyethylene glycol Uridine diphosphate

Introduction

HA, a natural, linear polysaccharide found in all living tissue, especially skin and cartilage, is particularly prevalent in the umbilical cord. It is constituted of alternating N-acetyl-d-glucosamine and d-glucuronic acid. At physiological pH, HA exists as hyaluronate, which gives tissues plasticity by synthesizing elastomeric hydrogels (Cowman et al. 2015). Given its soaring viscoelasticity and capability to hold a vast proportion of H2O, hyaluronan finds extensive use in the aesthetic and medicinal fields (Armstrong and Johns 1997). Considering respective utilization, the HA components and derivatives have a significant aggregate worth varying from 2000 to 60,000 US dollars per kilogram. Initially, the production of HA involved the removal of animal tissues, primarily rooster combs. Given the limited availability of animal tissue supplies, the dangers of virus contamination, and due to the exorbitant expenses implicated in the isolation and refinement of HA from animal sources, the viability of large-scale synthesis through microbial HA manufacturing has increased (Cooney et al. 1999; Gao et al. 2006). Additionally, the fabrication of HA using fermentation techniques enables the management of contextual conditions and genetic modification to enhance product quality and yield (Armstrong and Johns 1997). Streptococci are the most frequently employed microorganisms in the commercial synthesis of HA. These bacteria are sophisticated nutrient-required microorganisms with the restricted capability to manufacture specified vitamins and amino acids (Fitzpatrick and O’keeffe 2001; Hofvendahl and Hahn-Hägerdal 2000). There is also the nutritional need for organic nitrogen, a significant carbon source for their cells’ biosynthesis (Armstrong et al. 1997). Traditionally, the growth medium utilized for the bacterial production of HA

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comprises growth agents, peptones, yeast extract, mineral supplies, casein hydrolysate, and considerable amounts of complex nitrogen sources (Chong and Nielsen 2003; Armstrong et al. 1997). Biopolymers with therapeutic potentials, including alginate, chitosan, gums, HA, and beta-glucan, offer good potential as delivery methods or adjuvants for the creation of vaccine formulations and the design of vaccine delivery devices. Such biodegradable polymers will have crucial roles to play in the fight against diseases based on their antiviral efficacy and immunomodulatory capabilities (Mallakpour et al. 2021). Hyaluronan’s presence makes it easier for stem cells to move from their original niche to distant locations within growing organisms. Cell motility is facilitated by signal transduction pathways stimulated by HA (Turley et al. 2002; Entwistle et al. 1996). The negatively charged hyaluronan that makes room for cell movement is linked to this process (Turley 1992). Remarkably, the accountability of hyaluronan and its receptors in transformation and metastasis has been connected to the mechanisms of tumor cell invasion and motility. The collagen scaffolding and perhaps other elements of the ECM have been further interacted with by these entities. The viability of the tissue is stabilized by hyaloadherines, a group of peptides that could connect with hyaluronan. The cell membranes and a portion of these peptides are connected. When hyaluronan is present, these peptides operate as hyaluronan cell receptors and can activate several intracellular signal cascades (Schiller et al. 2003). By controlling epidermal dendritic cell maturation and migration, leukocyte trafficking into inflamed skin, and T-cell activation upon antigen presentation, HA performs crucial roles in immune responses (Mummert 2005). Due to its abundant plethora in nature, biodegradability, hydrophilicity, and tissuespecific interactions, HA is a desirable material for vaccine delivery applications. New vaccine compositions with enhanced antigen administration, specifically for mucosal immunization routes, have been developed because of current data targeted at improving the physicochemical characteristics of naturally occurring polymerbased nanoparticles. HA has been utilized in numerous forms to treat various disorders, including dermal filler, hydrogel, lotions, scaffolds, foams, films, intradermal injections, and gels. Numerous pharmacological effects of HA have been reported, including immunomodulatory skin mending (Narurkar et al. 2016), anti-inflammatory (Chen et al. 2018), (Fiszer-Szafarz et al. 1988), wound healing and tissue regeneration (Hussain et al. 2017), antidiabetic (NIu 2008), antiaging (Papakonstantinou et al. 2012), anticancer and antiproliferative (Safdar et al. 2018), and aesthetic qualities (Pavicic et al. 2011). HA has many functions in the body’s homeostasis and regulation of many biological processes. The HA macromers are synthesized using several methods, and their constituent parts are then processed to provide valuable materials for regenerative medicine. The biological processes involved in stem cell proliferation, differentiation, and other behaviors may be disrupted by HA-based hydrogels. Additionally, these distinctive materials transport biological substances, stem cells, and medications. Additionally, these materials are a strong contender for transitional applications because to their biocompatibility, safety, and efficacy.

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Nutraceuticals are oral diet elements that are naturally present in food and are thought to have health or medicinal benefits (Chauhan et al. 2013). The name was created in 1989 by Dr. Stephen Defelice, who merged the words “nutrition” and “pharmaceutical.” Important natural bioactive substances known as nutraceuticals give people health-promoting and therapeutic benefits. Nutraceuticals are now a multibillion-dollar industry due to the rising demand for augmented nutraceuticals for treating and preventing human disorders worldwide. However, the widespread usage of nutraceuticals is constrained by supply issues and the challenges associated with extracting ingredients from congenital resources like animals, plants, or fungi. Offering a more sustainable option, metagenomics through microbial equipment on board has improved the manufacturing of significant nutraceuticals from simple carbon feedstock. Microbes were able to create a variety of nutraceuticals by utilizing their metabolic networks, thanks to the possible genetic manipulation and the tolerance of heterologous enzymes. This chapter will summarize the microbial production of hyaluronan and its various applications in biomedicine as an adjuvant for drug delivery and nutraceuticals.

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Microbial Production

HA can be made through microbial fermentation or taken from rooster combs. Because HA is administered in combination with proteoglycans in tissues, obtaining elevated, high-molecular-weight HA is challenging (O’Regan et al. 1994). Moreover, there is a growing antagonism to employing biomolecules derived from animal sources for pharmaceutical purposes due to the possibility of a mixed breed viral infection and other unintentional agent contaminations. As a result, HA’s favored provenance gradually shifts from extraction to microbial synthesis. Shiseido was the first company to produce microbial HA in the 1980s industrially. The strain of S. zooepidemicus that is most frequently utilized to manufacture HA can produce a considerable amount of HA in the right growing conditions (Fig. 13.1). However, the HA production from S. zooepidemicus faces the following three difficulties: 1. At optimum HA concentration, the broth’s viscosity can approach 400–500 mPas, resulting in a low oxygen mass transfer rate and poor mixing, significantly restricting the amount of HA that can be produced. 2. For common precursors like UDP-glucuronic acid and UDP-Nacetylglucosamine, HA production and cell proliferation are in fierce conflict. 3. A significant by-product of HA fermentation is lactic acid, which significantly inhibits both cell growth and the fabrication of HA when it builds up. Streptococci are lactic acid bacteria that have strict dietary requirements and cannot manufacture some amino acids (Armstrong et al. 1997). A few amino acid supplements, like arginine and lysine, were beneficial for cell proliferation and HA synthesis (Liu et al. 2009b). S. zooepidemicus can be cultured on a chemically

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Fig. 13.1 Most common pathway utilized for production and purification of hyaluronic acid

defined medium (CDM) with some nutritional components necessary for growth and the same concentration and rate of HA synthesis as a complicated medium (Armstrong et al. 1997). In a medium created by Zhang et al. with starch as a carbon source, the concentration of HA was 6.7 g/L (Zhang et al. 2006). Significant impacts on microbial HA synthesis were also caused by the mineral ions and starting glucose content (Pires et al. 2010; Pires and Santana 2010). In contrast to the preliminary glucose concentrations, where another homolactic metabolic activity reigned supreme, the disappearance of glucose emanated in a mixed acid metabolism autonomous of the air supply (Pires and Santana 2010). Successful chemical mutagenesis and serial selection techniques have produced nonhemolytic and hyaluronidase-negative mutants (Kim et al. 1996). Spontaneous mutagenesis has also been proved feasible to develop cultures that synthesize high-molecular-weight HA (Stangohl 2000; Murano et al. 2011). The microbial HA generation is greatly influenced by the medium composition and growth environment (agitation, pH, temperature, dissolved oxygen, shear stress, aeration rate, and bioreactor type). S. zooepidemicus typically produced HA at a pH and temperature of 7.0 and 37, respectively (Johns et al. 1994; Kim et al. 1996). Since S. zooepidemicus produces HA through a generally viscous mechanism, oxygen mass transfer and mixing efficiency rate have a big jolt on hyaluronan production. The implications of agitation speed, aeration rate, dissolved oxygen, and shear stress on microbiological hyaluronan synthesizers have been the focus of countless investigations (Liu et al. 2009b; Johns et al. 1994; Kim et al. 1996; Duan et al. 2008; Duan et al. 2009; Huang et al. 2006; Liu et al. 2009a; Wu et al. 2009). An aerobic condition produces HA with a greater titer and molecular weight when contrasted to an anaerobic condition (Armstrong et al. 1997; Johns et al. 1994). The preceding can be used to describe how aeration stimulates HA production:

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1. Because streptococcal cells aggregated and were protected from oxygen metabolites by their HA capsule, oxygen might prompt the fabrication of HA. 2. The percentage of ATP produced can be increased by dissolving oxygen in the media, which results in switching the carbon flux toward acetic acid from lactic acid. Higher HA titers can be attained, thanks to the additional ATP produced during acetate production. 3. The aeration allows for diverting additional acetyl-CoA from the primary carbon metabolism to revitalize it to manufacture HA (Wu et al. 2009). Aeration rate and agitation speed rise together with HA formation; however, excessive agitation speed damages cells and lowers HA concentration (Hasegawa et al. 1999). HA has been produced using a variety of fermentation processes, including repeated batch, fed-batch, batch, and continuous culture (Blank et al. 2005; Chen et al. 2009; Huang et al. 2008; Im et al. 2009; Liu et al. 2008a; Vázquez et al. 2010; Cooney et al. 1999; Don and Shoparwe 2010). The primary method of production for HA is batch culture. Correlating to batch culture, fed-batch culture can speed up the cultivation process and produce more HA (Vázquez et al. 2010). For the initial hours, S. zooepidemicus was cultivated in a fed-batch method with a concentration of sucrose (1.0 g/L), and the following hours were spent in batch cultivation with a sucrose concentration of 15 g/L. For the synthesis of HA, it was discovered that the fed-batch and batch methods worked well together. Compared to batch culture, this two-stage culture approach increased HA fabrication by more than 30% (Liu et al. 2008a). It is simple to reach the operational limit of HA fermentation, 5–10 g/L, in a conventional batch and fed-batch culture during 6–16 h. Subsequently, the repeated batch culture was also utilized to manufacture HA, and the output of HA was much increased (Chen et al. 2009; Huang et al. 2008). Continuous cultivation can be advantageous to lengthen the culture duration when there is a limit to product concentration, decrease the increase in the culture duration when there is a limit to product concentration, and decrease the percentage of time required for bioreactor turnover. Continuous cultivation might also minimize peptide contaminations by prolonging the changeover into the stationary phase, which has been demonstrated to result in the flaking of cell wall proteins and the elimination of stationary phasecorrelated pathogen components (Mausolf et al. 1990; Leonard et al. 1998). Last but not least, the development in polydispersity perceived during HA batch manufacturing as a consequence of variations in growth rate could be prevented by continuous cultivation (Armstrong and Johns 1997). However, HA synthesis in a chemostat was challenging due to the volatile behavior of widely encapsulated streptococci strains that produce HA at a high dilution percentage. The most remarkable dilution rate for steady HA production in a chemostat culture was 0.4 h-1 (Blank et al. 2005). As a result, continuous cultivation is not possible to achieve HA industrial production (Chong et al. 2005). For researchers working on purification, obtaining highly purified HA has been difficult. In addition, some intricate procedures, such as diafiltration cycles or consecutive micro- and ultrafiltration, are made up of a single operation separated into phases. In-depth, multi-operation sequences make it possible to see how each

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operation in a process affects the outcome. Even though it was used at different stages of the procedures and with different HA sources, organic solvent precipitation produced more outstanding gains in purity (Rangaswamy and Jain 2008; Zhou et al. 2006). Proteins are the most significant impurity in the processes used to produce HA. Serum, non-plasma proteins, and collagen all contain HA produced from connective tissues. The contaminants that must be eliminated from the HA are later created from the protein in the fermentation medium. Precipitation using quaternary salts or organic solvents has been employed extensively to remove proteins (Balazs 2009; Murado et al. 2012; Schmut and Hofmann 1981). The filtration process relies on keeping particles on a porous membrane following their size. The fundamental drawback of traditional filtration is the fouling phenomena, where pore obstruction gradually increases pressure drop and flux resistance. By using the feed flow diagonally in the direction of the membrane rather than the vertical flow, tangential filtration gets over this restriction. Other methods for enhancing precipitation performance include adding filter aids or altering the diafiltration operation mode. A specific mode of operation called diafiltration involves supplying fresh water or a buffer to the storage tank to compensate for the fluid lost as permeate, whereas the retentate is constantly recirculated. This method increases the purification effectiveness by preventing overconcentration and membrane fouling (Oueslati et al. 2015; Howell et al. 2012). It has been widely documented that filtration is used in HA purification procedures to bargain its application for cell purification in fermented hyaluronan (Reddy and Karunakaran 2013; Schiraldi et al. 2010; Rajendran et al. 2016). The capacity of chemicals to be selectively retained on the surface of porous substances serves as the foundation for the adsorption process. Adsorption is typically utilized in batch mode to purify HA, followed by precipitation and filtration (Nimrod et al. 1988; Yang and Lee 2007; Han et al. 2009; Ünlüer et al. 2013; Akdamar et al. 2009; Pal and Nath 1974). Hands down, the most prevalent adsorbent is activated charcoal, which is widely available and inexpensive (Cavalcanti et al. 2019; Rajendran et al. 2016; Choi et al. 2014). Conversely, silica gel and resins frequently use the same processes and adsorbents (Rangaswamy and Jain 2008; Han et al. 2009; Schmut and Hofmann 1981). The generation of HA depends heavily on downstream processing, which also requires substantial investments. As a result, it is crucial to understand each operation’s underlying costs and effectiveness. A commercialized HA product’s molecular weight is a crucial quality criterion since it impacts the HA’s physiological reaction, rheological qualities, and appropriate applications (Armstrong and Johns 1997; Blank et al. 2008). High-molecularweight HA provides favorable properties in ophthalmology, orthopedics, wound healing, and cosmetics, such as moisture retention, good viscoelasticity, and mucoadhesion, while HA oligosaccharides or HA with relatively low molecular weight have been demonstrated to stimulate angiogenesis, trigger the release of inflammatory mediators, and prevent tumor development (Sheng et al. 2009).

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Vaccine Development

In order to disseminate vaccines, antigens and adjuvants can be placed on biomaterials as building blocks. The level and nature of the immune response induced can be adjusted to a considerable extent by including biomaterials in vaccine delivery platforms. Dendritic cells (DCs) can be directly activated and grown by the biomaterial. These biodegradable polymer immune-priming centers stimulate particular innate immunity at the puncture site by supplying all required cues to draw novice DCs, enhancing DC contact with antigen and encouraging DC expression of cytokines and co-stimulatory component overexpression to stimulate T cells (Leifer 2017). Inflammation is significantly influenced by ECM remodeling. As a result, the ECM is essential for modulating the cancer surveillance and immune system, affecting cancer proliferation and invasion (Leight et al. 2017). Immunotherapy is anticipated to enhance intratumoral T cell accessibility and functionality by suppressing key enzymes in matrix synthesis, including hyaladherin, for HA (Johansson et al. 2016). For innovative vaccine delivery devices, the MW size of hyaluronan is crucial. Studies, for instance, assessed the transport of various HA MWs between 7 and 741 kDa to the mice lungs. Concerning the pharmacokinetic characteristics of hyaluronan, while 67–215 kDa HA demonstrated prolonged lung survival, smaller HA MW demonstrated faster systemic distribution. Due to the quick absorption of HA beneath 67 kDa, minimal lung permeation, and mucociliary clearance of viscous formulations of HA bigger than 215 kDa, lung exposure appears to have peaked in this size range (Kuehl et al. 2016). The physicomechanical characteristics of HA viscosupplements can also be modified by the cross-linking degree (CD) and MW (Lee et al. 2014). Transcutaneous microneedles have been created using HA, an essential substance in healthy skin tissues. Sodium hyaluronate-based MicroHyala® microneedle patches have been utilized to deliver a variety of antigens from the influenza virus, diphtheria, tetanus, and diphtheria virus through the skin. Micromolding techniques created antigen-loaded microneedle patches intended to degrade after being inserted into the skin and distribute antigens to the stratum corneum. Similar IgG antibody titers were produced via subcutaneous injection and immunization with microneedles. In terms of safety, the biodegradable property of hyaluronan-based microneedles provides a superlative replacement to traditional microneedles, providing a straightforward, efficient, and noninvasive vaccine administration technology (Matsuo et al. 2012). Another benefit of HA is its capacity to inhibit tumor growth, especially colorectal cancer, which is thought to be mediated via dendritic cell activation (Alaniz et al. 2009). When dendritic cells were preconditioned with low-molecular-weight HA, the percentage of dendritic cell trafficking to tumorregional lymph nodes was significantly boosted in vivo. Compared to treatment with dendritic cells pulsed with tumor lysate alone, immunization of tumor-bearing mice with dendritic cells preconditioned with HA and pulsed with colorectal carcinoma tumor lysate produced significantly greater antitumor cytotoxic T cell lymphocyte respondents and much more outstanding protracted safety against tumor recurrence (Alaniz et al. 2011). HA has been researched in conjunction with several

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other components, such as liposomes, as a vaccine booster. Thiolated HA was conjugated with L-cysteine using the EDC/NHS technique to create powerful copolymer aggregates for vaccine formulation. With more significant IgG titers, this simulated model demonstrated improved colloidal stability, sustained antigen release, and a robust humoral immune response. When used for intranasal immunization in a mouse model, cationic liposome-HA hybrid nanoparticles are effective transporters of protein antigens and immunostimulatory substances both in vitro and in vivo (Bernasconi et al. 2016). First, before monitoring the increase in the expression of coactivator molecules like CD86, CD40, and MHC class II, which significantly contribute significant contribution to an enhanced specific T cell and antibody response after intranasal vaccination, these hybrid nanoparticle systems have been discovered to encourage dendritic cell (DC) maturation (Verheul et al. 2011). According to their antigenicity, influenza viruses are divided into three varieties and are members of the RNA virus family. The injectable influenza vaccines prescribed in clinics cause the blood to create immunoglobulin G (IgG), which defends against viral illnesses. Mucosal vaccination, which delivers immunoglobulin A (IgA) on the mucosal and triggers immunoglobulin G (IgG) in the blood, represents one of the most excellent strategies to prevent influenza infestations. In research, HA was modified with tetraglycine-L-octaarginine, and the modified HA was then used to create a mucosal adjuvant (Ukawa et al. 2022). The research team used a virus exposure to demonstrate the biopolymer’s effectiveness as a crossprotective adjuvant. Immunoglobulin ought to protect the host from infection. Mice subjected to infectious viruses after receiving an inactivated virus nasal inoculation experienced significant weight loss. Inactivated viral particles were used to immunize mice in the absence and presence of this biopolymer. Evaluation of viral infectious symptoms in mice injected with viral antigens and mitigated HA was challenging due to the significant activation of immunoglobulins, which can commune with the viruses. According to the findings, the host’s immunity and capacity to fend against viral infection are enhanced by this polysaccharide derivative. Considering this, the study demonstrated the suitability of this biopolymer as a mucosal adjuvant to prevent viral infection. The extracellular matrix, which is present throughout the body, contains the biodegradable biopolymer HA, a member of the glycosaminoglycan family, broken down by enzymes. Both cosmetics and medicine frequently use it. However, the nondegradable platform must be transformed into a degradable one for clinical usage. In a different study, peptides that can penetrate cells and degrade were created using HA derivatives. The HA-based platform was developed to prevent the formation of octaargininelinked poly(N-vinyl-acetamide-co-acrylic acid) that is poisonous and dangerous to humans. Studies conducted in vitro revealed that the HA derivative was less hazardous than poly(N-vinyl acetamide-co-acrylic acid) compounds. The safety of the HA-based adjuvant for mucosal influenza vaccination was generally supported by this investigation (Ukawa et al. 2019). In research using HA and polycaprolactone, microneedles for transcutaneous influenza immunization were developed, and their efficacy was evaluated. Prior to the dip-coating operation, the

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tips of HA were quickly frozen to cover the antigens on the surfaces with a protective layer. Microneedles can correctly release the medicine through skin penetration without breaking, according to ex vivo tests. As soon as they are injected, tips covered in a vaccine can be readily peeled away from the base (Choi et al. 2018). The use of HA in medicine delivery has grown during the past few years (Wickens et al. 2017; Guter and Breunig 2017; Dosio et al. 2016; Tripodo et al. 2015). In contrast, the use of HA for vaccines and gene therapy is still in its early stages. Researchers discovered increasing nanoparticle sustainability and cytocompatibility by exploiting HA’s anionic properties to shield the core polymer’s positive charge. Due to the selectivity of HA for the CD44 receptor, which is abundantly expressed in tumor or progenitor cells, these nanoparticles are also suitable for controlled delivery strategies. Through the endocytosis of HA-coated nanoparticles by dendritic cells through CD44 receptors, their components were released into the cytoplasm and endosomal vesicles. In comparison to particles without HA, the nanoparticles stimulated higher cellular and humoral immune responses in mice. For nanomaterials to be employed in diagnostics and therapeutics where immunological effects are potential problems, how they regulate dendritic cell maturation and the related adjuvant function must be considered (Babensee and Paranjpe 2005). Additionally, hyaluronan has been examined as an adjuvant to lessen the negative impacts of vaccinations. To see if the progressive infusion of HA could minimize the local withdrawal symptoms of the Bacillus Calmette-Guerin (BCG) immunization, a prominent medication for non-muscle invasive bladder cancer is needed. The rationale was that HA might be an anti-inflammatory agent by limiting leukocyte migration and immune complex adhesion, strengthening the urine-tissue barrier through ECM integration. The scientists developed a new method for minimizing BCG side effects when they saw a significant drop in side effects after administering HA after the BCG vaccine in a short clinical trial (Topazio et al. 2014). Adjuvants have also been created using HA, monophosphoryl lipid A, and aluminum salt for the hepatitis B vaccine (Moon et al. 2015).

13.4

Biomedicine

High levels of biodegradability, biocompatibility, hydrophilicity, and viscoelasticity are characteristics of HA (Xie et al. 2018; Luo et al. 2000; Sahiner et al. 2019). These characteristics make HA relevant in biomedicine, especially for in vivo endeavors. Since it is effortlessly connected to the blossoming of amphiphilic compounds with significant immobilization action and could degrade over controlled timeframes, this chemical is helpful in tissue engineering, drug delivery, and wound healing procedures (Wang et al. 2016; Chen et al. 2019; Montanari et al. 2013; Kim et al. 2018; Zhang et al. 2020). Sensing composites benefit from HA’s biocompatibility and moisture retention qualities, which are particularly advantageous in the biosensing industry. Additionally, when employed, HA has demonstrated a boost

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in electrical conductivity for biosensors (Kim et al. 2019b; Kim et al. 2019a; Cabral et al. 2016). Because of hyaluronan’s specified binding to cancer cell surface receptors and its biocompatibility and biodegradability, the use of HA in the intended medicine delivery of anticancer medication has advanced dramatically. This could combine with several other medications to create conjugates. To meet the timing and directional release criteria, the conjugates have a controlled drug release and organized system that can regulate the dissemination of several medications to diverse sick regions (Chen et al. 2014). Through the action of charges, cationic polymers can enter cell membranes and increase DNA stability while avoiding endosome destruction. These gene delivery vehicles do, however, suffer from poor targeting, low transfection rates, and significant cytotoxicity. HA-based gene medication delivery method demonstrated positive benefits (Saravanakumar et al. 2014). HA was initially employed for gene delivery when HA-adipic acid dihydrazide (ADH) hydrogels shielded DNA from catalytic disintegration and permitted uninterrupted DNA release (Shoham et al. 2013). Researchers also made HA microparticles colloid comparably, and DNA was added to the gel network for delivery (Yun et al. 2004). These hydrogel-based gene delivery techniques are often employed, particularly in tissue engineering. A warehousing system made of HA hydrogel was employed to manage gene distribution during tissue regeneration. HA gel systems administered siRNA (Lee et al. 2007). HA might be cross-linked by disulfide bonds intended to be broken down by glutathione in the cytoplasm. In comparison to cell lines with lesser CD44 expression, the overexpressed CD44 cell lines had a significantly better efficacy of gene silencing and cell absorption. Using bioconjugation technology, natural polymer molecules were recently synthesized, including HA and polyethylene glycol (PEG), which have been thoroughly exploited in creating biopharmaceuticals with favorable pharmacokinetics (Choi et al. 2011). According to reports, chemically altering PEG could decrease the immunological response, relieve enzyme breakdown in vivo, and limit renal dispensation of peptide and protein medicines, all of which would increase efficacy. PEGylation can potentially have a detrimental impact, though, according to reports. PEGylated liposome injections were administered repeatedly and the so-called rapid blood clearance resulted in a long-term cyclic reduction (Ma et al. 2012). Prodrugs created by covalently attaching small molecule anticancer medications to HA are known as HA-drug conjugates. In the blood, these covalent bonds are difficult to dissolve, but once they reach the target, they do so by hydrolysis or enzymolysis, allowing the drug to be released. In addition to improving a drug’s solubility, HA-drug conjugates can change a drug’s half-life and distribution in vivo, and boosting the osmotic retention mechanism will enhance the aggregation in cancer tissue (Fan et al. 2015). Amphiphilic HA analogs can self-assemble in water to produce nanostructures with a central core. Antitumor medications and contrast agents can be contained in the interior hydrophobic core of nanoparticles for diagnostic and treatment (Mayol et al. 2014). The hydrophilic coating inhibits pointless protein adsorption, preventing the endothelium reticular system from being taken up unintendedly – different levels of the hydrophobic moiety

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substitution control zeta potential and the particle size of self-assembled nanoparticles. Typically, when the hydrophobic moiety proportion rises, the inner core of the nanoparticles becomes more hydrophobic and their particle size decreases. As HA occurs naturally in the joint capsule, synovial fluid, and articular cartilage, orthopedics frequently uses it. This substance mainly treats joint conditions like osteoarthritis or rheumatoid arthritis. The most prevalent joint disease, osteoarthritis, causes significant impairment and a reduction in the well-being of life. This disruption results in an imbalance between the destruction and formation of articular cartilage, favoring the latter (Gupta et al. 2005). A reduction in GAG, a surge in proteoglycans and collagen-degrading enzymes, and increased stored water are possible intra-articular alterations in this state. Reduced molecular mass and concentration are necessary for changes in endogenous HA (Barbucci et al. 2002; Moreland 2003). The formation of reactive oxygen species, which oversees breaking down of collagen, laminin, and HA, increases due to inflammation (Bates et al. 1984). This mechanism generates HA fragments that fuel an endless cycle of heightened inflammatory reactions. By limiting the migration of inflammatory cells and chemotaxis, high molecular mass HA acts as an effective barrier to inflammatory procedures and safeguards against the impacts of free radicals. The critical question regarding the etiopathogenesis of OA relates to the inhibitory and stimulatory actions of HA on proteoglycan synthesis and chondrocyte death, respectively. HA directly affects the analgesic effect (Barbucci et al. 2002; Moreland 2003; Moore and Willoughby 1995; Ghosh et al. 1995; Balazs and Laurent 1998). Numerous investigations on animals have demonstrated the effects of administered intra-articular HA, which were seen in the articular cartilage repair processes. Even though HA by itself cannot completely regenerate articular surfaces, it does provide benefits by decreasing discomfort and, as a result, the need for nonsteroidal anti-inflammatory drugs, a benefit that is more than satisfactory. The introduction of autologous chondrocyte cultures, for which HA could be the optimal substrate, would be the future path of these treatments (Barbucci et al. 2002; Moreland 2003; Schiavinato et al. 1989; Moskowitz 2000). Patients are increasingly interested in oral HA formulations in addition to HA injections. On the other hand, oral formulations do not have any proven therapeutic benefits for OA. According to the research that is now available on oral HA formulations, oral supplementation may decrease pain and enhance the quality of life. The insufficient size of the study groups is what makes these conclusions poor. In other words, these research findings are biased. None of the investigations evaluated the impact of HA with morphological alterations, even though several did not reach statistical significance. Numerous uses for HA exist in ophthalmology, both from a conservative and practical standpoint. Due to its viscoelastic characteristics, it is widely employed as the “lubricant” component and frequently makes up many artificial tear formulations used to treat dry eyes. It soothes discomfort, hydrates the eye, and makes up for any sodium hyaluronate deficiency in the tear film. The symptoms of dry eye are significantly lessened by its noteworthy qualities, which include stabilizing the tear film, reducing friction during blinking, and preventing dangerous particles from

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adhering to the eye. More than 50% of respondents say they no longer want to wear contact lenses due to dry eye. Because most ophthalmic solutions contain artificial ingredients and preservatives, they leave residues on the eye’s surface, making it easy to distinguish treatments containing HA from other eye drops. Additionally, the fluid frequently does not disperse evenly on the eye’s surface, which can result in visible blurring and decreased vision. Because HA is hydrophilic and viscoelastic, it reduces friction and slows the evaporation of tears. Significantly, HA-based medicines do not enlarge the blood vessels in the conjunctiva, making them safe to use during the winter (Maltese et al. 2006; Kogan et al. 2007). HA is crucial in ophthalmic surgery as well. Its effects primarily depend on creating and maintaining cozy surroundings to aid in healing the postsurgical area. This is accomplished by stabilizing the tear film, shortening the healing process, lowering the danger of adhesions, reducing the production of free radicals, and restoring normal intraocular pressure. The anterior portion of the eye is frequently operated on during procedures like cataract removal, trabeculectomy, refractive surgery, glaucoma therapies, and corneal plastic surgery. Sodium hyaluronate is the most popular cosmetic filler currently. It has the purpose of enhancing and completing the extracellular tissue space. Cross-linked formulations are utilized to augment facial features and model the breast, the thorax, and the buttocks; meanwhile, liquid compositions use them to smooth out minor underlying wrinkling and provide skin softness and resilience. Minimizing or eradicating nasolabial creases and wrinkling, elevating eyebrows, positioning the nose, altering the contour and volume of the lips, sculpting the cheekbones and chin, and performing cosmetic procedures can all produce outstanding outcomes. More recently, it has been demonstrated that altering the morphology of the labia is similarly efficient (labiaplasty). The total effect lasts for around 6 months after intradermal or subcutaneous injections of small amounts of HA. Additionally, several lotions and oral formulations containing KH are available at pharmacies, but no randomized studies have yet demonstrated a beneficial, long-lasting smoothing impact on skin wrinkles when utilizing these techniques. In a combination of high-molecular-weight ingredients by generating a protective barrier that makes skin softer and smoother, HA is utilized in cosmetics that treat the face, eyes, neck, and body, as well as cellulite and stretch mark issues. KH, on the other hand, keeps the skin’s moisture level by covering the stratum corneum and not penetrating the skin’s deeper layers (Kogan et al. 2007; Van Beek et al. 2008; Kanchwala et al. 2005; Tezel and Fredrickson 2008; Washburn et al. 2013). Implantable biosensors are based on biological, chemical, or electrical signals converted by analyte-specific reactions at the junction of an enzyme-containing bioreceptor subunit (Karunakaran et al. 2015; Chalklen et al. 2020). The electrode, which could generate a detectable signal for later transformation and preparation into a visualizable representation to be commenced in connection with the preliminary analyte existence or quantity, is a crucial part of specific biosensor equipment (Karunakaran et al. 2015; Kim et al. 2019b). Damaged tissue development at the sensor side and peptide and cell adhesion to the electrode, both parts of the body’s natural response to foreign implantation, have all been demonstrated to degrade

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biosensor functionality and lessen its sensing capabilities (Kim et al. 2019b; Campbell and Wu 2018; Wellman and Kozai 2017). This is a common problem for implantable bioelectrodes. In order to maintain the electrical capabilities of the sensing complex while simultaneously providing the electrode surface with antifouling properties, HA was combined with polydopamine (PDA) and created an electrochemical bioelectrode. The hydrophilic property of hyaluronan, which permitted it to maintain and absorb a significant amount of moisture surrounding the electrode, is probably what gave HA its anti-fouling properties, whereas PDA allowed for enhanced immobilization by entrapping HA molecules. The authors list a reduction in unwanted fibroblast adhesion, protein adsorption, and scar tissue formation as advantages of such sensor electrode modification (Kim et al. 2019b).

13.5

Nutraceuticals

Tests conducted in vivo and in vitro have exhibited that hyaluronan has antioxidant properties. By removing ROS like the hydroxyl radical, the anti-inflammatory HA helps to start and regulate the inflammatory response (Liu et al. 2008b; Zhang et al. 2010; Li et al. 2015). The low-molecular-weight oligo-HA (oHA) is a more stable, bioavailable, and effective DPPH radical scavenger than conventional HA. Additionally, compared to the conventional native HA, oHA demonstrated several functional variations, including immunostimulatory and antiangiogenic properties (Ke et al. 2011; Ke et al. 2013). Using cutting-edge nanotechnology, the nutraceutical hyalurosome nano-food delivery system (CRHs) packed with curcumin and resveratrol (Cur, Res) was developed to increase the durability, accessibility, and antioxidant capacity of insoluble antioxidants. The oligohyaluronan-curcumin (oHC) polymers used to create the CRHs, also known as sustainable amphiphilic oligo-hyaluronan-curcumin, comprise both Cur and Res. Comparing CRHs to single formulations and liposomes, they showed better radical scavenging activity in a dose-dependent manner. Because of this, the new CRH nano-food has demonstrated potential properties for an improved gastrointestinal formulation and a novel nano-food delivery method using juice, yogurt, and dietary supplements (Guo et al. 2018). Utilizing glucosamine sulfate (20 mg/kg body weight per day), chondroitin sulfate (1200 mg/day), and HA (50–100 mg/day) as nutritional supplements can be beneficial for providing antioxidant, anabolic, and high anticatabolic properties with low, moderate, and high activity, respectively (Castrogiovanni et al. 2016). Future potential nutraceutical Aloe sterol encourages the production of type I and type III collagen in dermal fibroblasts while reducing the expression of MMP-2 and MMP-9 and perhaps raising HA levels. It enhances collagen production to improve skin suppleness, guard against the breakdown of collagen and ECM, and may be raise the amount of HA in the dermis (Souyoul et al. 2018).

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Conclusion

HA has been widely utilized in controlled release and targeted drug delivery systems. In vivo experiment reports are scarce, and most current investigations are in the in vitro experimental stage. However, it is anticipated that if new materials and technologies are discovered and developed, the potential for HA as drug carriers will grow even more. The most efficient method of illness prevention is vaccination, which the scientific community is researching and developing. In aesthetic dermatology, HA adjunct therapy increases tissue hydration and cellular rigidity to mechanical stress and has fewer side effects than the application of glucocorticoids and NSAIDs (non-steroidal anti-inflammatory drugs) in the diagnosis of osteoarthritic chronic diseases. This is because HA has lubricating properties for the corneal endothelium. Numerous studies suggested its function in medicines, liver illnesses, and tumor markers; nevertheless, more studies are required to reach firm conclusions. Additionally, there are still limitations in the development and application of drug carriers, biomedicine, and nutraceuticals that utilize diverse and targeted chemical modifications of HA. There is still a long way to go before HA is widely used in clinical settings and industrialized as a drug delivery system, enhancing its biomedicine properties.

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Molecular Docking in Drug Designing and Metabolism

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Shyamalima Saikia, Minakshi Puzari, and Pankaj Chetia

Abstract

The traditional drug discovery methods and analysis of drug target interactions and physiological responses through biological experiments are expensive and time-consuming, with higher failure rates in preclinical trials. The introduction of bioinformatics and new molecular modeling approaches to the field of drug discovery has led to drastic expansion and success of the drug discovery field by unmasking unifying principles and catalyzing the discovery of new biological insights that aid in analyzing, comparing, and understanding genomic data at the molecular level. Molecular docking is one of the most basic and critical computer-assisted drug discovery strategies that predict the interaction between the ligand and receptor of known three-dimensional structure to form a stable complex through different methods like computational screening, identification of hit, and optimization of lead molecules. It anticipates the favored orientation of the small molecule or drug candidate against the receiving molecule or drug target by evaluating a particular pose by predicting the strength of connection by counting several favorable intermolecular interactions and binding affinity utilizing scoring functions. This technique predicts the top binding conformations or poses of a ligand against the target of interest. The possible interactions of ligand-receptor are sorted via scoring functions to predict the structure most likely to be present in nature. The ultimate goals of molecular docking are to attain an optimized docked conformer of the interacting molecules with the highest affinity, to simulate the molecular identification process computationally, and to achieve a stable complex with minimized free energy of the whole system. Optimization of ADME (absorption, distribution, metabolism, and excretion) S. Saikia · M. Puzari (✉) · P. Chetia Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_14

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properties and toxicity analysis is the most challenging part of the drug discovery process calculation, which is essential to predict the behavior of a drug molecule inside the living body to facilitate the search for the most potential drug candidate. Keywords

Computer-aided drug design · Molecular docking · Computational tool · Drug target identification · Binding energy · Protein-ligand interaction

Abbreviations CADD CATH GPCR HTS LBDD MCODE MDS MMDB MSD NDB NMR PDB QSAR RCSB SBDD SBVS SMILES

14.1

Computer-aided drug designing Class, architecture, topology, and homologous superfamily database G protein-coupled receptors High-throughput screening Ligand-based drug design Molecular complex detection Molecular dynamic simulation Molecular modeling database Macromolecular structure database (MSD) Nucleic acid database Nuclear magnetic resonance Protein Data Bank Qualitative structure-based assessment relationship Research collaboratory for structural Bioinformatics Structure-based drug design Structure-based virtual screening Simplified molecular input line entry system

Introduction

Bioinformatics has created a considerable transformation in biological science and changed the global perspective by unmasking unifying principles and catalyzing the discovery of new biological insights that aid in analyzing, comparing, and understanding genomic data at the molecular level (Baxevanis and Ouellette 2004; Wong 2004). The successful representation of the molecular structure is closely related to advancements in nuclear physics and computer technologies. The three-dimensional (3D) models of the molecules can be predicted through molecular and electronic structures achieved by analyzing their physical and chemical properties through molecular modeling. The X-ray crystallographic data, fragment libraries, and two-dimensional sketching of the molecules, followed by 2–3-dimensional conversion, are the primary methods for generating three-dimensional structures of the

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molecules. Techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) have also contributed to understanding the atomic structures of proteins and their complexes. These advances allow the computational approaches to permeate all the aspects of drug discovery today through different methods like computational screening, identification of hits, and optimization of lead molecules (Baxevanis and Ouellette 2004; Wong 2004). New molecular modeling approaches with advances in computational technologies have led to drastic expansion and success of the drug discovery field. Molecular docking is an extensively used cheap tool that has solved the riddles of biological system operation. However, further precision and accuracy are required to confirm the correct conformation within the active sites of a given target molecule (Baxevanis and Ouellette 2004; Wong 2004). Recent technological advancements have incrementally contributed to an increase in the accuracy of predictions. Enormous research over the previous two decades has been pursued to analyze the different docking algorithms and pinpoint the residues involved in interaction with small molecules. The explosion of structural informatics, genomics, and proteomics plays a principal role in advancing modern drug discovery and development. CADD (computer-aided drug design) is categorized into ligand-based drug design (LBDD) and structure-based drug design (SBDD) based on the accessibility and employment of the target structure (Ferreira and Andricopulo 2018; Gschwend et al. 1996). Molecular docking is one of the most basic and critical computer-assisted drug discovery strategies that predict the interaction between a small molecule to a large macromolecule of known three-dimensional structure to form a stable complex (Bender et al. 2021; Ferreira et al. 2015). It anticipates the favored orientation of the small molecule or drug candidate against the receiving molecule or drug target by evaluating a particular pose by predicting the strength of connection by counting several favorable intermolecular interactions and binding affinity utilizing scoring functions. The orientation of bound molecules gives an idea of the binding energy, binding affinity, and stability of the bonds (Raval and Ganatra 2022; van Montfort and Workman 2017). The ultimate goal of molecular docking is to attain an optimized docked conformer of the interacting molecules, to simulate the molecular identification process computationally, and to achieve a stable complex with the minimized free energy of the whole system. Various docking programs come with packages that enable visualization of the docked complexes and calculating the docking score (Elokely and Doerksen 2013; Pantsar and Poso 2018). Final predicted binding free energy (ΔGbind) is predicted in terms of dispersion and repulsion (ΔGvdw), desolvation (ΔGdesolv), hydrogen bond (ΔGhbond), electrostatic interaction (ΔGelec), final total internal energy (ΔGtotal), torsional free energy (ΔGtor), and unbound system’s energy (ΔGunb) (Åqvist et al. 1994; Pantsar and Poso 2018).

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Computer-Aided Drug Design (CADD)

The computer-aided drug design (CADD) is the most promising computational approach employed for identifying drug targets of various diseases to find the most suitable ligand that binds strongly to the target. CADD combined with wet lab validation elucidates the search for new medicines and to design of novel drugs for unknown or known targets (Anderson 2003; Shulga et al. 2022). The traditional method of drug designing is time-consuming, laborious, and expensive, generally takes 20–25 years for an adequate drug to come to the market, and costs around 350 million USD. With the ever-increasing demands for new therapeutics, the concept of CADD draws greater attention due to its higher efficiency, selectivity, low toxicity, short time duration, and better fit with various pharmacokinetic parameters (Batool et al. 2019; Maia et al. 2020). CADD is well accepted for three main reasons: (a) in silico screening for identification of active drug candidates, (b) hit or lead optimization, and (c) design of novel compounds. Finally, the lead molecules obtained through docking analysis will have to face preclinical as well as clinical trials before getting approval from the regulatory bodies. In the process, a large number of databases of compounds are screened for searching binding capacity against targets, followed by selecting a set of compounds and suggested for in vitro testing. The purpose is to increase the hit rate of the drug compound by reducing the number of compounds to test experimentally. It requires minimal compound design or prior knowledge; however, it yields numerous hit compounds where promising drug candidates have been elected (Bruch et al. 2020; van Montfort and Workman 2017). The main aim of CADD is to screen, optimize, and evaluate the activity of compounds or ligands against the targets. CADD is based on target identification, structure prediction, active site or binding site identification, validation, understanding the molecular protein-ligand interaction, screening of compounds followed by molecular dynamic simulation based on physiological conditions, tallying with ADMET properties, and estimation of biological activity through QSAR (qualitative structure-based assessment relationship) required for the identification of better lead compounds (Chandrasekaran et al. 2018; Elokely and Doerksen 2013; Wang and Zhu 2016). CADD helps narrow down potential lead molecules having good pharmacokinetic properties from a vast ocean of molecule libraries. After screening out and filtering them, one can use the docking tools to understand the interaction of the molecules with target molecules and have insight into different pathways (Arjmand et al. 2022; Ferreira et al. 2015; Samsdodd 2005). CADD aims to use quantum mechanics, molecular mechanics, and statistical tools to make the drug discovery and development process faster by eliminating undesirable molecules and selecting candidates with more potential for success (Ojima 2008; Tang et al. 2006; Van Drie 2007). CADD is generally categorized into two main types: target- or structure-based drug designing (SBDD) and ligandbased drug designing (LBDD) (Ferreira et al. 2015; Kitchen 2017). In target-based drug designing, the target structure is known, but the ligand structure is unknown, like having a lock without a key, and the job is to design a key to open the lock. Whereas in LBDD, the structure of the ligand is known, but the

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target structure is unknown, like having the key of an unknown lock, and the main job is to find the lock for that specific key. SBDD is more reliable and robust in the drug lead discovery process directing the discovery of a lead drug molecule with the most negligible micromolar affinity for the drug target (Baxevanis and Ouellette 2004; Wong 2004).

14.2.1 Structure-Based Drug Designing (SBDD) Structure-based drug design is the approach used to either design an optimized molecule or identify a chemical structure from a million compounds suitable for clinical testing. The three-dimensional structure of the drug target is the fundamental prerequisite for SBDD. For drug targets whose structure is unknown, structure prediction tools can be used, such as the homology modeling tool ( Ferreira et al. 2015; Ferreira and Andricopulo 2018). The well-defined binding pockets of the targets are identified using specific tools by screening for appropriate target properties like hydrophobicity, number of hydrogen bonds, energy potential, volume, and solvent accessible surface area. Certain filters like Lipinski’s rule, ADMET filters, and QSAR models are used stepwise for the virtual screening of libraries of lead compounds of natural or synthetic origin ( Ferreira et al. 2015; Ferreira and Andricopulo 2018). After the success of X-ray crystallography in providing protein structures, the idea of using the macromolecule structure to design new therapeutic agents was rooted. One such example of the use of crystal structure to formulate a solution was the work of John Kendrew and Max Perutz on hemoglobin and myoglobin to understand their oxygen-carrying properties. The experiment sheds light on the molecular basis of sickle cell anemia and its potential cure options (Anderson 2003; Batool et al. 2019). The amino acid sequence and three-dimensional structure of insulin determined by Fred Sanger and Dorothy Hodgkin, respectively, led to the production of synthetic insulin for diabetes treatment. These novel studies epitomize how therapeutic exploitation is essential for understanding protein structure and function. The availability of the three-dimensional structures of therapeutically significant proteins allowed the identification of potential inhibitor binding sites that formed the foundation for structure-based drug design (SBDD) (Gschwend et al. 1996; Maia et al. 2020; Van Drie 2007). Additionally, the exponential increase in the number of macromolecular structures deposited in the PDB (Protein Data Bank) database stimulated the development of sophisticated software and the CADD process. SBDD is more advantageous because of the experimentally solved available structures of proteins or targets in the Protein Data Bank (PDB), which also serve as templates for homology modeling when the structure of interest is missing. SBDD analyze macromolecular target three-dimensional structural information, mainly of proteins and RNAs, to identify the target sites and interactions that are otherwise crucial for their respective biological functions (Kim et al. 2016).

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Structure-based virtual screening (SBVS) attempts to predict the most suitable interaction between the small molecules against a molecular target or macromolecule to form a complex. In this approach, compounds are elected and classified based on their affinity for the receptor site, with the most promising compounds at the top of the list. Molecular docking is noteworthy among the techniques of SBVS due to its low cost and better results. The evaluated molecules are sorted according to their affinity for the receptor site with a score function that verifies the likelihood of a binding site unfolding the association between the target and ligand (Ferreira et al. 2015; Zheng et al. 2013). It is a powerful strategy of drug designing with better efficiency and success rate in avoiding molecular obesity, which has been considered one of the main reasons for drug candidates’ high attrition rates and low productivity in the pharmaceutical industry. High molecular weight and high complexity are the main reasons for the failure of drug molecules. Polanski’s analysis revealed that the lesser the drug molecules’ complexity, the more the market success (Macalino et al. 2015; Maia et al. 2020; Meng et al. 2011; Van Drie 2007). Thus the drug candidates should be analyzed before structural simplification. Reducing the molecular complexity and molecular weight positively influences the pharmacokinetic and pharmacodynamic profile. The number of chiral structures present, the number of rings, and how they are linked are the key factors contributing to molecular complexity. SBDD can be defined as the design and optimization of the chemical structure based on the structure of its biological target (Ferreira et al. 2015; Kiriiri et al. 2020; Torres et al. 2019). In SBDD, with the help of computational chemistry tools, the structure of a target protein is used as the key to identifying potential drug compounds that could bind to the target in such a way that thereby inhibits the activity of the target protein. Molecular docking calculates the orientation and conformation of the docked compounds in the active pockets of the target, and a variety of scoring criteria is used to predict the most stable interaction and to design the best compounds against the target protein experimentally (Ferreira et al. 2015; Kitchen 2017; van Montfort and Workman 2017). The molecular dynamic simulation (MDS) model, the behavior of complex molecular systems based on the chemical entities, fundamental properties, and interaction between them provide a more dynamic view of interactions between compounds and their targets of SBDD (Bender et al. 2021; Maia et al. 2020; Meng et al. 2011; Pantsar and Poso 2018).

14.2.2 Ligand-Based Drug Designing (LBDD) Ligand-based drug design (LBDD) is the indirect approach of CADD that starts with either a single compound or a set of compounds potent against a target, the threedimensional structure of which is unavailable (Ajjarapu et al. 2022; Attwood et al. 2020). With the presence of ligand structure, this approach is based on the principle that molecules with similar structures are likely to have similar properties. In LBDD, the compounds are selected based on chemical similarity, and computational algorithms are used to predict the biological activity of the prepared model. Both

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structural and physicochemical molecular descriptors, like molecular weight, hydrophobicity, ring content, presence of rotational bonds, chirality, bond distance, atom types, aromaticity indices, etc., are considered. Statistical approaches are also used to correlate ligand activity to structural information (Ajjarapu et al. 2022; Baxevanis and Ouellette 2004; Ferreira and Andricopulo 2018; Wong 2004). The most common techniques include QSAR (quantitative structure-activity relationship), similarity-based search, and pharmacophore modeling. QSAR-based virtual screening approaches, combined with HTS (high-throughput screening), can also be used to extend the panel of bioactive compounds against the target of interest to increase the overall hit rate (Gautam et al. 2022; Huang et al. 2010).

14.3

Identification of Drug Targets

The drug discovery begins with identifying a possible biological target and elucidating its role in the disease. A target is a biochemical entity (a protein, RNA, or gene) to which a drug can bind and elicit a physiological change. A target must possess an active site or binding pocket to which a potential drug can attach. An optimal target should be druggable, safe, efficient, and able to fulfill commercial requirements (Baxevanis and Ouellette 2004; Floris et al. 2018). Targets can be identified using public databases like DrugBank, TTD, or literature search. Traditionally, G protein-coupled receptors (GPCRs) and kinases have served as excellent drug targets for many years. These days, we see the emergence of new modalities for treating disease, including previously less tractable targets (Alhosaini et al. 2021). One example is the recent advances in cancer therapy by approving the first selective KRAS inhibitor, sotorasib, in 2021 for treating tumors with the KRAS G12C mutation (Arjmand et al. 2022). Also, bromodomain inhibitors have emerged as a promising class of anticancer agents. The concept of protein degradation by heterobifunctional small-molecule proteolysis-targeting chimeras (PROTACs) has also gained much interest in recent years, as well as gene therapy (e.g., the use of CRISPR technology) which can offer personalized treatments for patients. A drug’s polypharmacological profile can lead to desirable or adverse effects. Thus, drug target interaction profiling is crucial in drug discovery to maximize the therapeutic action and minimize the undesirable effects on a patient’s body (Rask-Andersen et al. 2011; Wong 2004). The conventional methods of identification of drug target interactions using biological experiments are expensive and time-consuming, due to which they are substituted with computational approaches. With the advance in network pharmacology and systems biology, drug discovery approaches have been shifted from linear mode (one drug, one target, one disease) to network mode (multidrug, multitarget, and multi-disease approach) (Floris et al. 2018; Torres et al. 2019). A detailed understanding of molecules and mechanisms involved in drug pathophysiology is essential for correctly identifying drug targets. However, the search for new targets with pharmacological importance is limited due to the availability of limited information about disease pathogenesis (Gashaw et al. 2012). Identifying drug targets is

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the initial step of drug discovery, followed by target validation, hit generation, and lead development. The characterization and identification of drug targets start with identifying a particular gene or protein in the disease through the analysis of their role by molecular mechanism study (Floris et al. 2018; Gashaw et al. 2012). Genetic-driven drug target identification and validation is a powerful tool enhancing the success of the drug discovery program started in 2003, with the discovery of the reason behind autosomal dominant hyperglycemia. Gain of function of the gene PCSK9 encoding for pre-protein convertase subtilisin protein causes autosomal dominant hyperglycemia, whereas loss of function of the same leads to a considerable decrease in plasma level of low-density lipoprotein cholesterol, which plays a crucial role in the reduction of coronary heart disease (CHD) (Floris et al. 2018; Zdrazil et al. 2020). Thus, therapeutic inhibition of PCSK9 gene could decrease the concentration of low-density lipoprotein and thereby prevent CHD. Around 73% of projects implemented to increase drug discovery success, able to discover therapeutic compounds with a genetic link between the target and disease, successfully succeed in phase II trials. Genome-wide association study increases the success of drug development from 2% at the preclinical stage to 8.2% among approved drugs (Attwood et al. 2021; Gashaw et al. 2012; Zdrazil et al. 2020). Anderson et al. (2011) identified 435 effective drug targets encoded by the human genome modulated by 989 drugs through 2242 drug target interactions (Rask-Andersen et al. 2011). The cell surface receptors or extracellular proteins serve as the most suitable drug targets. Most launching antibodies developed for cancer and inflammatory disease treatment antibody-drug conjugates belong to this class. Identifying target properties comes from RNA or protein expression in the target tissue or healthy tissue comparison. Focused proteomics, like activity-based protein profiling (ABPP), identifies differential enzymatic activity on diseased versus targeted or normal tissue. Other sources of novel target ideas are informed on genetic alteration, phenotypic knockout mice, somatic mutations, etc. Key factors contributing to target identification include gene expression profiling, phylogenetic analysis, functional screening, genetic association, etc. (Floris et al. 2018; Gashaw et al. 2012; Rask-Andersen et al. 2011). Since the year 2014, the US National Institute of Health termed Illuminating the Druggable Genome (IDG) have made a strategic effort focusing on exploring the current understanding of druggable proteins to find the association of compounds, targets, monoclonal antibodies, and diseased which are mapped, resulting in developing target level which classifies the drug targets according to the depth of investigation. The greater the number of targets hit by a drug, more is the probability of the drug causing an adverse effect. Thus highly selective targeting governs the success rate of the drug discovery process (Zdrazil et al. 2020). The G protein-coupled receptors (GPCR) constitute 800 genes, covering about 2% of the human genome, and 40% of drugs target GPCR. As of 2020, out of 1500 drugs that have been approved by FDA (Food and Drug Administration), around 460 drugs target GPCR. About 94% of the drugs target Class A GPCR, followed by B = 4%, class C 2%, and class F 2%. For example, most antidiabetic drugs, like pramlintide, sitagliptin, albiglutide, etc., for treating type 1 and type 2 diabetes, target GPCR receptors. Similarly, alpha and beta androgenic receptors are targeted

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by most of the antiplatelet and cardiac drugs. In addition to small molecules, GPCR can be targeted by monoclonal antibodies, like several monoclonal antibodies that have been generated and treated as drug candidates, like mogamulizumab in leukemia treatment. GPCR nanobodies are also generated against chemokine receptors ten times smaller than the antibodies, only with a heavy chain (Alhosaini et al. 2021). A detailed understanding of the structure, property, and identity of the drug targets encoded by the human genome is of great importance. In 1966, Drews and Ryser estimated the number of potential drug targets in the human genome, which was 5000–10,000 for the first time. They listed 483 drug targets in the ninth edition of the pharmacological basis of therapeutics after analyzing drugs and drug targets. After sequencing the human genome, Hopkin and Groom estimated 120 targets for marketed small molecule drugs by analyzing the databases of the investigational drug and pharma projects. The publically available drug bank database was launched in 2006 (Rask-Andersen et al. 2011). Properties of an ideal drug target are the following: 1. The target should have a crucial role in the pathophysiology of the disease. 2. Modulation of the target should be less critical under physiological conditions or in the disease. 3. Target should not be evenly distributed throughout the body. 4. The target has favorable assay ability enabling high-throughput screening. 5. The target’s three-dimensional structure should be available to assess druggability. 6. The target should possess an excellent toxicity profile, and potential adverse effects can be predicted using phenotypic data. 7. The proposed target should have a favorable intellectual property (IP) status (applicable for pharma companies). Network-based approaches predict the novel drug target genes or drugs from multiple algorithms. The network-based methods are more reliable, fast, and independent of the three-dimensional structure of the drug target as compared to molecular docking-based methods (Baxevanis and Ouellette 2004). Recently, a new technique called thermal proteome profiling has emerged for target identification. This method uses quantitative mass spectroscopy to monitor the changes in the targets or proteins across the proteome. It is based on the principle that when proteins are heated, they denture first and become insoluble. Thermal proteome profiling (TPP) is a cellular thermal shift assay based on mass spectroscopic-based proteomics and determines the thermal stability of each protein in a biological system (Baxevanis and Ouellette 2004). The interaction of a protein with a ligand can change its thermal stability by monitoring proteome-wide thermal stability; it is possible to see which protein changes in thermal stability when tissues are treated with a drug. This method is performed in cells and proteins maintained in the natural environment (Baxevanis and Ouellette 2004). Target validation is an essential step in drug discovery. The molecular target validation ensures that a drug target is directly involved in a disease progression or

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mechanism, modulation of which is likely to achieve a therapeutic effect or desired result. Target validation consists of determining the structure-activity relationship, genetic manipulation, knockdown, and overexpression, generating a drug-resistant mutant, and altering the signaling pathways downstream of the presumed target (Baxevanis and Ouellette 2004). Earlier, most drug targets were obtained from the literature that gives insights into the molecular pathways and genetic determinants linked to the disease. With recent advancements, identifying novel drug targets is possible through genomics, functional genomics, machine learning, and artificial intelligence (Tsuji et al. 2021). The genomic data reveals the participation of essential genes and pathways in the progression of the disease but cannot fully depict what is happening inside the cells, tissues, organs, or the whole body. Functional genomic approaches are prevalent in searching for novel drug targets (Gashaw et al. 2012). Functional genomics allows us to more fully understand the relationship between genotypes and phenotypes. It underpins the identification and validation of novel targets, elucidating the mechanism of pathways and biological pathways affecting the disease. Technological advances made it possible to carry out the multi-omic characterization of tissues from thousands of patients, including genomic, transcriptomic, proteomic, metabolomic, and lipidomic analysis, together with detailed clinical data leading to the preparation of phenotypic fingerprint of the disease state and patient population (Baxevanis and Ouellette 2004; Wong 2004). From this, we can uncover the pathways and targets responsible for the disease progression, along with the shared mechanisms underpinning conditions with complex, often overlapping phenotypes.

14.3.1 Macromolecular Databases The proteins are the most complex macromolecules exhibiting variety and irregularity in shape, size, and mobility. The origin of bioinformatics lies in the most exciting and applied field of structure analysis, also called structural bioinformatics. With the help of bioinformatics, it becomes easier to analyze, visualize, predict, and evaluate the structure of proteins (Baxevanis and Ouellette 2004). Hierarchical is the most preferred method for describing protein structure, where proteins are visualized at different structural levels with increasing structural complexity (Baxevanis and Ouellette 2004). Only two known experimental techniques can reveal a protein’s comprehensive structural information at atomic resolution: X-ray crystallography and NMR spectroscopy (Baxevanis and Ouellette 2004). Protein crystallography is a challenging and complex process because it only allows the determination of large macromolecular structures, like large cytoplasmic or membrane-bound proteins (Baxevanis and Ouellette 2004). The first X-ray crystallographic structure of myoglobin protein was determined in the late 1950s, and from that point, more than 20,000 protein structures have been selected by X-ray crystallography technique. NMR spectroscopy is much newer and more reliable than X-ray crystallography; the first structure was solved in 1983. NMR is unique because it allows the determination of the structure and dynamics of molecules in a liquid state or in a physiological

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environment to generate multiple configurations of the experimental protein, all of which can be superimposed on each other to produce a blurrogram using computerbased constraint minimization of defined parameters (Baxevanis and Ouellette 2004; Berman 2005). The Protein Data Bank, initially set up at the Brookhaven National Library by Walter Hamilton in 1971, started with only seven protein structures and was the first electronic protein structure database. In 1998, PDB (Protein Data Bank) was moved from Brookhaven to the Research Collaboratory for Structural Bioinformatics (RCSB). It is the open-access US data center for the global protein DataBank archive of experimentally determined three-dimensional structure data for biological macromolecules (proteins, nucleic acids, and carbohydrates and their complexes) (Baxevanis and Ouellette 2004; Berman 2005). PDB archive currently contains more than 1 TB of structure data for proteins, DNA, and RNA (Kim et al. 2016). Apart from PDB, many secondary or curated databases take raw data from PDB. The macromolecular structure database (MSD), Molecular Modeling Database (MMDB), PDBsum Database, and TargetDB are examples of protein structure databases (Baxevanis and Ouellette 2004). For visualization of protein structure, G.N. Ramachandran et al., in 1963, developed a new approach for visualization of a protein considering x, y, and z coordinates that give a clear-cut idea about the protein properties. This approach discovered before computer visualization software is also called Ramachandran plot (Baxevanis and Ouellette 2004). Modern high-speed computers with high-end graphic cards made the high-quality protein model creation process much simpler and more accessible (Baxevanis and Ouellette 2004). RasMol (RASter MOLecule), introduced in 1993, was a significant breakthrough in software-driven three-dimensional protein structure interpretation. WebMol, Cn3D, SwissPDB-Viewer, and Deepview are examples of popular macromolecular visualization packages (Baxevanis and Ouellette 2004). Class, architecture, topology, and homologous (CATH) superfamily database, created by J. Thornton et al. in 1993, is a protein structure classification database that contains hierarchical domain classification of protein structures available in the PDB database (Baxevanis and Ouellette 2004). CATH’s latest version includes 1,14,215 domains, 2178 homologous superfamilies, and 1110 folds. This database categorized the proteins into groups based on their secondary structure content, sequence similarity, and fold. The SCOP (Structural Classification of Proteins) database is a hierarchically structured database like CATH developed in 1995 by Murzin et al. (Baxevanis and Ouellette 2004; Berman 2005). It provides comprehensive information about proteins’ structural and evolutionary relationship with known structures available in PDB. On the other hand, Nucleic Acid Database (NDB) is a comprehensive database that provides experimentally determined three-dimensional structures of nucleic acids and complex assemblies (Coimbatore Narayanan et al. 2014).

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14.3.2 Metabolic Pathway Databases Understanding the metabolic pathways is the most important in the drug discovery field. A metabolic pathway can be defined as a series of interlinked reactions of reactants and intermediates or metabolites, which are modified into products that, in turn, can be used in different cellular activities for growth and maintenance (Labena et al. 2018). Understanding the working mechanism of a cell at the molecular level by mapping cellular and molecular interaction networks is a significant challenge for biologists. However, these metabolic interactions or pathways are necessary to emphasize metabolic or signaling pathways, reaction participants, and the relationship between genes, enzymes, and reactions in detail (Wittig 2001). In the drug development process, the compounds are screened for desirable gene expression or inhibition, enzymatic activity, or signal transduction (Lee et al. 2022). The genetic and metabolic pathways help identify the target’s role in disease and identify critical intermediates or enzymes that can serve as potential targets. The signaling cascade serves as an off-switch in metabolic processes. The most commonly used pathway databases include WIT, KEGG, pathway commons, MetaCyc, Ecocyc, PathDB, UM-BBD, ExPASy biochemical pathways, etc. (Baxevanis and Ouellette 2004; Likić 2006; Wittig 2001). The accepted dogma for establishing the correlation between genotype and phenotype is that in metabolism, the peptide molecules act as signaling switches in the final gene expression (Wittig 2001). For example, several phenomena are associated with carbohydrate metabolism, including glycolysis, Kreb cycle, glycogenesis, glycogenolysis, ATP synthesis, etc., through several interconnected intermediates, conversion of which are catalyzed by several enzymes, with the recruitment or evolution of energy in the form of ATP. The deficiency of one such enzyme, glucose-6-phosphatase, causes glycogen storage disease type I (GSD-I), also known as von Gierke’s disease, an autosomal recessive disorder caused by the deficiency of microsomal activity of glucose-6-phosphatase (G6Pase) enzyme (Froissart et al. 2011). Clinical manifestations include short stature, hepatomegaly resulting from glycogen and fat accumulation in the liver, hypoglycemia, hyperuricemia, and lactic acidemia. The enzyme glucose-6-phosphatase, a membrane-bound enzyme, plays a significant role in the breakdown of stored liver glycogen into glucose during starvation; deficiency caused by mutation of the glucose-6-phosphatase enzyme coding gene leads to hypoglycemia or low blood sugar, resulting from blockage of the last step of glycogenolysis or gluconeogenesis (Froissart et al. 2011). Hyperlactacidemia and glycogen storage is due to excess glucose-6-phosphate, which cannot be metabolized into glucose. Ultimately, fructose, glycerol, and galactose contribute to hyperlactacidemia. Increased synthesis of acetyl-CoA through the malonyl-coenzyme A pathway leads to hyperlipidemia and aciduria by inhibiting carnitine palmitoyltransferase I (Froissart et al. 2011). Most cancer cells exhibit an increased rate of glycolysis and use this as the main pathway of ATP generation as the primary energy source (Arjmand et al. 2022). This phenomenon is called the Warburg effect. P53 is a tumor suppressor gene, activation of which is induced by DNA damage, a number of stress signals, oxidative stress, and activation

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of oncogenes. The p53 protein acts as the transcriptional activator of p53-regulated genes that lead to cell cycle arrest, cellular senescence, or apoptosis (Arjmand et al. 2022; Ling and Huang 2020). Loss of function of tumor suppressor gene or gain of function of oncogene modulates the p53-regulated gene functions and modulates the well-balanced environment earlier maintained by P53, thereby increasing uncontrolled cell growth and glucose uptake. Therefore, since P53 can explain the cancer metabolic phenotype, the interconnected metabolic pathway involving P53 can serve as potent anticancer drug targets.

14.3.3 Computational Interaction Networks and Identification of Alternate Drug Targets All biological system works in a complex and integrated manner. The genomic and proteomic approach introduced a new term, “protein interactome,” which represents the cell’s entire set of protein-protein interactions (Wong 2004). This new approach is helpful in the drug target discovery process as it provides a comprehensive description of the protein-protein interaction network and helps identify new genes or proteins by revealing their association with the functional partners. The genes and proteins correlated with expression serve as potential drug targets of new therapeutics (Wong 2004). The Biomolecular Interaction Network Database (BIND) is one of the largest hubs of freely available molecular interaction databases that stores protein-protein interactions, protein-DNA interactions, and genetic interactions of yeast Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans (Baxevanis and Ouellette 2004). The STRING is another notable database for the prediction of protein-protein interaction, which includes both physical and functional associations that store information from computational calculation, knowledge transfer between organisms, and various interactions aggregated from other databases. Currently, the STRING database covers 2,45,84,628 proteins from 5090 organisms, interactions of which are driven by genomic context prediction, high-throughput lab experiments obtained from various sources such as experiments, systematic co-expression analysis, curated databases, shared selective signals across genomes, scientific literature mining, and gene ontology-based exchange of knowledge in several animals (Baxevanis and Ouellette 2004). The predicted functional partners for the query protein are listed by this tool and estimated by confidence score, indicating the interaction between nodes connected to each other through different pathways. The recently available large number of complete genome sequences allowed for finding sequence-based partners that correlate with protein interactions. The gene neighborhood approach was the first to predict protein interaction networks from a genomic context. The phylogenetic profiles, gene fusion, and in silico two hybrid strategies are the ultimate forms of gene co-localization prediction algorithms for molecular interactions (Baxevanis and Ouellette 2004). On the other hand, biologists used network and pathway visualization tools to map biomolecular interaction networks to a graph, representing biomolecules as

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Fig. 14.1 Overview of protein-protein interaction network showing functional partners in differently colored nodes. (Source: STRING Database)

nodes and their interaction as edges (Baxevanis and Ouellette 2004). Cytoscape is a freely available network visualization and analysis tool that helps in finding closely connected gene clusters in a network based on different topological parameters (Baxevanis and Ouellette 2004). The MCODE (Molecular Complex Detection) app of Cytoscape software was used for clustering analysis, finding highly interconnected regions in a network based on an algorithm separating locally dense areas. BioLayout, Osprey, and VisANT are examples of network visualization tools (Baxevanis and Ouellette 2004). Baishya et al. (2019) identified 21 highly associated genes from 1679 functional partners that play a significant role in carbapenem resistance in carbapenem-resistant Enterobacteriaceae (CRE) that could serve as potential drug targets for designing new antimicrobial drugs (Baishya et al. 2019). Puzari et al. (2020) identified potential alternate drug targets from a gene-gene interaction network analysis of efflux pump proteins in Shigella species (Fig. 14.1).

14.3.4 Functional Annotation Study Thousands of proteins have been characterized experimentally in the laboratory, and their functions in different cellular and metabolic processes are recorded in protein databases. Functional annotation and enrichment analysis have been widely used in omic research (Holt and Yandell 2011). Functional annotation can be defined as the

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approach in which biological information is attached to the sequence and structures of genes and proteins. On the other hand, functional enrichment analysis determines the classes of genes and proteins in a large group of proteins and gives insight into their relation with disease phenotype (D. W. Huang et al. 2009). This approach is based on a statistical method that determines significantly enriched groups of genes. The preparation of the network and identification of functional partners alone cannot establish an identified drug target as a potent one (Baishya et al. 2019; D. W. Huang et al. 2009; Zhou et al. 2022). Therefore, predicting the function of all associated genes or proteins obtained from the interaction network is vital for understanding the interaction mechanism through gene co-expression and functional association. ProtoNet and PANDORA are two bioinformatic tools that deal with different aspects of protein annotation and functional predictions. Several gene annotation and functional enrichment analysis softwares or databases employed for predicting functions of genes include DAVID, KEGG, STRING, Cytoscape, Onto-Express, MAPPFinder, GoMiner, EASE, GeneMerge, FuncAssociate, Uniprot, etc. (Zhou et al. 2022). The Database for Annotation, Visualization, and Integrated Discovery (DAVID) is an extensive database with a comprehensive set of functional annotation tools that help investigators uncover the biological meaning behind a large set of genes (Puzari et al. 2020). It identifies GO terms, visualizes genes on BioCarta and pathway KEGG maps, clusters redundant annotation terms, identifies other functionally-related genes not included in the search list, and many more. FunRich database is a human-only database that supports enrichment analysis of biological processes, cellular components, molecular function, site of expression, and transcriptional factors (Baxevanis and Ouellette 2004; Wong 2004; Zhou et al. 2022).

14.4

Structure and Activity of the Drug Target

The membrane or intracellular receptors, different enzymes playing a pivotal role in metabolic processes, ion channels, and carrier molecules served as the primary targets for drug action. The drugs bind to their receptors in a precise manner. Drugs belonging to an individual class can bind only to certain specific targets, and individual targets also recognize only particular classes of drugs (Baxevanis and Ouellette 2004). For example, drugs inhibiting enzyme actions belong to the monoamine oxidase inhibitor group, which are also used in treating exceptional depression cases untreatable with other agents. In reality, no drugs are entirely specific in their actions, which is the reason for the superfluous side effects of commonly used therapeutics. Similarly, statins are another example of enzyme-inhibiting drugs employed globally for the treatment of cardiovascular prophylaxis, and their mechanism of action is now well understood (Li et al. 2021). Li et al. (2021)) explained two main target identification methods for natural compounds: the chemical probe approach and the non-probe approach. The activity-based protein profiling (ABPP) method and compound-centered chemical proteomics (CCCP) fall under the chemical probe approach, whereas omic-based techniques and computational prediction using biological data fall under the non-probe approach. CCCP is a growing field in

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target identification that can identify drug targets or macromolecules without affecting the biological activity and function of the protein (Li et al. 2021).

14.5

Databases of Small Molecules

The advent of computational chemistry and high-throughput screening (HTS) leads to the development of well-designed syntheses of candidate compounds through organic chemistry or by computational-derived methods (Hersey et al. 2015; Tang et al. 2006). Computer analogs of ligands are generated and stored in virtual libraries containing molecules physically unavailable; however, they can be achieved through combinational chemistry. A ligand is an ion, a molecule of any size, or a molecular group that binds to a specific macromolecule at one particular site through non-covalent forces, thereby altering the biochemical activity of the target or receptor (Hersey et al. 2015; Wong 2004). Drug response results from chemical interaction between the macromolecule and ligand at the binding site of the therapeutic target. The ligand’s reversible or irreversible binding either activates or antagonizes the receptor upon binding (Salahudeen and Nishtala 2017). The concentration of ligand or drug at the receptor site determines the effect of the drug effect’s intensity. When a ligand or small molecule binds to a receptor of noninterest, it is called nonspecific binding, linearly proportional to unbound ligand concentration. Most of the receptors are activated by dimerization caused by the binding of ligands leading to autophosphorylation of the cytoplasmic domain, thereby triggering a cascade of reactions (Salahudeen and Nishtala 2017). The ligands or drugs can be both hydrophilic and hydrophobic. Lipophilic or hydrophobic ligands can easily cross the cell membranes, whereas hydrophilic drugs require channels or pores to cross the membrane bilayer (Larrañeta et al. 2018). Structure-based virtual screening has gained so much popularity in recent years; however, the need for a unified database of small molecules to screen against the targets is a barrier to this approach (Chaudhary and Mishra 2016). To overcome this situation, Irwin and Shoichet prepared the ZINC library of 727,842 commercially available molecules, each with a three-dimensional structure, in numerous file formats, including SMILES, DOCK flexibase, mol2, and three-dimensional SDF format (Baxevanis and Ouellette 2004; Wong 2004). SMILES (Simplified Molecular Input Line Entry System) is a chemical notation that allows the users to represent a chemical structure in a way that can be read and used by the computer (O’Boyle 2012). PubChem is an open database or repository for information on chemical molecules, and its biological action was launched in 2004. For the last 18 years, it has grown as an essential chemical information resource for scientific communities serving many areas like medicinal chemistry, chemoinformatics, chemical biology, and drug discovery (Kim et al. 2016). PubChem is currently the world’s most extensive collection of freely accessible databases of chemical information, consisting of three interlinked databases: PubChem Compound, PubChem Substance, and PubChem Bioassay. The naturally occurring chemical compounds with a wide range of activities are vital sources of new drug development. A large number

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of bioactive compounds have been identified by pharmaceutical companies and drug discovery centers (Li et al. 2021). The collection of available small molecule or compound databases for biological research is depicted in Table 14.1.

14.6

Pre-docking Screening of Ligands

14.6.1 In Silico Screening of Ligands for Physicochemical and Pharmacokinetic Properties The ability of a chemical compound to elicit a therapeutic effect depends on the physicochemical properties of the ligand. The effect of the administered drug is the result of the molecular interaction between the drug molecule (ligand) and its target (site of action) (Wong 2004). The spatial arrangement of the ligand’s atoms and the way they interact with the particles of the target site are responsible for the drug’s pharmacological activity. These diversified physicochemical properties of the drug administer the various pharmacological effects of the drugs (Chandrasekaran et al. 2018). The biological behavior of a drug depends on a number of physicochemical properties, which include melting point (MP), boiling point (BP), water solubility, and partition coefficient (LogP), as well as the bioconcentration factor (BCF) (Jambhekar and Breen 2013). Prediction of the drugs’ physicochemical properties and their behavior after administration using computational tools is crucial in the drug discovery process to minimize the failure rate. Lipophilicity is one of the prime physiochemical properties of drugs because of its significant influence on pharmacokinetic properties, including absorption, permeability, distribution, and excretion or route of drug clearance (Blake 2000; Malan and Chetty 2002). The lipophilicity of a drug or ligand has a direct relation to the passive diffusion of the molecule across the hydrophobic cell membrane. A drug candidate should pass the membrane through passive diffusion without the consumption of ATP at a faster rate. Lipophilicity is also related to the solubility, metabolism, and toxicity of the compound. The intracellular macromolecules or targets of the neurotransmitter pathway require drugs with considerable lipophilic nature to accomplish the desired outcome. At the same time, the drug candidate should possess aqueous solubility, which is another fundamental property related to the drug efficiency of drug candidates (Blake 2000; Chandrasekaran et al. 2018; Malan and Chetty 2002). Aqueous solubility affects the uptake, transfer, and elimination of the drug from the body. Drugs with poor solubility eliminate quickly before entering the blood circulation. The permeability of drug molecules is an important physicochemical property described by hydrogen bonding parameters. Permeable drugs cross the biological membrane and blood-brain barrier by passive diffusion along the concentration gradient (Jambhekar and Breen 2013). Besides these, molecular size, hydrogen bond acceptors/donors, and charge are essential physicochemical properties of a drug candidate for efficient binding. Therefore, before molecular docking, the ligands are prepared, and their drug-like behavior is predicted, determining the drug candidate’s success. Lipinski’s rule of five, also known as the rule of five

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Table 14.1 A list of widely used ligand databases Databases PubChem

CheMBL

ZINC

Dr. Duke’s phytochemical and ethnobotanical database DrugBank

SureChEMBL

GLIDA

3DMET

LIGAND

ChemDB

Description World’s most extensive collection of the freely accessible chemical information database, maintained by the National Library of Medicine and National Institutes of Health Chemicals from the European Molecular Biology Laboratory, a curated database of bioactive molecules with drug-like properties, bringing together chemical, bioactivity, and genomic data for developing effective new drugs Freely accessible database of commercially available compounds for virtual screening, provided by Irwin and Shoichet laboratories, University of California An online database developed by James A. Duke that stores details of phytochemicals with biological activity with toxicity data A freely accessible, comprehensive, global online database that stores information on drugs and drug targets. It was developed by the University of Alberta and the Metabolomics Innovation Centre, Canada This is a publicly available largescale database of bioactive molecules with drug-like properties maintained by the European Molecular Biology Laboratory GPCR ligand database for chemical genomic drug discovery, a GPCR-related chemical database, was founded in 2008 Three-dimensional structure database of natural metabolites Composite database of chemical compounds and reactions of biological pathways, released in 1998 A freely available database of small molecules, founded in 2005

Features Consist of three interlinked databases, compound, substance, and bioassay

Current release ChEMBL31, with 2,331,700 distinct compounds and 15,072 targets

Contains 230 million purchasable compounds in ready-to-dock, three-dimensional conformation

A user-focused tool with a large number of plants with chemical profiles Information on clinical drug data which is free for academic users

Provide open access to 17 million compounds

The web interface of this database has a GPCR search page and a ligand search page Contain three-dimensional structures and physical properties of natural metabolites This database stores information about metabolites, chemical compounds, and enzyme molecules Chemical database containing nearly 5 M commercially available ligands

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(RO5), formulated by Christopher A. Lipinski, in 1997, evaluate the drug likeness behavior of a compound and determines whether a chemical compound with pharmacological properties can be used as an orally active drug by human (Lipinski et al. 2012). Consideration of this rule is essential in drug discovery when an active lead structure is optimized to enhance the drug-like compound’s activity and selectivity. Another important consideration in the development of bioactive molecules as therapeutic targets includes the high oral availability of the compound (Veber et al. 2002). Lipinski’s rule states that a compound can be considered an orally active drug if the compound has a molecular mass of less than 500 daltons, not more than five hydrogen bond donors, ten H-bond acceptors, and a calculated partition coefficient (log P) value do not exceed 5. In addition to the molecular properties described by Lipinski, the other critical molecular properties of oral bioavailability include a number of rotatable bonds and polar surfaces (Veber et al. 2002). Therefore prior to molecular docking, the bioactive compounds are screened for drug-like behavior through Lipinski’s and Verber’s rule filters using different tools.

14.6.2 Calculation of ADMET Properties Optimization of ADME (absorption, distribution, metabolism, and excretion) properties and toxicity analysis is the most challenging part of the drug discovery process. The calculation of ADMET properties of ligands is essential to predict the behavior of a drug molecule inside the living body (Chandrasekaran et al. 2018). Besides showing efficacy, a drug molecule should show appropriate ADMET properties at a therapeutic dose. ADMET profiling parameters of active biomolecules include the rate of absorption, deposition, and metabolism of the drug candidate in the target tissue and in the body as a whole (Ferreira and Andricopulo 2019). The calculation of ADMET (absorption, distribution, metabolism, elimination, toxicity) facilitates the search for the most potential drug candidate and gives emphasis to the site of the drug being absorbed (absorption), the rate of distribution (distribution), time taken for the drug to metabolized, mechanism of action, how the drug is excreted and rate of excretion (elimination), and the toxic effect of the drug to the body (toxicity) (Ferreira and Andricopulo 2019; Guan et al. 2019). The absorption parameter illustrates the movement of a drug from the site of administration to the site of action, distribution emphasizes the movement of the drug through the bloodstream to the whole body, and metabolism portrays how the drug is metabolized and broken down into an easily excretable product whereas toxicity predicts the toxic behavior of the drug molecule. A drug’s distribution volume (DV) can be defined as the amount of drug present in the body fluid at the same concentration as measured in plasma (Chandrasekaran et al. 2018; Zhang and Tang 2018). A drug molecule with an elevated volume of distribution has a higher tendency to leave plasma and enter extravascular compartments of the body, indicating that higher drug concentration would be required to achieve a given plasma concentration and vice versa. The drug distribution from plasma to target tissue can be affected by several factors, including high molecular weight and PPB

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(plasma protein binding) (Jambhekar and Breen 2013; Malan and Chetty 2002). Under physiological conditions, the BBB (blood-brain barrier) plays a crucial role by controlling both the influx and efflux of biological substances and maintaining a highly controlled environment to promote normal functioning of the central nervous system (CNS) to maintain homeostasis. The BBB comprises capillaries of endothelial cells that protect the brain from harmful compounds (Kadry et al. 2020). These capillaries are tightly packed, preventing molecules from entering through passive diffusion. Numerous tight junctions and adherens junctions like claudin and occludin allow the selective transport of molecules into the brain (Kadry et al. 2020). The traditional technique for calculating ADMET properties took place in 1863. In the last few years, computerized prediction of ADMET properties of compounds with drug-like behavior has given a new dimension to computer-aided drug designing and drug discovery (Chandrasekaran et al. 2018; Guan et al. 2019). Gastrointestinal absorption of a drug is a major obstacle due to the impact of different factors, including physiological, physiochemical, and formulation effects such as pKa, solubility, gastrointestinal pH, gut wall metabolism, particle size, dosage forms, and many more (Blake 2000; Chandrasekaran et al. 2018). Estimating ADME properties gives a deeper insight into the correlation of ADME parameters with molecular structure and properties. The success of an oral drug depends on its readiness to dissolve in the gastrointestinal tract, absorption rate through the gut wall, plasma protein binding, penetration to the blood-brain barrier, CYP2D6 binding, and hepatotoxicity (Chandrasekaran et al. 2018; Malan and Chetty 2002). Drugs can be metabolized by oxidation, reduction, hydrolysis, condensation, and isomerization, and the liver is the primary site of drug metabolism (Blake 2000). Drug metabolism aims to convert the drug into a compound that can be easily excreted from the body. The enzymes involved in drug metabolism are generally present in the liver. The overall process of drug metabolism occurs in two main phases. In the first phase, the chemical structure of the drug is altered by oxidation, reduction, or hydrolysis. The cytochrome P450 oxidase enzyme (CYP450) catalyzes the drug molecule’s oxidation and converts it to a polar compound. In the next phase, the conjugation reaction occurs through methylation, acetylation, sulfation, glucuronidation, or glutathione conjugation (Malan and Chetty 2002; Zhang and Tang 2018). Oxapenem, the active metabolite of diazepam conjugated with glucuronide and became physiologically inactive and excreted out from the body without further modification, is an example of phase II metabolism (Veber et al. 2002; Zhang and Tang 2018). Biotransformationally, drug metabolism often converts lipophilic to hydrophilic molecules, readily excreted from the body. Conjugation makes drugs more soluble. A number of tools are available today for the prediction of ADMET, such as ADMET predictor, ADME suite, ADMEWORKS predictor, QikProp, DataWarrior, StarDrop, MetaTox, ADMETlab, AdmetSAR, MetaSite, and Tox suite for toxicity prediction.

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Molecular Docking and Virtual High-Throughput Screening

Molecular docking is the final step to finding an active drug compound against a target with an available three-dimensional structure. This method can predict the binding affinity and structure of the protein-ligand complex (Raval and Ganatra 2022). The first algorithm for molecular docking was developed by Kuntz et al. in 1980. This technique aims to predict the top binding conformations or poses of a ligand against the target of interest (Salmaso and Moro 2018). The possible interactions of ligand-receptor are sorted via scoring functions to predict the structure most likely to be present in nature. Molecular docking is a mathematical tool that anticipates a compound’s conformation to its target with the highest affinity. Docking falls into two main categories: blind docking and direct docking. If the active site of the macromolecule is unknown, the search for the binding site of the protein and recognition of the specific binding site of the ligand is called blind docking, while if the binding site is known, the docking of ligands into the active site is called direct docking (Ferreira et al. 2015; Salmaso and Moro 2018). The docking method can be subdivided into three types based on the degree of flexibility of the molecules that include rigid docking, flexible docking, and semiflexible docking (Agrawal et al. 2019; Gschwend et al. 1996; Salmaso and Moro 2018). In the case of rigid docking, both ligands and proteins are regarded as rigid entities, comparable to the “lock and key” model used primarily for protein-protein docking, considering three transitional and three rotational degrees of freedom only. The flexible docking considers multiple degrees of freedom with a scoring function of numerous coordinates. The basic requirements to perform molecular docking include one or a set of prepared ligands, an optimized and prepared target, and a platform to perform the docking process. Different softwares available for docking are depicted in Table 14.2. The initial step in molecular docking is the optimization and preparation of the protein by deleting ions, water molecules, and other small ligands from the binding pocket before docking. The target is then prepared by adding hydrogen atoms and energy minimization for efficient ligand binding (Meng et al. 2011). The binding pocket or active site identification of the target is the most important step in molecular docking. Once the protein is prepared, the selected, prepared ligands are docked against the binding sites of the receptor (Meng et al. 2011). The efficient binding conformation is predicted by considering the most negative binding energy. The higher the negative binding energy, the lower the energy and better the stability of the interacting complex.

14.8

Binding Energy Analysis

The binding of a ligand or drug-like molecule to a therapeutic target provides the molecular basis of the activity of the ligand. Thus, accurate in silico prediction and optimization of protein-ligand binding affinity have significant importance in drug

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Table 14.2 List of some computational molecular docking software Program name AutoDock GOLD DOCK BetaDock EAdock

GLIDE

BIOVIA Discovery Studio

Description Automated docking of ligand to a macromolecule, developed in 1990, the Scripps Research Institute Combined mechanistic tool for ligand-protein interaction, developed in 1995, UK Based on geometric matching algorithm, developed in 1988, USA, University of California – San Francisco Based on the theory of beta-complex, developed in 2011, South Korea uses the AMBER+ AutoDock scoring function A powerful tool for drug development with accuracy, suitable for high-throughput screening, developed in 2007 in Switzerland, uses CHARM force field for energy calculations Software for conformational, orientational, and positional space of docked ligand, developed in 2004, USA, Columbia University Brings world-class in silico techniques into a common environment, molecular modeling, and simulation software developed by the Dassault Systems BIOVIA

License type Open Open Free for academic use Open Commercial

Commercial

Commercial

discovery (Wang and Zhu 2016). The ligand molecule forms a non-covalent stoichiometric complex with the macromolecule at thermal equilibrium with the unbound molecules present in the solution (Åqvist et al. 1994; Pantsar and Poso 2018). The free energy difference between the unbound and bound points can be computed to estimate the absolute free energy of binding, considering the energies of all microstates or configurations. The introduction of molecular mechanic force fields speeds up the energy evaluation with great accuracy (Åqvist et al. 1994; Pantsar and Poso 2018). The strength of protein-ligand binding is related to the intramolecular interaction of protein-ligand complex, solvent effect, and dynamics. Molecular docking utilizes scoring functions to estimate the binding affinity. The scoring function plays a key role in pose selection, which differentiates the correct binder and binding modes from non-binders (Salmaso and Moro 2018). Empirical scoring function, force fieldbased scoring function, and knowledge-based scoring functions are three types of scoring functions used in molecular docking. The force field is a concept of molecular mechanics that estimates the potential energy of a system by considering bonded (intramolecular) and nonbonded (intermolecular) components (Pantsar and Poso 2018; Salmaso and Moro 2018). In molecular docking, intermolecular components are considered for a ligand-protein complex. The nonbonded intermolecular components include electrostatic and van der Waals interactions. The force field is the sum of bonded and nonbonded energy (Pantsar and Poso 2018; Salmaso and Moro 2018). The empirical scoring functions consider hydrogen bond, electrostatic interaction, van der Waals interaction, entropy, desolvation, hydrophobicity, and many more, which are optimized to produce binding affinity data. The LUDI scoring function, GlideScore, and Chemscore are examples of empirical scoring

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functions. Knowledge-based methods depend upon the statistical data derived from complex structures of ligand receptors (Pantsar and Poso 2018). Scoring functions estimate the binding affinity, which has a direct relation to Gibbs’ free energy of binding (ΔGbind). The binding energy can be explained as the summation of the change in solvation and desolvation energy (ΔGsolvent), the change in energy due to the interaction of ligand and receptor (ΔGint), the change in energy of ligand and receptor due to complex formation (ΔGconf), and the change in energy due to rotational, vibrational, and translational motion (ΔGmotion) (Pantsar and Poso). The binding energy includes conformational changes and changes in interaction (entropic effect) of the protein-ligand complex. The direct polar interaction between protein-ligand and solvent is enthalpic in nature that greatly depends on ionic interactions, hydrogen bonding, and Lennard-Jones type of functions (Pantsar and Poso 2018; Salmaso and Moro 2018). Formations of strong hydrogen bonds are required for the high affinity of the ligand to the receptor. Force field scoring functions also consider the distance and angular parameters during binding energy calculations. CHARMM (Chemistry at Harvard Macromolecular Mechanics), OPLS3, and AMBER (Assisted Model Building With Energy Refinement) force fields are the most commonly used binding energy calculation force fields with better hydrogen bonding description (Cournia et al. 2017; Pantsar and Poso 2018). The bond angle, bond length, torsion angle, and dihedral angle are also considered during binding energy calculations. ðΔGbind Þ = ðΔGsolv Þ þ ðΔGint Þ þ ðΔGconf Þ þ ðΔGmotion Þ

14.9

MD Simulation

Docking can give an idea of the interactions between different protein targets and ligand molecules. However, in a biological system, such interactions might not be stable owing to different parameters inside a body, such as temperature, pressure, and water molecules, which might interfere with the bond stability. Therefore, docked complexes are simulated in an artificial system that resembles certain parameters of the biological system. The docked complex is subjected to energy minimization and put in a solvent system. Pressure and temperature are applied to the system to check the stability of different bonds under different conditions. Any macromolecule can be simulated alone or in complex with a small molecule to study either the stability of the backbone of the protein or the stability of interactions between the two molecules. Molecular dynamic simulation can throw insight into changes in the different protein chains before and after subjecting them to energy minimization and other temperature and pressure factors. The steps in any molecular dynamic simulation of a complex include the following (Lemkul 2018): 1. Preparation of protein topology 2. Preparation of ligand topology

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Preparation of an appropriate box to which the complex fits properly Choosing a solvent system Addition of ions to the system Energy minimization of the system System equilibration Final MD run Post MD analysis

At the beginning of any simulation run, we need to ensure that the two molecules have been docked properly, and there are no unwanted heteroatoms in the system. It is important to choose a suitable force field, such as CHARMM, AMBER, GROMOS87/GROMOS96, OPLS-AA, etc., for the run that is compatible with our system. Once protein topology is obtained, the next phase is the crucial part of generating a compatible ligand topology for which, again, there are different force field options. There are automated tools for ligand topology generation, such as Antechamber, acpype, CGenFF, PRODRG, ATB, Topolbuild, LigParGen, etc. After generation of appropriate topology files for the system, a proper box shape and size need to be defined. Too small box would not cover our entire molecule and a too large box would mean unnecessary space filled with solvent and more timeconsuming run. Visualization tools such as VMD can be used to monitor to changes at each step of the run. Once the complex is inside the solvent-filled box, ions are to be added to the solvated system in case it had any net charge. Once that is done, the system is ready for energy minimization. Equilibrating the energy-minimized solvated system includes applying position restraints to the ligand and applying temperature (e.g., 300 K) and pressure (e.g., 1 bar) to the system. The equilibrated system is subjected to MD run for a definite time (e.g., 100 ns) depending on the requirement, size of the system, and the capacity of the computational system. After the MD simulation run, it is essential to perform various analyses on the simulated system to understand the changes. Post-simulation analysis provides an idea of the radius of gyration, solvent accessible surface area, hydrogen bond formation, root mean square deviation, root mean fluctuation of residues, binding energy, etc.

14.10 Scopes and Limits of CADD The ultimate goal of molecular docking is to identify the new lead drug molecules. Molecular docking is a simple, low-cost, and safe technique for structure-based drug designing. However, there are challenges that need to be addressed before performing the docking process. The overlapping of the binding site of the cofactor with the ligand, the presence of crystal water molecule at the binding site mediating binding between ligand and receptor, the presence of metal ions, and the covalently bound ligand may show false binding results. Therefore, if an active compound shows significant binding energy against the target, validation is required before considering the molecule as a drug.

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At the same time, any potential drug target in CADD must be investigated to achieve the desired outcome. Target identification and validation with the help of genomics, functional genomics, and transcriptomics plus artificial intelligence have the potential to transform the whole drug discovery process; however, the true power lies in the synergies. Although the impact of molecular docking is well established, the identification of compounds with better efficacy, robust and reliable scoring function, and software is one of the challenges in molecular docking that needs to be addressed in the future.

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Recent Advances in PGPRs and Their Application in Imparting Biotic and Abiotic Stress Tolerance in Plants

15

Babita Joshi, Satya Narayan Jena, S. R. Joshi, and Brijmohan Singh Bhau

Abstract

Quality plant products may be a significant concern from fields to practical shelves, particularly for perishable crops. There is an immediate requirement for modern eco-friendly strategies, including using microorganisms that will provide a long-term solution in maintaining the quality of soil and plant produce. Bacterial activity in the rhizosphere of many plant species grown around the world offers opportunity to research and use these microorganisms for plant health promotion. PGPR (plant growth-promoting rhizobacteria) are organism that support growth and development of plants by increasing nutrient uptake, improving resistance to biotic stress factors, regulating hormone levels and protecting plants from phytopathogens. Through a mutualistic association in which both parties (plants and bacteria – PGPR) coexist, they build up several interactions with plants and other soil-dwelling species. PGPR is thought to operate as organic control organisms within the rhizosphere soil. Investing in

B. Joshi Plant Molecular Genetics Laboratory, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India S. N. Jena Plant Molecular Genetics Laboratory, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India S. R. Joshi (✉) Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India B. S. Bhau Department of Botany, Central University of Jammu, Rahya-Suchani (Bagla), Jammu and Kashmir, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_15

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investigating agriculture and horticulture is one of the more promising approaches for enhancing plant growth without hampering the ecosystem. Applications of nanobiotechnology are also gaining significant momentum. Nano-fertilisers accelerate development compared to conventional fertilisers; the plants quickly absorb them at the same time. Combining current developments in the study of plant-microbiome interactions with cutting-edge omic strategies may soon enable us to create a comprehensive plan to approach plants against unfavourable circumstances. This chapter summarises the use of PGPR in different plant species under constrained crop productivity exposed to various biotic and abiotic stresses. Concerns about the application of PGPR and its effects on ecosystems, as well as potential difficulties in commercialising these PGPRs as biostimulants to enhance crop yield under various environmental restrictions of plant development, are also discussed in this chapter. Keywords

Soil quality · Plant growth · Biotic stress · Abiotic stress · Rhizosphere · Bacteria

15.1

Introduction

In many developing nations, agriculture provides jobs and a sizeable portion of national income and ensures food security (Singh et al. 2015; Hanson and Boland 2020; Christiaensen et al. 2021). The world’s surface area is estimated to be around 6.38 billion hectares harbouring around 7.41 billion of population, and 1.3 billion of peoples depends on agriculture (Gouda et al. 2018). Since the world’s population is increasing at an alarming rate (estimated to be 8–9 billion by 2030), dependency of human on agricultural sector is increasing drastically (Egamberdieva et al. 2017). In order to meet the demand of food for next 40 years, a 60% increment in agricultural productivity may be necessary. In most of the developing countries, agriculture provides employment, food security and a significant portion of the national income and export profits in many developing countries (Singh et al. 2015). Cameroon depends on agriculture for food and subsistence, exploiting natural resources as the primary engine of the economy’s expansion and development. The agricultural sector is responsible for ensuring that the local food production meets the needs of both the rural and urban inhabitants, thereby ensuring food security. Detection of plant stress is considered to be one of the vital aspects for enhancing crop yields, particularly at given climatic condition. A variety of abiotic and biotic factors including salt, temperature, water, light, air, soil, nutrient availability, agrochemicals, pests and diseases affects the development and growth of the plants. With this expanded panorama in mind, the choice of technology is diverse to handle biotic and abiotic stress. Looking at the spatial scale, the effects of stress (biotic and abiotic) on plants can even modify genetic material (DNA), in the cell and then to the entire plant and eventually to the entire field. The diversity of microorganisms is essential because of their distinctive properties, which can be used to increase or

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Fig. 15.1 Plant growth-promoting rhizobia showing direct and indirect mechanisms of action for plant benefit

produce crops. Several types of microorganisms/microbes present in the soil and other elements contribute to the rhizosphere ecosystem and the development of plants. This ecosystem’s operation is controlled by microbiological dynamics (Kennedy and Smith 1995). According to estimates by different microbiologists, it has been discovered that the soil can hold ten billion thousand different types of microbial species. The main objective of the research on rhizosphere microbial diversity is to determine its role in many spheres. In general, microorganisms were present in all plant components, including from the root to the plant’s apical shoot, the leaf/shoot’s surface (phyllosphere) and the plant’s interior (endophytes). Many of these microorganisms benefit plants by assisting in their healthy growth and development. Among all of these, PGPR (term coined by Kloepper and Schroth in 1978) plays a significant part and holds a key position in crop productivity and management. The diversity of rhizospheric microbes contains many species that benefit the plant environment. The region of soil surrounding the roots, known as the rhizosphere, is home to a diverse array of microorganisms including bacteria, fungi, insects and nematodes. Among them, the bacteria residing in the rhizosphere commonly known as plant growth-promoting rhizobacteria (PGPR) enhance plant growth. These bacteria influence the plant growth through various direct and indirect mechanisms such as nitrogen fixation, phytohormone synthesis, phosphate solubilisation, increased iron availability and ACC deaminase. Indirect mechanisms include siderophore production, antibiosis and acquired and induced systemic resistance response (Fig. 15.1). However, the specific effects of these mechanisms may change depending on the plant species and the bacterial strains involved. Studies have also shown that the presence of PGPR on the root surface can lead to an increase in ion fluxes, allowing for direct absorption of minerals by the plants (Cleyet-Marcel et al. 2001).

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It has been observed that a wide variety of bacterial species, including Bacillus, Burkholderia, Azospirillum, Azotobacter, Rhizobium and Pseudomonas, are PGPR, with Bacillus, Rhizobium and Pseudomonas being the most well-known species. Various economically important plants, including chickpeas, maize, pea, peanut, rice and others, have been subjected to PGPR and found to be very effective. One of the most incredible endowments of these organisms (especially PGPRs) is communication between plants and them leading to the signal complexity and evolution of life on this planet (Ma et al. 2016a, 2016b). To defend themselves from biotic and abiotic pressures, plants have developed a variety of biochemical and structural defence mechanisms because they cannot flee in unfavourable environmental conditions. Developing symbiotic, mutualistic and commensal connections between plants and microbes and effective communication could increase plant immunity (Badri et al. 2009). Similarly, bacteria, fungi and other microorganisms emit various chemical signals that can change the composition of root exudates, which may adversely affect plant’s physiology (Pritchard and Birch 2011). Historically, the root system has been primarily viewed as a means of anchoring and transporting nutrients and water. However, in addition to serving as an anchor, root exudates also provide the soil with carbon (such as polymerised sugars, amino acids and organic acids) and micronutrients which can compete with neighbouring pathogenic microbes (Badri et al. 2009; Glick 2014). It also contains a variety of primary and secondary metabolites which support the soil microbial population and are also essential for plant maintenance and growth (Kumar et al. 2014). Endophytic microorganisms within the shoot system of plants can also produce essential secondary metabolite products. Furthermore, the interaction between plants and microbes may provide a new bioremediation possibility for cleaning the soil ecosystem of toxic elements. Numerous studies and applications about consortiums of microorganisms promoting plant growth can be found in literature, yet the scientific community does not delve deeply into the in-depth knowledge of these microbes or their potential for improving food quality, increasing nutritional security and managing disease. PGPRs play a crucial role in sustainable agriculture, carbon sequestration and bioremediating soils contaminated by pesticides, organic chemicals and heavy metals, as well as producing biomass and biofuels. This assessment evaluates the ability of plants to grow under various biotic and abiotic stresses by utilising PGPRs.

15.2

Different Types of Biotic Stress and Their Impact on Plants

Plants are the primary allies and closest buddies of humans on Earth. Eighty percent of our daily caloric intake comes from plants, which also provide 98% of the oxygen we breathe. Despite this, we often overlook their crucial roles and fail to understand the importance of maintaining their health. Each year, up to 40% of food crops are lost due to plant pests and diseases, significantly impacting the poorest communities whose livelihoods depend on agriculture. These pests and diseases are widespread and can easily spread to new locations in a more globalised and connected society.

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Climate change accelerates this spread by creating favourable conditions for pests and diseases in new regions. For example, pests such as the red palm weevil, fall armyworm, fruit fly, desert locust and emerald ash borer have already expanded their host range or distribution due to climate change. The proliferation of pests also poses a significant threat to the environment as they can lead to a substantial loss of biodiversity, losses in crop yields and reduced farmers’ revenues. This raises the question of which plant diseases are the most prevalent and how climate change impacts their spread? Various sources of biotic stress on plants include weeds, nematodes, bacteria, viruses and arachnids. Many of these biotic stresses can harm the plants by reducing their resistance against different pathogens and even cause plant death (Gull et al. 2019). Additionally, they result in the alteration of various metabolic processes and lead to the decline in productivity. Plant growth, development, yield and biomass production can be negatively impacted by biotic stress and require prompt action to be addressed through various green technologies (Liliane and Charles 2020).

15.3

Different Types of Abiotic Stress and Their Impact on Plants

According to Yang et al. (2019), global climate change, characterised by rising air temperatures and increased atmospheric CO2 levels, is expected to intensify in the future, modifying rainfall patterns and distribution. Cohen et al. (2021) also note that while lack of rainfall is often the primary cause of drought stress, high temperatures, intense light and dry winds can also cause soil moisture to evaporate, exacerbating the stress. Drought stress conditions, caused by climate change, affect large areas globally. In addition to drought, salt stress caused due to water scarcity is also a major abiotic stress to many plants (Mostofa et al. 2018; Adnan et al. 2020), and Tariq et al. (2020) highlight that salt stress caused by water scarcity is also a major issue. The effects of global warming on soil and plants include a loss of water as a result of increased transpiration. This exacerbates existing water shortages in many agricultural regions worldwide (Sultan et al. 2019). According to Ray et al. (2019), the rise in the temperature by 2 °C in next few decades suggested that nearly 20% of the global population could face severe water shortages. Anthropogenic activities such as industrialisation, deforestation and urbanisation have a significant impact on rainfall patterns and plant water availability (Fatima et al. 2020). Rainfall patterns are becoming more sporadic and frequent during early spring and winter, with early fall and summer experiencing drier and hotter conditions with less or no rain. Drought stress on plants is particularly severe during the summer due to increased atmospheric water demand, higher evaporation and transpiration rates and reduced rainfall. Variations in rainfall distribution and intensity play a critical role in controlling water supplies for plants and causing drought conditions (Leal 2010; Karandish and Šimůnek 2016). Modifying the duration of the rainy season can help in overcoming the current and future water shortage/excess problems in specific

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regions (Leal 2010). Two strategies for addressing these fluctuations are proper management and crop planning. Excessive soil salinity is a major environmental challenge for crops because it can cause metabolic, ionic toxicity and oxidative stress, as well as osmotic stress in plants. Salinity has always been the most challenging abiotic stress as it causes both osmotic and water stresses that significantly reduces crop yields. This can lead to reprogramming of gene expression, which is critical for a plant’s adaptability towards salt stress. High salinity in soil can negatively impact plant survival by causing water stress and excessive uptake of ions (sodium, chloride, etc.), leading to cytotoxicity and nutritional imbalances. Also, oxidative stress due to salinity causes the production of reactive oxygen species (ROS), thereby affecting plant growth and development (Hernandez et al. 2001). Farmland, crops and farmers’ livelihoods are all susceptible to severe harm from moving and standing water. Although damage from moving water is frequently limited to relatively small geographical regions, it can be severe. The speed of the water and the quantity of turbulence impact the material that it can carry, deposit and dislodge. This ability results in several issues, but soil erosion may be detrimental to the soil’s present and potential future production. The soil’s topmost productivity layer is eroded entirely or partly due to flooding. Field-grown plant debris can also be carried by moving water. As water flow slows, soil and residue build-up may obstruct water drainage channels and smother vegetation. When plants are at their largest, floods in the summertime often cause additional damage from moving water. Because water is so heavy, its power can shatter or lodge crop plants. Standing water’s impact on soil oxygen levels is the main reason the soil becomes oxygen-free when water builds up. For survival and growth, oxygen is necessary for soil microbes and plant roots. Water can carry 25 times more oxygen than air. Therefore, standing water prevents the passage of carbon dioxide out of the soil and oxygen into the soil. The result is hypoxia or oxygen levels below what is required for plant growth and production. Due to low oxygen availability, submerged plants typically perish within two or three days. Because they are young and short, this frequently hazards seedlings and young plants. Larger plants with stems and leaves that protrude above the water may endure for more than three days, but plant functions and yield potential can be severely harmed. No matter how tall a plant is, oxygen is necessary for healthy root growth. Although many crops and crop types are more susceptible to flooding than others, if standing water persists for more than a few days, all plants will suffer, except for rice. Due to flooding, plant productivity is severely influenced due to the non-availability of oxygen for a long time. Standing water and the resulting low oxygen content will cause physical and chemical changes in the soil. In standing water, soil aggregates and healthy soil structures may disintegrate. The soil structure may be further impacted by individual clay and silt particles that may settle from water into soil pores. Some soil bacteria can utilise the minerals in the soil to produce energy instead of oxygen. The conversion of nitrates to gaseous forms of nitrogen is a typical reaction. This process, known as denitrification, can drastically reduce the nitrogen concentration of soils. Microbes

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under flooding also alter the availability of other mineral nutrients, such as sulphur, iron and magnesium, which impact plant growth. If roots are in stranded water, leaf stomates close. Water uptake happens through the plant halts when the stomates are closed and not functional. In submerged conditions, with the water, the plant takes several mineral nutrients, including nitrogen. Corn growing under submerged conditions develop yellow leaves, a sign of an N shortage. Rhizobia are among the beneficial bacteria for soybean farming in the field. These bacteria fix nitrogen by converting gaseous forms of nitrogen into ammonium that plants may use. Lack of oxygen prevents rhizobia nodulation and causes less fixation in existing nodules. Soybean leaves will also display signs of a nitrogen shortage. Plant roots under submerged conditions cannot generate energy through normal respiration without sufficient oxygen. Instead, they make use of oxygen-free processes. Unfortunately, these interactions produce toxins, including ethanol and formaldehyde, which can harm plant tissues. Farmers have little choice but to wait to repair the damage caused by flooding and standing water. At least some damage will be restored once the water is drained from the soil and oxygen has returned to the soil pores. Although plant death is irreversible, plants that survived are likely to yield at least some. Plant species have different responses to temperature throughout their life cycles, which are primarily related to different developmental stages. The range of maximum and minimum temperatures sets the limits for visible growth for each plant species. As temperatures reach the optimal range for a particular species, vegetative development (node and leaf appearance rate) accelerates. The ideal temperature for vegetative growth is often higher than the ideal temperature for reproductive growth for most plant species. Crop production is the indicator of climate change’s effects that is most obvious because it is this characteristic that both farmers and consumers are most concerned about. If crop production is mainly constant, the growth cycle length is of little relevance. The yield of different crop species responds differently to temperature based on their cardinal temperature requirements. Climate changeinduced temperature rises will affect both crop yield and plant growth and development. Research shows that the heavy metals in soil can harm plant growth and metabolism by changing various physiological and biological processes in plants (Nagajyoti et al. 2010). Essential trace elements, such as Zn, Fe, Ni, Mo, Cu and Mn, are required by plants in small amounts for proper growth of plants, and if present in high concentration, they can also act as cofactors in several metabolic activities. However, other elements, such as Cr, Cd, Pb and As, when present in soil, can cause harmful effects on plants, even at very low concentrations. Even though essential and necessary, metals immobilised in plant tissues are considered the most dangerous and harmful for agricultural crops, such as Zn, Pb, Ni, Cd, Mo and Cu; however, the biological and molecular mechanisms of metal toxicity are yet not well known. The combination of different stressors can be considered a new stress to observe different defence and adaptive responses in plants. Environmental changes, including solar radiation, are responded to by plants through complex molecular and

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biochemical signalling processes that determine the plant’s tolerance or sensitivity at the whole plant level. High-energy particles or waves, such as ionising radiation, alpha, beta, gamma, neutron and x-rays, in solar radiant energy can cause significant damage to plants. UV-B radiation, in particular, is a known stressor that causes DNA, protein and membrane damage (Yoon et al. 2021).

15.3.1 PGPR The plant rhizosphere, a hub for various microbial activities, is a confined area of soil around the root systems of plants (De la Fuente Canto et al. 2020). This area is mostly inhabited by a variety of microbial taxa such as bacteria, viruses, fungi, nematodes, protozoa, arthropods, etc. Among these, bacteria and fungi are the most prevalent groups and perform fundamental ecological functions as they secrete several regulatory products that support plant growth (Kalam et al. 2017). These compounds aid in improved nutrient uptake by host plants, defend against phytopathogenic microorganisms and foster tolerance to a variety of abiotic stresses, thus impacting the overall health of plants (Backer et al. 2018). Different PGPR strains can improve seedling emergence, stimulate nodulation in legumes, display biocontrol, increase resistance to foliar diseases and increase crop yields (Vejan et al. 2016; Swarnalakshmi et al. 2020). PGPRs have been reported in various genera such as Caulobacter, Delftia, Azotobacter, Azospirillum, Arthrobacter, Frankia Agrobacterium, Bacillus, Acinetobacter, Azoarcus, Aeromonas, Allorhizobium, Azorhizobium, Bradyrhizobium, Burkholderia, Chromobacterium, Enterobacter, Flavobacterium and many more (Vessey 2003; Goswami et al. 2016; Parray et al. 2016; Kalam et al. 2020a, 2020b). PGPRs play a major role in promoting plant growth by acting as biofertilisers, improving the resistance to biotic and abiotic stress and providing essential nutrition to host plants. They also protect plants in various ways, including colonising the root systems, positively impacting plant growth and development, promoting biofertilisation, inducing systemic resistance and controlling phytopathogens, all of which ultimately contribute to enhanced plant growth. A comprehensive understanding of the mechanisms of PGPR is crucial for manipulating rhizosphere microbes to optimise processes that significantly enhance plant productivity. Traditionally, the mechanisms of PGPR have been categorised into two mechanisms, that is, direct and indirect mechanisms as mentioned in earlier sections. Indirect mechanisms occur outside the plant and do not affect the plant metabolism, whereas direct mechanisms occur inside the plant with an immediate impact on plant metabolism. In case of indirect mechanism, two important processes, that is, enhancement of systemic resistance to pathogens causing biotic stress and safeguarding against detrimental environment conditions in plants, take place during defensive metabolic processes on receiving signals from the bacteria (Gouda et al. 2018). While in case of direct mechanisms, microorganisms hinder the balance of the plant growth regulators and also act as a sink for hormones that are released by the plant (Glick 2014; Gouda et al. 2018).

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15.4

439

Role of PGPR in Overcoming Abiotic Stress

Climate change has led to an escalation in the intensity, frequency and duration of various abiotic stresses, which leads to detrimental effects on agricultural productivity. Some of the most important abiotic stresses are drought, high or low temperature and salinity, and heavy metal has drastically reduced the quality of agricultural land. Thus, using an environmentally friendly approach based on PGPR is the most promising technique to minimise the negative effects of these stresses. PGPRs induce local and systematic resistances and also undergo various osmotic and oxidative stresses in order to provide defence against heavy metals, thereby enhancing the ability to withstand salinity, drought and metal toxicity (Table 15.1; Fig. 15.2). Processes such as acidification, chelation, redox reactions, portioning, immobilisation, precipitations, complex formation, etc. may lead to the change in the bioavailability of heavy metals in the soil. Many PGPRs also secrete phytohormones, synthesise siderophores (iron chelates), provide nutrients, promote biological N2 fixation and increase the availability of trace elements, which eventually leads to the improvement in plant growth (Glick 1995; Khan et al. 2018). Additionally, the microbes are essential for reducing the negative impacts of drought stress and increasing plant output (Khan et al. 2020). The microorganisms can help prevent oxidative damage in plants cultivated under various environmental challenges, allowing grains to withstand dry conditions. The impact of drought stress on agriculture can lead to significant financial losses and harm to plant growth and survival. In order to survive in drought condition, plants close their stomata (which reduce water loss), which will eventually lead to plant death due to the decrease in CO2 uptake and low photosynthetic activities. Thus, to adapt to drought, plants employ strategies such as producing reactive oxygen species, synthesising stress hormones, altering root and shoot architecture and activating antioxidant defence systems (Murali et al. 2021). Injection of PGPR is reported to reduce the effects of drought stress and also can boost drought tolerance in dry settings by forming colonies in the root zones in many crops (Gontia-Mishra et al. 2016; Niu et al. 2018). They can also solubilise micronutrients, making them absorbable by plants, and act as biofertilisers/biostimulants (Etesami et al. 2017; Kalam et al. 2020a, 2020b; Khan et al. 2020). Maize plants inoculated with Bacillus species produce solutes which help plants to overcome drought stress (Alamgir 2018). Arun et al. (2020) showed that the inoculation of phyllosphere bacteria can reduce the abiotic stressors in rice plants. The increase in biomass, plant homeostasis, growth and drought tolerance index is reported by the inoculation of bacterial strains such as Bacillus amyloliquefaciens, Azospirillum brasilense, Rhizobium leguminosarum and Mesorhizobium cicero (Kumar et al. 2020). Similarly, Trichoderma sp. has been observed to be helpful for rice drought tolerance (Khadka and Uphoff 2019). Other mechanisms to combat drought conditions in plants include production of exo-polysaccharides, volatile organic compounds, changes in antioxidant activities, regulating hormonal balances, etc. (Cohen et al. 2015; Saleem et al. 2018; Jabborova et al. 2020).

7.

6.

5.

4.

3.

2.

Sr no. 1.

Bacillus megaterium, Acinetobacter lwoffii, B. marisflavi, Bacillus tequilensis and Bacillus subtilis

Endophytic Lysinibacillus fusiformis, Bacillus subtilis, Brevibacterium halotolerans, Bacillus pumilus, Achromobacter xylosoxidans and Pseudomonas putida Endophytic Bacillus spp. strain SLS18

Microbes Achromobacter piechaudii AVR8 Pseudomonas pseudoalcaligenes and Bacillus pumilus Ion exchange resins and Arthrobacter scleromae SYE-3 Endophytic Bacillus amyloliquefaciens SB-9

Phytolacca acinosa Roxb. (pokeberry) and Solanum nigrum L. (black nightshade) Sarjoo 52 rice variety

Salt stress mitigation in vitro and in particular plant biomass increase

PGPR activity of isolate leads to accumulation of biomass

Cd and Mn stress

Isolate and characterise salttolerant 1-aminocyclopropane-1carboxylic acid deaminase possessing PGPR for 09 agro-

PGPR activity of isolates

Salt and drought stress

Vitis amurensis ‘Changbai 9’ V. vinifera ‘Cabernet Sauvignon,’ and V. labruscana summer black root Rhizospheric soil and roots of Prosopis strombulifera

Enhanced indole-3-acetic acid and d 1-aminocyclopropane-1carboxylate deaminase activity Enhanced growth and upregulation of melatonin synthesis

Mode of action Enhancement of ACC deaminase activity Cell caspase-like enzymatic activity increased

Salinity stress

Salinised greenhouse cultivation site

Salinity (5–25 gm NaCl L-1)

Abiotic stress Salinity using 43 mM NaCl

Lettuce,radish andcabbage

Plant used Lycopersicon esculentum Mill cv. F144 Oryza sativa GJ-17 cultivar – salinity sensitive

Table 15.1 Application of PGPR to overcome abiotic stress in different plant species

Misra et al. (2017)

Luo et al. (2012)

Sgroy et al. (2009)

Jiao et al. (2016)

Reference Mayak et al. (2004) Jha and Subramanian (2014) Hong and Lee (2017)

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Hartmannibacter diazotrophicus E19T y isolated from the rhizosphere of Plantago winteri Neotyphodium coenophialum – endophyte

13.

EI ryegrass (Lolium perenne L. cv. Justas)

Hordeum vulgare L.

Microorganism-free Saccharum spp. cv. SP70–1143 (shoot)

Cadmium tolerance

Salinity (seedlings exposed for two h to 200 mM and 400 mM NaCl stress)

Water-deficit assay

Salinity – ACC deaminasecontaining bacteria were isolated from the rhizosphere soil of rice

Salinity (salt stress conditions (0.17 M–0.86 M NaCl))

Tall fescue – Festuca arundinacea

Oryza sativa cv. Ratna

Cold tolerance

Arabidopsis thaliana

Capsicum annuum

Enhanced tillering and reduced leaf elongation. Genes reported

Protected the rice seedlings under salt stress by decreased stress ethylene, ROS, enhanced seed germination and the seed vigour index Activate ABA-dependent signalling genes, ABA and ET signal transduction downregulated Induced ACC deaminase activity and detected microbes in the roots using FISH

Accumulation of pigments and reduced expression of RbcL and COR78 prevented the plasmalemma disruption PGPR activity of isolates

PGPRs produce siderophore and exhibited IAA production

(continued)

Ren et al. (2006)

Suarez et al. (2015)

Vargas et al. (2014)

Sarkar et al. (2018)

Kapoor et al. (2017)

Su et al. (2015)

Patel et al. (2017)

Recent Advances in PGPRs and Their Application in Imparting Biotic. . .

15.

14.

Gluconacetobacter diazotrophicus strain PAL5

Enterobacter ludwigii isolated from the rhizospheric soil of Cynodon dactylon Enterobacter spp. strain P23

Bacillus spp., Alcaligenes sp., Proteus sp. and Aneurinibacillus aneurinilyticus Endophytic PGPR, Burkholderia phytofirmans strain PsJN (Bp PsJN)

12.

11.

10.

9.

8.

climatic zones of Uttar Pradesh, India Salinity stress in pot soil supplemented with 50 mM NaCl

15 441

Endophytic fungus Piriformospora indica

Endophytic fungus Piriformospora indica

Pseudomonas koreensis AGB-1

19.

20.

Microbes Endophytic Bacillus subtilis, bacillus amyloliquefaciens, Pantoea agglomerans, Agrobacterium sp. Root endophyte Piriformospora indica (PiHOG1) Endophytic Arthrobacter, Bacillus and Microbacterium,

18.

17.

16.

Sr no.

Table 15.1 (continued)

Miscanthus sinensis (roots)

Arabidopsis thaliana Columbia (Col-0), gl1 (SAIL_1149_D03) and gl1-HKT1::AtHKT1;1 no. 17–3 Brassica campestris ssp. Chinensis cabbage

Zn, Cd, As and Pb

Drought (solution of 20% polyethylene glycol 4000)

Salinity (0–150 mM NaCl)

Osmotic stress

Salinity stress of 200 mM NaCl

Oryza sativa (roots)

Sweet pepper Capsicum annuum L. ‘Ziegenhorn Bello’

Abiotic stress P. agglomerans from teosinte was tested for salinity

Plant used Eight corn varieties and teosinte

Reduced and delayed phosphorylation of the PiHOG1 protein Increased biomass and stress relief, reduced upregulation or even downregulation of the stress-inducible genes CaACCO and CaLTPI Increases plant biomass, lateral root density and chlorophyll content under salt stress Increase in KAT1 and KAT2 gene transcript levels Upregulation of droughtrelated genes DREB2A, CBL1, ANAC072 and RD29A and enhanced peroxidases, catalases and superoxide dismutases in the leaves of the Chinese cabbage The application resulted in biomass increase by 54% and chlorophyll content by 27%

Mode of action Increase the tolerance of tropical corn to saline conditions

Babu et al. (2015)

Sun et al. (2010)

Abdelaziz et al. (2017)

Sziderics et al. (2007)

Jogawat et al. (2016)

Reference Gond et al. (2015)

442 B. Joshi et al.

Endophytes Pseudomonas fluorescens, Bacillus sp., Pseudomonas protegens, Pseudomonas putida Soil-isolated Pseudomonas vancouverensis OB155-gfp P. frederiksbergensis OS261gfp

Chryseobacterium humi ECP37 and Ochrobactrum haemophilum ZR3–5

Sinorhizobium meliloti

Staphylococcus arlettae NBRIEAG-6

Thalassobacillus denorans and Oceanobacillus kapialis isolated from salt mines

22.

24.

25.

26.

27.

23.

Pseudomonas pseudoalcaligenes and Bacillus pumilus

21.

Salinity (0–2 g NaCl l-1)

Helianthus annuus – sunflower

Arsenic stress in soil

Salinity (NaCl- 0–150 mM)

Medicago sativa (Aragón’ or ‘N4’ × Sinorhizobium meliloti 102F78) Brassica juncea n.Var. R-46

Rice – Basmati385

Moderate drought conditions

Chilling treatment

Phosphate solubilisation

Different levels of salinity

Seeds of Solanum lycopersicum cv. Mill

Pisum sativum var. early onward

Oryza sativa GJ17 cultivar (salinity sensitive)

and total protein content 0f M. sinensis by 28% Increased tolerance to salt by reducing reactive oxygen species toxicity of ROS by reducing plant cell membrane index Enhanced expression of gcd genes and different strains solubilise phosphate at various concentrations Cell membrane damage and ROS levels reduced, enhanced antioxidant activity and overexpression of cold acclimation genes LeCBF1 and LeCBF3 Enhanced plant biomass and accumulation of K+, Mg2+ and Ca2+. Reduced Na + concentration in tissues Enhanced succinate and superoxide dismutase genes (sod) FeSOD Soil properties changed, enhanced arsC gene expression and arsenic reductase activity Enhanced salt tolerance in plants

Recent Advances in PGPRs and Their Application in Imparting Biotic. . . (continued)

Shah et al. (2017)

Srivastava et al. (2013)

Naya et al. (2007)

Pereira et al. (2016)

Subramanian et al. (2015).

Oteino et al. (2015)

Jha and Subramanian (2014)

15 443

29.

Sr no. 28.

Microbes Trichoderma hamatum DIS 219b isolated from infected pods of Theobroma gileri Trichoderma harzianum TH-56

Table 15.1 (continued)

Oryza sativa genotypes PSD-17, PB-1 and KNMK3131

Plant used Theobroma cacao Var. comun

Drought

Abiotic stress Drought

Improves drought tolerance when treated with the microbe, enhanced expression of DHN/AQU and malonaldehyde genes

Mode of action Delayed drought response

Pandey et al. (2016)

Reference Bae et al. (2009)

444 B. Joshi et al.

15

Recent Advances in PGPRs and Their Application in Imparting Biotic. . .

445

Fig. 15.2 PGPR mediated different actions (second ring) and mode of influence (outer ring)

Synthesis of antioxidant molecules (flavonoid and phenolic compounds) and UV-B screening molecules help in combating ultraviolet (UV) stress in younger leaves of Brassica oleracea L. var. acephala. Also, the production of UV-B pigments, increasing antioxidant enzymes, and induction of pathogenesis-related (PR) proteins at the gene level play a major role in plant defence mechanism. In plants, energy for the metabolism in plants is transferred through the solar radiation which helps in the synthesis of photosynthetic assimilates and vaporisation of different metabolic components in the plant through stomata (Yoon et al. 2021). Despite decades of studies, the mechanisms of Na + and Cl- uptake into roots remain one of the most complex issues in plant salt stress research. Crop tolerance is one of the multifaceted solutions needed to address the increasing salinisation of arable land. Exploiting genetic diversity can help achieve this goal, but it is more successful when accompanied by a thorough understanding of the molecular mechanisms underlying tolerance. While there has been significant progress in recent years, many fundamental mechanisms that influence salt tolerance are still

446

B. Joshi et al.

only partially understood. A better understanding of the role of other minerals may allow us to reduce salt stress by adjusting and distributing these nutrients (Isayenkov and Maathuis 2019).

15.5

Role of PGPR in Overcoming Biotic Stress

Plants are susceptible to a range of biotic stresses from seed germination to harvest, with known and emerging pests, nematodes and microorganisms being major threats to crop quality and quantity. Plant pathogens can lead to crop losses of over 30%, resulting in a decline in global agricultural production and financial difficulties for farmers and countries. In the past, these plant diseases were controlled with agrochemicals, but their unrestricted use increases costs and harms human health, soil and the environment. As the dangers of agrochemicals become better understood and the benefits of organic farming and food safety become more apparent, microbial biofertilisers, which act as biopesticides and promote plant growth, are becoming a preferred, environmentally friendly alternative. The need for chemical-free control of plant pests and diseases, maintaining healthy agricultural ecosystems and protecting human and animal health, is pressing. The use of PGPR to manage biotic stress is effective in controlling plant diseases and provides induced systemic resistance and systemic acquired resistance, thereby reducing crop losses and gaining attention from researchers, farmers, policymakers and consumers. Biological control through beneficial microorganism against plant pathogen and pests has gained importance since it is a very cost-effective, environmentally friendly and sustainable approach for protecting plants worldwide (Azcon-Aguilar and Barea 1997). However, more intensive research is needed to better understand the complex interactions between plants, the environment and pathogens. Maintaining optimal populations of beneficial microbes in the rhizosphere is crucial for preventing pathogen growth. Rhizosphere bacteria such as Bacillus and Pseudomonas species are mostly widely used biopesticides/PGPRs. Georgakopoulos et al. (2002) reported that the use of Pseudomonas antagonists by soaking or seed coating in the peat medium showed satisfactory results in controlling damping-off of sugar beet and cucumber. Microorganisms found in soil and the rhizosphere of plants have the ability to act as entomopathogens (Lacey et al. 2015). Among all, the most well-studied entomopathogenic species for the organic control of insect pests is B. thuringiensis, which has been reported to be more effective against bugs than traditional pesticides used in microbial pest management. According to Lacey and Goettel (1995), B. thuringiensis produced insecticidal proteins that specifically target insect gut and do not harm non-target organisms. Different strains of B. thuringiensis are commonly used to control against lepidopteran pests and armyworms and diamondback moth larvae in agriculture and forestry (McGaughey and Johnson 1992; Zhao et al. 2021). Additionally, the soil bacterium Bacillus cereus, as described by Sevim et al. (2010), has been found to be pathogenic in a number of different insect species.

15

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Bacterial isolates from Agrotis segetum were found to be responsible for 60% insect mortality after 8 days of application; these pathogenic isolates include B. cereus (Ags1), Bacillus sp. (Ags2), B. megaterium (Ags3), Enterobacter aerogenes (Ags4), Acinetobacter calcoaceticus (Ags5), Enterococcus gallinarum (Ags8) and Stenotrophomonas maltophilia (Ags9) (Sevim et al. 2010). Species isolated from predatory larvae of the antlion species Myrmeleon bore (such as Klebsiella, Morganella morganii, Serratia marcescens and B. cereus) were reported to be working against cutworms Spodoptera litura (Nishiwaki et al. 2007). PGPRs have the ability to undergo a process known as induced systemic resistance (ISR), which is similar to systematic acquired resistance (defence mechanism that occurs in response to primary infection caused by pathogen). PGPR-based ISR was first reported by Van Peer et al. (1991) and Wei et al. (1991) in carrot (Dianthus caryophyllus) and cucumber (Cucumis sativus), respectively. In carrot, ISR shows decreased susceptibility to wilt diseases caused by Fusarium sp., while in cucumber, it showed lower susceptibility to foliar disease caused by Colletotrichum orbiculare. These PGPR-mediated ISR are mostly the free living rhizobacterial strains, but endophytic bacteria are also reported to show ISR activity. For instance, ISR was induced by P. putida and P. denitrificans against Ceratocystis fagacearum in oak (Brooks et al. 1994), Bacillus pumilus species against F. oxysporum on pea roots (Benhamou et al. 1996), P. fluorescens against F. oxysporum species on tomato (M’Piga et al. 1997), P. fluorescens strains against red rot diseases caused by Colletotrichum falcatum on sugarcane (Jayakumar et al. 2007) and Verticillium dahliae on tomato (Nazar et al. 2018) (Table 15.2, Fig. 15.2).

15.6

Molecular Mechanism of PGPRs in Control of Biotic and Abiotic Stress

Crop plants in today’s world are facing extensive biotic and abiotic stress due to increasing climatic change and human activities. In addition to organisms that live in the rhizosphere, PGPRs also include different strains of rhizobia that can form nodules on the roots of certain plants (legumes) and endophytes that can live within a plant’s internal tissues (Doty 2018). Nowadays, several PGPRs are available for purchase as biocontrol agents or biofertilisers (Zehra et al. 2021). Despite of extensive research on the effects of PGPRs on plant growth, function, disease resistance, productivity enhancement, phytoremediation and soil quality enhancement, very less is known about the molecular mechanism involved in such plant microbe interactions. However, due to advancement in sequencing technologies (next-generation sequencing), the study of gene expression in different plants species associated with PGPRs is possible in large scale. These technologies enable to understand how PGPRs affect plant growth and gene functions. For example, strain of Pseudomonas fluorescens undergoes gene regulation of 97 genes in roots of Arabidopsis thaliana but not in leaves (Verhagen et al. 2004). Another study found that the P. putida strain MTCC5279 altered the expression of genes that are

Bacillus subtilis strains BBG127 and BBG131

Bacillus amyloliquefaciens 937a, Bacillus subtilis 937b and Bacillus pumilus SE34 Bacillus amyloliquefaciens strain SN13

Bacillus cereus strain AR156, Bacillus subtilis strain SM21 and Serratia sp. strain XY21 Bacillus cereus MJ-1, Bacillus macrolides CJ29 and Bacillus pumilus CJ-69 Bacillus fortis strain 162 and Bacillus subtilis strain 174

2.

3.

5.

7.

6.

4.

Microbe Azospirillum brasilense strain Sp245

S. no. 1.

Soil fertility

Fusarium wilt pathogen (Fusarium oxysporum f. sp. Lycopersici)

Capsicum annum L. Seminis

Tomato

Sweet pepper Capsicum annum L. variety grossum

Pepper blight disease caused by Phytophthora capsici

Tomato mottle virus induced disease using vector Bemisia argentifolii Virulent Rhizoctonia solani

Tomato cultivar Agriset

Susceptible cultivar of rice (Narayan)

Conidia of Botrytis cinerea strain 630

Biotic stress Rhizosphere fungi (Rhizoctonia spp.)

Grapevine variety 41B

Plant Prunus cerasifera L. clone Mr.S 2/5

Table 15.2 Application of PGPR to overbiotic stress in different plant species

Suppressed the fusarium wilt disease in treated plants by polyphenol oxidase, phenyl

Mode of action Imparted enhanced innate resistance of the plant against pathogen Cyclic lipopeptides produced by PGPR-induced local resistance against the pathogen Induction of ISR (induced systemic resistance) leading to reduced disease frequency Rice plants developed resistance again the fungal pathogen by enhancing the growth and health of plant. It also helped to maintain a balance of ROS and modified the cell wall. Increased the plant vigour, yield and fruit taste by supressing the pathogenic fungi present in the soil Enhanced gibberellin production fresh weight of plants Akram and Anjum (2011)

Joo et al. (2005)

Zhang et al. (2019)

Srivastava et al. (2016)

Murphy et al. (2000)

Farace et al. (2015)

Reference Russo et al. (2008)

448 B. Joshi et al.

Paenibacillus lentimorbus MEN2 and Bacillus subtilis strains ALB629, UFLA285, UFLA168, UFLA246, UFLA373, UFLA116 and UFLA29 isolated from cotton rhizosphere Bacillus subtilis (UFLA285)

9.

Bacillus subtilis

12.

Triticum aestivum

Tomato (Lycopersicon esculentum cultivar Peto-86)

Saccharum officinarum L.

Gossypium hirsutum cultivar Deltapine Acala 90

Lycopersicon esculentum (cultivars Daniela and Brillante) and Capsicum annum (cultivars Roxy and Antonio) Common bean cv. Pérola (Phaseolus vulgaris)

Colletotrichum falcatum and Fusarium moniliforme isolated from sugarcane and Fusarium oxysporum, Rhizoctonia solani, Macrophomina phaseolina and Pythium splendens Fusarium oxysporum f. sp. lycopersici

Rhizoctonia solani AG4

Yellow variant of Curtobacterium flaccumfaciens pv flaccumfaciens

Experimental field, sand and hydroponic comparison

Production of plant growth hormones (gibberellins) and reduced disease severity

Application of PGPRinduced resistance in plants against fungal infection and enhanced expression of many resistance gene osmotic regulations Application of PGPR leads to the reduction of disease caused by multiple pathogens by enhanced production of IAA, ACC deaminase and proline

Application of PGPR reduced the bacterial wilt infection by enhancing growth and phenolic and lignin content

(continued)

Abd-Allah et al. (2007)

Xia et al. (2020)

Medeiros et al. (2011)

Martins et al. (2013)

Lucas et al. (2004)

Recent Advances in PGPRs and Their Application in Imparting Biotic. . .

13.

Bacillus xiamenensis strain PM14

11.

10.

Bacillus licheniformis (B2; CECT 5106)

8.

ammonia-lyase and peroxidase production Maturation period of plants shortened and root colonisation of PGPR observed

15 449

Kluyvera cryocrescens strain KUDC1771 and Brevibacterium iodinum strain KUDC1716 isolated from the rhizosphere of Elymus tsukushiensis Pseudomonas fluorescens 89B-61, Bacillus pumilus T4, Bacillus pasteurii C-9, Bacillus subtilis GB03, Bacillus amyloliquefaciens IN937a, Serratia marcescens 90–166 and Enterobacter cloacae JM22 Pseudomonas stutzeri Pr7 and Bacillus toyonensis Pr8

15.

17.

16.

Chickpea, pea cultivar CDC Mozart and lentil cultivar milestone

Enterobacter sp.

14.

Prunus domestica

Arabidopsis thaliana

Pepper Capsicum annum L.

Plant

Microbe Beijerinckia fluminensis strain BFC-33

S. no.

Table 15.2 (continued)

Verticillium wilt disease causing pathogens Verticillium dahliae and Fusarium oxysporum f. sp. melonis

Fungal disease

Biotic stress Alternaria alternata, Rhizoctonia solani and Fusarium oxysporum, Ustilaginoidea Fusarium avenaceum, Rhizoctonia solani CKP7 and Pythium sp. strain p88-p3 Grey leaf spot disease fungal pathogen Stemphylium lycopersici

Application of Pr7 and Pr8 helped in acclimatisation of grapevine and peach rootstock. PGPR also induced resistance against tomato verticillium wilt disease.

PGPR treatment led to the release of volatile compounds (2,3-butanediol and acetoin) resulting in enhanced growth and induced resistance against pathogen

Phosphate solubilisation, production of IAA and siderophores and suppressed the grey leaf spot disease

Mode of action Enhancing phenyl ammonium lyase (PAL), beta-1,3 glucanase and polyphenol oxidase PGPR properties exhibited by the microbes and showed potential as biofertiliser

Essalimi et al. (2022)

Ryu et al. (2003)

Son et al. (2014)

Hynes et al. (2008)

Reference Al-Shwaiman et al. (2022)

450 B. Joshi et al.

22.

21.

20.

19.

18.

Pseudomonas fluorescens strain PFV, Pseudomonas fluorescens strain PFP, Pseudomonas fluorescens strain PSV, Bacillus subtilis strain BSV and Bacillus subtilis strain BSP Pseudomonas fluorescens (ATCC13525), Bacillus subtilis (DSM1088) and Rhizobium leguminosarum Bacillus pumilus strains T-4, SE-34, SE-49, SE-76 and INR-7 S. marcescens strain 90–166, P. fluorescens strain 89B-27, P. putida strain 89B-61, Curtobacterium flaccumfaciens strain INR-5, Burkholderia gladioli strain IN-26 and Enterobacter asburiae strain JM-22 Pseudomonas fluorescens strains UTPF68 and UTPF109; Rhizobium etli strain RH5 and R. leguminosarum strains RH3, RH4, RH6 and RH7 Pseudomonas fluorescens AUR6 and Chryseobacterium balustinum AUR9

Arabidopsis thaliana col 0

HCN, IAA, siderophore and chitinase production leading to the enhanced biocontrol activity against pathogen

Application of PGPR leads to the induction of systematic resistance in A. thaliana.

Damping off (Rhizoctonia solani strain AG-4)

Pseudomonas syringae – leaf spot pathogen pv. Tomato DC3000

Recent Advances in PGPRs and Their Application in Imparting Biotic. . . (continued)

Ramos Solano et al. (2008)

Samavat et al. (2011)

Park and Kloepper (2000)

Induction of ISR (induced systemic resistance) is linked to the PR-1a activity and PGPR

Tobacco necrosis virus

Tobacco (Nicotiana tabacum L. cv. Xanthi-nc)

Common bean (Phaseolus vulgaris)

Mohamed et al. (2019)

Saravanakumar et al. (2007)

Application of PGPR + AFM resulted in decline of stem rot disease incidence

Foliar application of P. fluorescens Pf1 reduced disease incidence and increased yield

White rot (Sclerotium rolfsii)

Tea blister blight disease (Exobasidium vexans)

Common bean (Phaseolus vulgaris L.)

Camellia sinensis (L) O. Kuntze

15 451

Pseudomonas migulae 8R6

Fluorescent Pseudomonads sp. strains R62 and R81

Pseudomonas spp.

Rhizobium leguminosarum biovar phaseoli, Glomus mosseae and G. fasciculatum Rhizobium strains

Stenotrophomonas maltophilia

24.

25.

26.

27.

29.

28.

Microbe Pseudomonas fluorescens strain PTA-CT2

S. no. 23.

Table 15.2 (continued)

Triticum aestivum

Common bean

Common bean

Groundnut

Fungal pathogen Fusarium graminearum

Sclerotium rolfsii

Fusarium semitectum, Fusarium graminearum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii Macrophomina phaseolina (charcoal rot) Root rot (Sclerotinia sclerotiorum)

Flavescence dorée phytoplasma (yellows disease of grapevine)

Catharanthus roseus

Tomato

Biotic stress Botrytis cinerea strain 630

Plant Grapevine cv. Chardonnay clone 7535

Production of HCN, siderophores and antibiosis Stimulation nitrogen fixation in plants and reduced infection rate IAA and siderophore production ACC deaminase (ACCD), gibberellic acid, indole acetic acid (IAA),

Mode of action PGPR-treated plants exhibited enhanced expression of phytoalexins and glutathione – S transferase. Production of 1-aminocyclopropane1carboxylate (ACC) deaminase enzyme after application of P. migulae helps the plant regulate the stress-related hormone ethylene level and induced systematic resistance. Application of PGPR leads to the decline in the disease incidence.

Volpiano et al. (2018) Singh and Jha (2017)

Gupta et al. (2002) Aysan and Demir (2009)

Sarma et al. (2011)

Gamalero et al. (2017)

Reference Gruau et al. (2015)

452 B. Joshi et al.

Pseudomonas putida and P. stutzeri

Bacillus aryabhattai strain SRB02

30.

31.

Tomato seeds of four Korean cultivars (IT 252842–13, IT 252869–14, IT 260627–16, IT 259462–15

Cucumis sativus L.

Fusarium wilt (Fusarium oxysporum f. sp. lycopersici)

–

siderophore and inorganic phosphate solubilisation Application of PGPR strains enhance sugar and proline production leading to the better osmoregulation PGPRs lead enhanced production of amino acids and reduced infection rate. Higher concentration of SA and JA in PGPR-treated plants reduces the effects of Fusarium wilt disease in tomato Shahzad et al. (2021)

Nawaz and Bano (2020)

15 Recent Advances in PGPRs and Their Application in Imparting Biotic. . . 453

454

B. Joshi et al.

involved in genome integrity and hormonal signalling in the leaves of A. thaliana (Srivastava et al. 2012). Additionally, the endophytic PGPR Herbaspirillum seropedicae, when applied to rice roots, was found to regulate the defence-related proteins (PBZ1) and enhance the genes involved in the auxin and ethylene pathway (Brusamarello-Santos et al. 2012). This shows that various PGPRs can affect plant growth in different ways, and understanding these pathways could make it easier for researchers and industries to use PGPRs to boost plant development and develop new products. Furthermore, PGPRs in the rhizosphere inhabit a variety of microbial environments, since they include bacteria that can colonise internal root tissues in addition to saprophytic soil bacteria that infiltrate the rhizosphere. This indicates that the distinction between saprophytes lacking plant-beneficial effects (particularly plant commensals) and vertically inherited endophytes or plant endosymbionts is not always straightforward. Additionally, many bacteria exhibit different ecological niches, and occasionally some may operate as PGPRs and exhibit different sets of gene expressions. Plants have the ability to recognise pathogenic microorganisms through MAMPs (microbe-associated molecular patterns), which are small conserved molecular motifs found on the surface of pathogens. This recognition is made possible by the presence of PRRs (pattern recognition receptors) on the surface of the plant. Studies have shown that this interaction between the plant and pathogen is a longstanding one and initiates PTI (pathogen-triggered immunity). The systemic activation of resistance, known as ISR, is thought to be caused by the translocation of a distant chemical or electrical signal through an interaction between PTI and MYB72 (transcription factor). Additionally, NPR1 (non-expressor of pathogenesis-related genes 1) plays a significant role in plant defence mechanisms against pathogens, regulating both ISR and SAR pathways. This regulation is controlled by DNA binding transcription factors, with MYC2 being a key regulator of the JA signalling pathway. The activation of defence-related genes, such as reactive oxygen species (ROS), is also triggered by these transcription factors to further elicit a resistance response. Thus, studies on PGPR-based ISR and MAMPs have highlighted the importance of various molecules such as lipopolysaccharides (LPS), pyoverdine, salicylic acid and, iron-regulated metabolite involved in defence mechanisms (Pieterse et al. 2014; Dong 2004; Lopez-Vidriero et al. 2021). SAR is characterised by an increase in the salicylic acid hormone in systemic tissues. This hormone activates the expression of PR (pathogenesis-related) genes, a large group of genes involved in plant defence responses, through the redoxregulated protein NPR1 (non-expressor of pathogenesis-related genes 1) (Van Loon et al. 1998; Sayahi et al. 2022). In contrast, ISR (induced systemic resistance) is independent of salicylic acid pathway that confers resistance against biotrophic diseases and is mediated by jasmonic acid and ethylene pathway. This pathway works without activating PR genes and ensure resistance to both necrotrophic pathogens and herbivorous pests (Bari and Jones 2009). The effectiveness of PGPR-assisted phytoremediation is greatly influenced by several factors, including rhizospheric activity, plant development, metal tolerance and heavy metal bioavailability. Among these, rhizospheric activity and metal

15

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455

bioavailability have a significant impact on the plant’s ability to perform phytoremediation. The process of metal absorption in plants varies depending on plant species and types of heavy metals. It is generally absorbed by the aerial parts of leaves through processes such as translocation, accumulation, exclusion and osmoregulation (Qin et al. 2017; Ma et al. 2016a, 2016b). The process of root uptaking of the heavy metals includes recognition of heavy metals through selectively permeable root cells and then binding of metal ions and then finally transporting it to aerial parts of the plants. This process involves the plant’s vascular system and a few transporter proteins (Suman et al. 2018; Mahajan and Kaushal 2018). The efficiency of phytoremediation, phytostabilisation and phytovolatilisation is increased by the production of organic acids, siderophores, EPS and biosurfactants by PGPR strains which in turn changes the nature and mobility of the heavy metals through processes such as chelation, acidification, precipitation, immobilisation at the rhizosphere and oxidation-reduction reactions (Manoj et al. 2020). Uptake and transport of heavy metals in plants are governed by the large number of genes, ensuring that all the tissues receive adequate amount of nutrients and metals that are necessary for important cellular functions. Heavy metals often enter roots via the nutrient transport pathway, aided by membrane transporter proteins and genes due to their structural resemblance to other essential nutrients. Many genes and gene families necessary for transporting and accumulating metals have been identified in plants, including low-affinity cation transporters (LCT), natural resistance-associated macrophage protein (NRAMP), cation diffusion facilitator (CDF), calcium cation exchangers (CCX), ZIP (ZRT- and IRT-like protein), ABC transporter, etc., all of which play a role in the transport and accumulation of heavy metals, either directly or indirectly (Sasaki et al. 2012; Nakanishi et al. 2006; Takahashi et al. 2012). Plant when inoculated with strains of PGPR significantly regulates the genes that are involved in metal transportation through various specific and non-specific plant growthpromoting activities. For example, the inoculation of a B. amyloliquefaciens strain that produces volatile chemicals affects the expression of the FRO2, IRT1 and FIT1 genes which lead to the accumulation of Cd and Fe in Arabidopsis tissues (Zhou et al. 2017). Pan et al. (2016) have demonstrated that the endophytic bacterial strain SaMR12-infected S. alfredii plants have increased Cd accumulation by increasing the expression of the transporter gene of Cd. A study by Jebara et al. (2018) found that a higher level of Cd toxicity enhances the expression of F-box and PCS genes, indicating that these genes play a major role in Cd tolerance in the Sulla coronaria plant. The F-box protein plays a major role in chelating Cd ions and facilitates their easy transport, which eventually promotes plant growth (Brunetti et al. 2011). Similarly, Azospirillum brasilense inoculation enhances Cd tolerance in T. aestivum by controlling the Tatm2the 0 gene expression (Ghassemi and Mostajeran 2018). PGPR isolates are highly recognised for their ability to solubilise phosphates in addition to nitrogen fixation. These processes are generally done by membranebound glucose dehydrogenase and its enzymatic cofactor pyrroloquinoline quinine (PQQ). These enzymes are expressed by pqq operon system containing six core genes, that is, pqqA, pqqB, pqqC, pqqD, pqqE and pqqF. Species such as

456

B. Joshi et al.

Francisella tularensis, Morganella morganii, Pseudomonas cepacian, Burkholderia cepacia, etc. all contain phosphate-solubilising genes, that is, acpA, phoC, gabY, napD and napE genes, respectively (Alaylar et al. 2020). Another important aspect of PGPR is the production of siderophores, which facilitate iron solubilisation and transport by generating soluble Fe3+. Reports have shown that PGPR causes the said gene to be upregulated, which produces siderophores. By modulating the expression of genes linked to metabolism, stress response, defence and phytohormones, PGPR alters gene expression in plants (Ovaa et al. 1995). Treatment of cells with MBE02 strains showed that the expression of 1260 genes was differentially altered, with 979 genes showing an upregulation and 281 genes showing a downregulation. Most of the differentially regulated genes were linked to hormonal homeostasis and induced systemic resistance (ISR) in peanut. In Arabidopsis thaliana, PGPR was found to alter the gene expression of the nitrate and ammonium absorption genes (Calvo et al. 2019). miRNAs (microRNAs), which are non-coding RNA molecules with 20–24 nucleotides, play a role in controlling the expression of many genes that are responsible for stress at the transcriptional and posttranscriptional levels. Gene regulation by these miRNAs depends on the complementary relationship between miRNAs and the target mRNAs which lead to translational repression and cleavage of mRNA, thereby, affecting its expression (Sunkar et al. 2012). Pu et al. (2019) have briefly described about the direct and indirect regulatory mechanisms used by miRNAs to control gene expression. This natural approach allows miRNA to regulate target gene translation in the cytoplasm by binding to the 30 UTR and 30–50 UTR regions of the target mRNA. Nowadays, scientists are more focussing on identifying stress-related miRNAs in plants through PGPR inoculation (Jatan et al. 2019). This miRNA-based regulation technology is a rapidly developing field of study, not only in the field of bacterial-assisted phytoremediation but in all areas of life science research, controlling target gene translation in the cytoplasm (Table 15.3).

15.7

Prospects of PGPR Application in Crop Improvement

It is well established that PGPRs have huge ability to improve crop productivity through various methods and techniques. However, the performance of PGPRs can vary greatly depending on environmental conditions. Factors such as climate change, soil characteristics and the combined activity of the soil’s native microbial flora can affect plant growth and functions (Gupta et al. 2015). Contemporary tools and methods from biotechnology and nanotechnology, including biosensors and nano-fertilisers, have been used to increase agricultural productivity and yields in agriculture and related fields. Additionally, PGPRs have been shown to have mutualistic interactions with plants and help in nutrient uptake by increasing tolerance towards biotic and abiotic stresses, mineral solubilisation and nitrogen fixation, leading to plant development. For example, the use of salinity-tolerant PGPRs (ST-PGPRs) could be a useful and

Azospirillum brasilense

Bacillus thuringiensis NEB17

Kluyvera ascorbata SUD165 Kluyvera ascorbata SUD165 Variovorax paradoxus

Pseudomonas brassicacearum

Kluyvera ascorbata SUD165 Pseudomonas asplenii AC Pseudomonas brassicacearum Am3

2.

3.

4.

7.

8. Abiotic Abiotic

Abiotic

Heavy metal stress (cu 2+)

Heavy metal stress (cd 2+)

Heavy metal stress (cd 2+)

Abiotic

Heavy metal stress (cd 2+)

Abiotic

Abiotic

Heavy metal stress (cd 2+)

Heavy metal stress

Abiotic

Abiotic

Abiotic

Abiotic

Biotic/ abiotic Abiotic

Heavy metal stress

Heavy metal stress

Drought

Drought

Stress Drought

Gene encoding ACC deaminase (acdS) Gene encoding ACC deaminase (acdS) Gene encoding ACC deaminase (acdS)

Gene encoding ACC deaminase (acdS)

Gene encoding ACC deaminase (acdS) Gene encoding ACC deaminase (acdS) Gene encoding ACC deaminase (acdS)

Genes for Thuricin 17

Genes involved ACO, ACS, PR1, MYC2, SOD, CAT, APX, GST, DREB1A, NAC1 and LEA DHN Nod genes

Pisum sativum L.

Lycopersicum esculentum Mill Phragmites australis Pisum sativum L.

Brassica juncea L.

Brassica juncea L.

Brassica juncea L.

Brassica napus

Glycine max

Phaseolus vulgaris

Crop plant involved Cicer arietinum

(continued)

References Tiwari et al. (2016) Burdman et al. (1996) Prudent et al. (2015) Burd et al. (1998) Burd et al. (2000) Belimov et al. (2005) Belimov et al. (2001) Burd et al. (2000) Reed et al. (2005) Safronova et al. (2006)

Recent Advances in PGPRs and Their Application in Imparting Biotic. . .

11.

10.

9.

6.

5.

PGPR Pseudomonas putida MTCC5279

S. no. 1.

Table 15.3 Molecular mechanism of various strain of PGPRs controlling of biotic and abiotic stress

15 457

Enterobacter cloacae

Kocuria rhizophila Y1

Enterobacter cloacae PM23 Paenibacillus polymyxa B2

12.

13.

14.

Pseudomonas putida

Bacillus amyloliquefaciens SQR9 Pseudomonas simiae

17.

18.

19.

Bacillus megaterium H3

16.

15.

PGPR Alcaligenes xylosoxidans

S. no.

Table 15.3 (continued)

Heavy metal stress

Abiotic

Abiotic

Abiotic

Heavy metal stress (cd 2+)

Heavy metal stress

Abiotic

Abiotic

Abiotic

Abiotic

Abiotic

Biotic/ abiotic

Heavy metal stress

Drought

Salinity

Salinity

Heavy metal stress

Stress

Glycine max

Zea mays L.

NHX and H+ - PPase

POD and CAT

Nicotiana tabacum

Zea mays L.

Arabidopsis thaliana

Zea mays L.

Zea mays L.

Lycopersicum esculentum L.

Crop plant involved

CzcA, CzcB and CzcC

arsC, aioA and arsM

ERD15

ZmGR1 and ZmAPX1, ZmNHX1, ZmNHX2, ZmNHX3, ZmWRKY58 and ZmDREB2A ItuC, sfp, srfAA, APX and SOD

Gene encoding ACC deaminase (acdS)

Genes involved Gene encoding ACC deaminase (acdS)

Vaishnav et al. (2016)

Ali et al. (2022) Timmusk and Wagner (1999) Wang et al. (2018) Nesler et al. (2017) Chen et al. (2016)

References Belimov et al. (2001) Grichko and Glick (2001) Xiaozhou (2020)

458 B. Joshi et al.

Pseudomonas aurantiaca ST-TJ4 produced volatile compounds Paenibacillus lentimorbus strain B-30488 Klebsiella pneumoniae strain JCK-2201

Bacillus xiamenensis strain PM14

Bacillus velezensis strain F21

Bacillus velezensis strain SQR9 Bacillus licheniformis strain BL06

22.

25.

26.

27.

28.

24.

23.

21.

Trichoderma harzianum isolate ITEM908 Streptomyces yangpuensis strain CM253

20.

Fungal wilt pathogen Fusarium oxysporum f. sp. cucumerinum Phytophthora sojae and Phytophthora capsici

Colletotrichum falcatum and Fusarium moniliforme causing red rot disease Fusarium wilt (Fusarium oxysporum f. sp. niveum)

Sclerotium rolfsii causing southern blight disease in tomato Ralstonia solanacearum causing bacterial wilt

Invasion of Meloidogyne incognita Corm rot in saffron (Fusarium oxysporum, F. solani, Penicillium citreosulfuratum and P. citrinum) Verticillium dahliae VDAG-02273 (hydrophobin gene) was downregulated

Biotic

Biotic

Biotic

Biotic

Biotic

Biotic

Enhanced expression of genes involved in MAPK signalling pathway and phytohormone signalling pathway degQ gene and SQR9 expression enhanced after PGPR application Pathogenesis-related protein gene NbPR1 and NbPR2 expression got

Enhanced expression of SIMKP4, SIMLO, RBOHF and RBOHD genes PGPR application leads to expression of LePR1, LePR2, LePR5, LePR3 and PI-II genes in the salicylic acid and jasmonic acid signalling acds gene expression increased

Biofilm from genes upp, rfbBC, efp, aftA, pssA, pilD, fliA and dhaM got enhanced expression

Biotic

Biotic

PR1

Biotic

Xu et al. (2019) Yuan et al. (2022)

Cucumber and tomato Nicotiana benthamiana

Recent Advances in PGPRs and Their Application in Imparting Biotic. . . (continued)

Amna et al. (2020) Jiang et al. (2019)

Kim et al. (2022)

Dixit et al. (2018)

Ni et al. (2022)

Leonetti (2017) Tian et al. (2022)

Saccharum officinarum L. (sugarcane) Watermelon (Citrullus lanatus)

Tomato

Verticillium dahliae mycelia radial growth and biomass Tomato

Lycopersicum esculentum L. Saffron (Crocus sativus L.)

15 459

Bacillus amyloliquefaciens strain HK34 Bacillus amyloliquefaciens strain wild-type and FZB42Δsfp mutant

29.

30.

PGPR

S. no.

Table 15.3 (continued)

Pseudomonas syringae pv. Tomato DC3000

Phytophthora cactorum causing foliar and root blight

Stress

Biotic

Biotic

Biotic/ abiotic Genes involved increased in addition to NbPAL gene Genes PgPR10, PgPR5 and PgCAT got upregulated on PGPR application Jasmonic acid (JA) signallingrelated genes are upregulated Arabidopsis thaliana Col-0

Korean ginseng (Panax ginseng)

Crop plant involved

Xie et al. (2018)

Lee et al. (2015)

References

460 B. Joshi et al.

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important tool for reducing salinity and enhancing global food supply. Utilising multi-omic approaches such as metabolomics, metagenomics, transcriptomics and meta-transcriptomics, microbiome engineering and rhizosphere engineering can classify potential bacterial strains based on their performance in the field. There is currently much discussion about the use and commercialisation of beneficial phytomicrobiome components as they improve the quality and quantity of soil organic matter in nutritionally deficient agrosystems. Additionally, it can also reduce carbon emissions and aid in the fight against climate change. There has been a significant interest in using eco-friendly plant-microbial combinations for heavy metal contamination remediation in recent years. Many in situ and ex situ studies have been conducted to evaluate the effectiveness of this combination (plant and microbes) strategy. Roles of transcriptional factors (TF)/regulatory genes involved in metal transformation in PGPR-associated plants are not widely studied; however, better knowledge is required to understand the pattern of gene regulation so that we can use PGPRs in multi-component phytoremediation system. Recently, genetically modified PGPR strains have also been introduced in phytoremediation as they are more effective, but their effects on ecosystems need to be studied before they can be commercialised since PGPR formulations frequently fail when tested in the field rather than in the lab (Manoj et al. 2020). Despite the significant benefits that PGPRs can provide to the host plant, using them for enhancing agricultural products has several difficulties/challenges. To fully understand the rhizospheric chemistry and identify powerful rhizospheric bacteria and microbial communities for effective formulations, multidisciplinary research is needed.

15.8

Conclusion

The toxic effects of biotic and abiotic stress on the environment have garnered global attention, leading to the development of effective methods for removing these contaminants. The use of bacterial fertilisers had a significant impact on soil health, plant productivity and growth. PGPRs can stimulate plant growth in two different ways: directly and indirectly. They also support plant development by suppressing phytopathogens that can inhibit growth and yield. Factors such as the age of the plant and the biological, physical and chemical characteristics of the soil can be greatly affected with the inoculation of PGPR. The use of PGPRs may lead to the replacement of pesticides/chemical fertilisers for sustainable maintenance of the environment. PGPR strains regulate the expression patterns of primary metal transporters and associated genes, which control metal chelation, uptake and compartmentalisation. PGPRs help in the mobilisation and solubilisation of nutrients and are highly versatile in transforming the rhizosphere soil which in turn helps in increasing soil fertility, thereby having positive influences in plant productivity and ecological functioning. As the demand for food continues to increase with the increase in the population, the use of PGPRs as a means of supporting sustainable agriculture has been suggested for the past four decades. Although many scientists have been gaining

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more comprehensive research, an in-depth understanding of the mechanisms used by PGPRs to promote plant growth has been needed. However, a deeper understanding of the mechanisms of PGPRs may aid in the development of a more versatile strain that can be used in various challenging environments which could result in the availability of a new PGPR strain in the rhizosphere. A lot of potential PGPRs has been identified, but they are yet to be successfully commercialised. In conclusion, researchers have a good understanding of the fundamental workings of PGPRs. However, more extensive research and considerable efforts are required for better understanding of the interactions between the microbes and plants and the use of PGPRs as an effective technique for sustainable agriculture.

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Microbial Hyaluronidase: Its Production, Purification and Applications

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Sandip P. Patil, Kiran S. Dalal, Leena P. Shirsath, and Bhushan L. Chaudhari

Abstract

Hyaluronidase (hyase) is an enzyme from glycosidase family that degrades hyaluronic acid (HA) and other associated glycosaminoglycans. It precisely cleaves the β,1–4 glycosidic linkages in hyaluronic acid substrate. Generally pathogenic Gram-positive bacteria produce hyases; where enzyme serves as virulence factor that facilitate the spreading of bacteria in host tissues by degradation of hyaluronic acid present in connective tissues. The optimization of nutritional and physical parameters for hyaluronidase synthesis carries high importance in enzyme yield improvement. The purification of extracellular microbial hyase from a protein mixture could be achieved by a series of purification steps like solvent and salt precipitation, membrane technology and various chromatographic methods. The present chapter summarizes hyase production, purification and its applications. The interest in hyaluronidase is increasing due to its various applications. Keywords

Hyaluronidase · Hyaluronic acid · Production · Purification · Characterization

S. P. Patil · L. P. Shirsath Department of Microbiology and Biotechnology, R. C. Patel Arts, Commerce and Science College, Shirpur, India K. S. Dalal · B. L. Chaudhari (✉) Department of Microbiology, School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_16

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Introduction

The enzyme hyaluronidase (hyase) is from glycosidase family that has an ability to degrade hyaluronic acid (HA) in majority while it can also degrade chondroitin sulphate, chondroitin, dermatan sulphate and other associated glycosaminoglycans (Stern and Jedrzejas 2006). The attention of scientific community in hyaluronidase research is increasing greatly due to its usefulness in physiological and pharmaceutical fields towards betterment of life (Nermeen et al. 2010). These enzymes are usually distributed in venoms (snakes, fishes, lizards, bees, scorpions, wasps and spiders); mammalian tissues and organs; body fluids (tears, blood, seminal fluid), microorganisms like bacteria, fungi and yeast; and invertebrate animals (leeches, crustaceans) (Kreil 1995). Several bacterial species were reported to produce enzyme hyaluronidase, namely, Streptococcus, Micrococcus, Peptostreptococcus, Propionibacterium, Streptomyces, Staphylococcus, Bacteroides, Clostridium, etc. (Sahoo et al. 2008), whereas species of Penicillium are also reported to produce enzyme hyase (Bakke et al. 2011). The enzyme hyaluronidases are extensively exploited in surgeries, orthopaedics, ophthalmology, dermatology, gynaecology and cancer (Farr et al. 1997). Since the acquaintance of knowledge on the new biomolecule hyaluronidase, newer roles of hyases are in search continuously where role in fertilization was also observed (Gmachl and Kreil 1993), as well as inflammation, growth and metastasis of tumour cells (Csoka et al. 1997), human embryogenesis, mammalian cell migration and differentiation and healing of wounds (Weigel et al. 1986). The bacterial hyases serve as the virulence factors which facilitate the spread of pathogenic bacteria in host tissues by the degradation of polymeric substrate hyaluronic acid present in connective tissues (Nukui et al. 2003). The breakdown of hyaluronic acid results in increasing permeability of extra cellular matrix (ECM) which eases entry of pathogenic microbes. The action of hyaluronidases on ECM plays a crucial role in dissemination of toxins and pathogens in various diseases like meningitis, synovitis, gas gangrene, nephritis, hyperplasia, diseases of teeth including infections and inflammation, mycoplasmosis, lung infections, mastitis of cows, sexually transmitted disease syphilis and septicemia (Sutherland 1995; Hynes and Walton 2000; Spellerberg 2000; Jedrzejas 2001; Jedrzejas 2004; Makris et al. 2004). The substrate HA-degrading activity of microbial species supports adhesion, establishment by colonization and extraction of nutrients for their growth and development (Hynes and Walton 2000).

16.1.1 History The enzyme hyase activity was identified by Duran-Reynals as the “spreading factor” in the mammalian test extracts which helped in spreading the injected vaccines in the body (Duran-Reynals 1928). Shortly thereafter, this enzyme could be detected in suspension of bacteria (Duran-Reynals 1933). Then this term “hyaluronidase” was coined to name the enzymes which specifically denature the

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substrate HA (Hobby et al. 1941; Chain and Duthie 1940). Karl Meyer categorized this enzyme into three classes depending on analysis of products obtained upon enzymatic reaction (Meyer 1971). Meyer was able to classify the three types of hyases including one prokaryotic lyase type of glycosidase and eukaryotic endoglycosidase hydrolases having two classes.

16.1.2 Natural Biological Role The earlier findings suggest that mammalian testis extracts show hyase activity; they play vital role in a process of fertilization. The enzyme hyase is generally present on the posterior head of mammalian sperm. During fertilization, mammalian sperm must first cross a cumulus cell layer that surrounds the egg. These cells are located in a hyaluronic acid-rich matrix (Salustri et al. 1992) that may be degraded by enzyme hyase. Naturally, hyase activity is also detected in the venom of fishes, snakes, wasps, bees, scorpions, spiders, etc. These enzymes can serve as “spreading factor” by depolymerizing hyaluronic acid, which facilitates the diffusion of constituents of venom (Ramanaiah et al. 1990). The entry and mechanisms of hyaluronidase enzyme by means of various virulence factors are associated with tissue dissection in gas gangrene, as a treponema spread in syphilis, and the penetration of the skin and gut by nematode parasite (Frost et al. 1996). The depolymerization of substrate HA by hyase significantly affects the integration of ECM and impedes its activity as a reservoir of cytokines, growth factors and various enzymes involved in the process of signal transduction (Girish et al. 2009). Hyaluronidase also plays a crucial role in cancer metastasis and angiogenesis (Paiva et al. 2005; Boregowda et al. 2006; McAtee et al. 2014).

16.1.3 Mechanism of Action The bacterial hyases (hyaluronate lyase) belong to a group of lyases (EC 4.2.2.1) which selectively cleave β,1–4-glycosidic linkage in substrate HA (Fig. 16.1). These enzymes are β-endoglycosidases that are incorporated in the unsaturated bond by β-elimination process (Stern and Jedrzejas 2006; Jedrzejas 2004). The mechanism of enzyme involves the proton acceptance and donation between enzyme and substrate. The detailed catalytic mechanism (Ponnuraj and Jedrzejas 2000; Li et al. 2000; Li and Jedrzejas 2001) comprises of several sequential steps which are stated below in brief: • The substrate HA binds into the enzyme cleft. • An enzyme Asn residue brings acidification of C-5 atom of a glucuronate residue. • The enzyme His amino acid takes out C-5 carbon proton, followed by an unsaturated bond formation between C-4 and C-5 of the glucuronate on the reducing side of the glycosidic bond.

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Fig. 16.1 Hyaluronidase cleaves the β,1–4-glycosidic bond between D-glucuronic acid and N-acetyl D-glucosamine

• Cleavage of the glycosidic bond occurs after a proton is contributed from the Tyr of the enzyme. • The enzyme balances the hydrogen ions in aqueous medium by leaving the disaccharide product. • Subsequently, the enzyme is ready for another offset of substrate catalysis.

16.2

Nomenclature and Classification of Hyaluronidases

Karl Meyer and his associate John Palmer purified an unidentified biological substance from the vitreous body of cows’ eye (Meyer and Palmer 1934). The biochemical analysis showed that the substance was comprised of two different sugar molecules, of which one was uronic acid. Hence, for convenience, they suggested the name “hyaluronic acid”. In 1940, Karl Meyer introduced the term “hyaluronidase” to designate the enzyme which have an ability to degrade substrate hyaluronic acid. Meyer classified enzyme hyaluronidases on the basis of their mode of action, biochemical analyses and reaction end products (Meyer 1971). The advances in amino acid sequences and protein structural data of hyases revealed that the classification scheme of Karl Meyer was remarkably accurate which does not require any modification till today. He classified hyases into three different groups and their peculiarities are stated in Table 16.1.

Class Hyaluronate 4-glycanohydrolase

Hyaluronate 3-glycanohydrolase

Hyaluronate lyase

Name Mammalian hyase

Leech hyases

Microbial hyases

EC 4.2.2.1

EC 3.2.1.36

EC number EC 3.2.1.35 End products Tetra- and hexasaccharides

Tetra- and hexasaccharides

Unsaturated disaccharides

Bond cleaved β-1,4-glycosidic bond

β-1,3-glycosidic bond

β-1,4-glycosidic bond

Table 16.1 Classification of hyases by Karl Meyer (1971)

β-Elimination

Hydrolytic

Activity Hydrolytic and transglycosidase

HA, chondroitin, dermatan, dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate

HA

Substrate specificity HA, chondroitin, dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate

Sources Yak Bos grunniens testis hyase, bee Apis mellifera venom hyase, spider Vitalius dubius venom, Indian cobra Naja naja venom, scorpion Tityus serrulatus venom Hirudo medicinalis, Nephelopsis obscura, Erpobdella punctata, Desserobdella picta, Placobdella ornata Streptococcus agalactiae, Streptococcus pneumoniae, Arthrobacter globiformis, Bacillus niacin, Streptococcus equi, Streptococcus uberis

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The first group is hyaluronate 4-glycanohydrolases, also known as mammalian hyaluronidase (EC 3.2.1.35) that degrades substrate hyaluronic acid by random cleavage of β-1,4-glycosidic bond. The major end products of the reaction include tetra- and hexa-saccharides with N-acetyl-D-glucosamine at the reducing ends. These glycosidase enzymes reveal hydrolytic as well as transglycosidase activity and have the ability to depolymerize HA, chondroitin, chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate. The well-known enzymes from this class are bee venom hyases, testicular hyases, lysosomal hyases and snake venom hyases (Cramer et al. 1994). The second group is comprised of hyases found in the salivary glands of leeches and hookworms (Hotez et al. 1992). These enzymes are hyaluronate 3-glycanohydrolases, also known as leech hyases (EC 3.2.1.36) which degrade substrate HA by cleavage of the β-1,3-glycosidic bond. These enzymes yield tetrasaccharide and hexa-saccharide end products with D-glucuronic acid at the reducing end. But these enzymes are inert towards other related glycosaminoglycans and show limited substrate specificity. The mechanism of action of these enzymes bears a resemblance to the vertebrate hyases than the microbial hyases. The third group represents microbial hyaluronidases, also known as hyaluronate lyases (EC 4.2.2.1). They catalyse substrate HA by β-elimination process to yield unsaturated disaccharide unit 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-D-glucose as the main end product. Unlike other two groups of hyases, this group of enzymes never utilizes hydrolysis activity for degradation of HA. The microbial hyases were purified and characterized from various microorganisms, namely, Micrococcus, Streptococcus, Clostridium, Bacillus, Arthrobacter, Propionibacterium, Staphylococcus and Streptomyces, that differ in substrate specificity (Zhu et al. 2017; Suzuki et al. 2002; Guo et al. 2014; Ahmed 2014; Pritchard et al. 2000; Tyner and Patel 2015).

16.3

Diversity of Hyaluronidases

16.3.1 Human Hyaluronidases Six distinguished hyase gene sequences are present in the human genome having significant homology (~40%) with each other (Jedrzejas and Stern 2005), but the expression of each gene is associated with different tissues. The major hyaluronidases expressed in somatic cells of human for the depolymerization of substrate HA are Hyal-1 and Hyal-2 (Csoka et al. 2001). Hyal-1 is a single subunit glycoprotein molecule that shows maximum activity in acidic pH range having molecular weight of 57 kDa. The enzyme Hyal-2 is also active in acidic pH range, and it is attached to the plasma membrane by a glycosylphosphatidyl-inositol (GPI) linkage (Lepperdinger et al. 1998, 2001). Hyal-3 is expressed in the testis, bone marrow and chondrocytes, while its expression in tissues increases when fibroblasts undergo the differentiation of chondrocytes (Nicoll et al. 2002; Flannery et al. 1998). The Hyal-4 is also a GPI-anchored protein

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like Hyal-2 and PH-20 (Prehm 1984; Lin et al. 1994) and is expressed in the placenta and muscle. PH-20 is the GPI-anchored hyaluronidase found on mammalian sperm, which is also known as SPAM-1 (sperm adhesion molecule-1) associated with sperm surface, and in the lysosome-derived acrosome. All these human hyases have significant amounts of sequence conservation, which reveals the reason of their common catalytic and structural properties. The amino acid sequences of these hyases are quite uniform in their chain length and range.

16.3.2 Bovine Testicular Hyaluronidases In 1928, Duran-Reynals detected the hyaluronidase enzyme activity in the extracts of mammalian testes as a “spreading factor”, which facilitated the diffusion of dyes and antiviral vaccines injected subcutaneously (Duran-Reynals 1928). The enzyme PH-20 was found as the predominant hyaluronidase form present in bull testis extracts as a membrane-bound protein (Meyer et al. 1997). This endoglycanohydrolase (EC 3.2.1.35) enzyme degrades substrate hyaluronic acid by the cleavage of β-1,4 glycosidic bond.

16.3.3 Venom Hyaluronidases Hyaluronidases are frequently detected in almost all types of animal venoms. The hyase enzyme activity has been detected in several snake venoms (Kudo and Tu 2001; Girish et al. 2004; Kemparaju and Girish 2006; Zhong et al. 2010; Wahby et al. 2012; Bordon et al. 2012; Bhavya et al. 2016), bees (Gmachl and Kreil 1993; Reitinger et al. 2001), scorpions (Ramanaiah et al. 1990; Pessini et al. 2001; Morey et al. 2006; Feng et al. 2008), stonefish (Poh et al. 1992), spiders (Rash and Hodgson 2002; da Silveira et al. 2007; Nagaraju et al. 2007; Sutti et al. 2014), hornets (Lu et al. 1995), caterpillars (da CB Gouveia et al. 2005), wasps (Kreil 1995; Jacomini et al. 2013) and lizards (Tu and Hendon 1983). The venom hyaluronidases specifically depolymerize the HA present in extracellular matrix of soft connective tissues that facilitates the diffusion of toxic venom constituents through the body of host (Girish et al. 2004; Kemparaju and Girish 2006). Among venom hyases, the bee venom enzyme has been studied in detail, as this is the first eukaryotic hyase cloned via cDNA (Reitinger et al. 2001).

16.3.4 Leech Hyaluronidases This hyase is obtained from the heads of medicinal leeches or tropical Asian leeches (Budds et al. 1987; Hovingh and Linker 1999). Leech hyaluronidases (EC 3.2.1.36) are hyaluronate 3-glycanohydrolases that degrade substrate HA by cleavage of the

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β-1,3-glycosidic bond yielding tetra-saccharides and hexa-saccharides with glucuronic acid at the reducing end. A novel HA-degrading enzyme has been isolated, purified and well characterized from leeches Nephelopsis obscura, Hirudo medicinalis and Erpobdella punctata (Hovingh and Linker 1999).

16.3.5 Microbial Hyaluronidases Microbial hyaluronidases (hyaluronate lyase, EC 4.2.2.1) belong to the class of glycosidase enzymes that degrade hyaluronic acid of the ECM. A variety of microorganisms are reported to produce enzymes having capability of degrading hyaluronic acid. In humans, a ground substance of connective tissues offers a defence against many pathogenic bacteria. The viscous nature of such a ground substance generally works as a barrier for the entry of pathogens and their extracellularly secreted products, whereas some bacteria have potential to penetrate the connective tissues. Several pathogenic bacterial strains are able to establish and spread infections at the mucosal or skin surface by producing the enzyme hyase as their potent virulence factor (Hynes and Walton 2000). This virulence factor of pathogenic microbes plays an important role in the development of several life-threating diseases, such as synovitis, gangrene, meningitis, mycoplasmosis, hyperplasia, periodontal disease, nephritis, mastitis, pneumonia, septicemia, syphilis, toxic shock syndrome and wound infections (Sutherland 1995; Li et al. 2000; Spellerberg 2000; Makris et al. 2004; Matsushita and Okabe 2001). The polymeric form of substrate hyaluronic acid plays a crucial role in immune system functions (Laurent and Fraser 1992). The breakdown of substrate hyaluronic acid by the hyaluronidases of pathogens can modulate immune system, facilitating growth of pathogen in the host (Hynes and Walton 2000). The microbes able to produce enzyme hyaluronidase belong to the species of Staphylococcus, Streptococcus, Propionibacterium, Peptostreptococcus, Clostridium and Streptomyces (Canard et al. 1994; Berry et al. 1994; Gunther et al. 1996; Girish et al. 2009), whereas it has also been detected in various species of Candida, including C. tropicalis, C. albicans, C. krusei, C. guilliermondii and C. parapsilosis (Shimizu et al. 1995). The causative agents of syphilis—Treponema pertenue and Treponema pallidum—are also reported to produce enzyme hyase (Fitzgerald and Gannon 1983). Veterinary pathogens, S. dysgalactiae and S. uberis—causative agents of mastitis—are also reported to produce enzyme hyaluronidase (Schaufuss et al. 1989; Matthews et al. 1994; Calvinho et al. 1998). Recently, hyases were also reported as important virulence factors of group B Streptococcus (GBS) responsible for ascending vaginal infections in pregnant women that lead to increase the foetal injuries, foetal demise and preterm birth defects (Vornhagen et al. 2016; Surve et al. 2016).

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481

The Sources of Enzyme Hyases

Hyaluronidases are ubiquitously detected in nature, which are found in vertebrate mammals, invertebrate animals (leeches, insects and crustaceans), pathogenic fungi (Streptomyces, Candida and Penicillium), bacteria and viruses especially bacteriophages (Kreil 1995) (Table 16.2). The significant activity of hyases was detected in various mammalian tissues and body fluids (blood, tears, seminal fluid) (Bollet et al. 1963). It was also found in the testes (Senn et al. 1992) and in various mammalian tissues, for example, lymphatic system, kidney, skin and liver. The hyases were also predominantly found in venoms of animals like snakes, spiders, lizards, scorpions, caterpillars, stonefishes, social wasps and bee venoms (Tan and Ponnudurai 1992a, b). The amino acid sequences of several prokaryotic hyases have been deciphered (Hynes and Walton 2000; Suzuki et al. 2002). The hyaluronate lyases from Streptococcus pneumoniae and Streptococcus agalactiae were well known and characterized among the bacterial hyases (Pritchard et al. 2000; Jedrzejas and Chantalat 2000; Jedrzejas et al. 2002). Among all of the hyases, only the microbial hyases have potential to produce it on large and commercial scales. In case of hyases of animal origin, it needs to sacrifice the animal while the quantity is also very small. Hence, microbes being convenient to grow on large scale in short span in affordable cost are preferred. There is again some diversity in the physicochemical characteristics of microbial hyases depending on the type of organism.

16.5

Hyase Production

The production process of enzyme hyase is significantly influenced by different physicochemical parameters like pH, inoculum size, temperature, duration of incubation and agitation rate and nutritional parameters like carbon and nitrogen sources. The optimization of these physical and nutritional parameters for enzyme hyaluronidase production plays an important role in increasing the yield of an enzyme. The optimization of hyase production from Streptococcus equi was studied by one-factor-at-a-time approach (Sahoo et al. 2007), Streptococcus mitis (Sahoo et al. 2008), Streptococcus pyogenes (Lukyanenko 2011), Streptococcus mitis MTCC 2695 (Mahesh et al. 2012), Brevibacterium halotolerans (Patil et al. 2021) and Bacillus sp. LA_04 (Abed et al. 2012) where other factors remain unchanged. However, Ahmed (2014) studied the hyase production and optimization by Staphylococcus aureus through statistical approaches of Plackett-Burman (PBD) and Box-Behnken designs (BBD). The optimum pH range for the production of microbial hyase has been found to be between 5 and 7 (Sahoo et al. 2007; Sahoo et al. 2008; Lukyanenko 2011; Mahesh et al. 2012; Abed et al. 2012; Patil et al. 2021). The temperature of 37 °C is most suitable for maximum hyase production by bacterial strains (Sahoo et al. 2007; Sahoo et al. 2008; Abed et al. 2012; Ahmed 2014). The most preferred carbon source and nitrogen source for maximum hyase production are glucose, sucrose,

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Table 16.2 Sources of hyases Common name of S. no. organism Bacterial hyases 1. Bacteria 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Fungal hyases 1. Fungi 2. 3. 4. 5. 6. 7. 8. 9. 10. Leech hyases 1. Leech 2. 3. 4. 5. 6. 7.

Scientific name of organism Streptococcus dysgalactiae Streptococcus zooepidemicus Streptococcus equi Streptococcus uberis Streptococcus agalactiae Streptococcus equi Arthrobacter globiformis Peptostreptococcus sp. Bacillus niacini Bacillus sp. A50 Streptococcus dysgalactiae Streptococcus pneumoniae Streptococcus pneumoniae Staphylococcus aureus Clostridium difficile Clostridium chauvoei Propionibacterium acnes Arthrobacter globiformis Staphylococcus aureus

Reference Sting et al. (1990) Sahoo et al. (2007)

Schaufuss et al. (1989) Ozegowski et al. (1994) Hill (1976) Zhu et al. (2017) Tam and Chan (1985) Kurata et al. (2015) Guo et al. (2014) Hamai et al. (1989) Akhtar and Bhakuni (2003) Nukui et al. (2003) Skalka (1985) Hafiz and Oakley (1976) Princewill and Oakley (1976) Tyner and Patel (2015) Zhu et al. (2017) Ahmed (2014)

Candida albicans Candida guilliermondii Candida parapsilosis Candida tropicalis Candida krusei Paracoccidioides brasiliensis Pseudozyma aphidis Cryptococcus laurentii Penicillium purpurogenum Penicillium funiculosum

Shimizu et al. (1995)

Haemopis marmorata Nephelopsis obscura Erpobdella punctata Desserobdella picta Placobdella ornate Glossiphonia complanata Helobdella stagnalis

Hovingh and Linker (1999)

De Assis et al. (2003) Smirnou et al. (2015) Bakke et al. (2011)

(continued)

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Table 16.2 (continued) Common name of S. no. organism 8. Venom hyases 1. Indian cobra 2. Snake 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Scientific name of organism Hirudo medicinalis

Reference

Naja naja Agkistrodon blomhoffii ussurensis Cerastes cerastes Crotalus durissus terrificus Bungarus caeruleus

Girish et al. (2004a, b, c) Zhong et al. (2010)

Honeybee Honeybee Caterpillar

Buthus martensii Palamnaeus gravimanus Tityus serrulatus Rhopalurus junceus Hippasa partita Loxosceles intermedia Vitalius dubius Apis mellifera Apis dorsata Lonomia obliqua

Feng et al. (2008) Morey et al. (2006) Pessini et al. (2001) Garcia-Gomez et al. (2011) Nagaraju et al. (2007) da Silveira et al. (2007) Sutti et al. (2014) Reitinger et al. (2001)

Stonefish Social wasp

Synanceia horrida Polybia paulista

Egyptian horned viper Rattlesnake Indian common Krait snake Indian black scorpion Chinese red scorpion Scorpion Funnel web spider Spider

Wahby et al. (2012) Bordon et al. (2012) Bhavya et al. (2016)

da CB Gouveia et al. (2005) Poh et al. (1992) Jacomini et al. (2013)

starch (Lukyanenko 2011; Ahmed 2014) and ammonium chloride (Sahoo et al. 2008; Abed et al. 2012; Ahmed 2014) (Table 16.4), respectively. The optimization by statistical approaches (PBD and BBD) elevated the hyase production up to 492 U/ mL (Ahmed 2014).

16.6

Hyase Purification Approaches

The purification of extracellularly produced microbial hyaluronidase from a mixture of heterogeneous proteins has been achieved by solvent precipitation (ethanol, acetone) and salt precipitation (ammonium sulphate), which is followed by various membrane technologies employing ultrafiltration and chromatographic techniques like affinity chromatography (DEAE-cellulose, CM-cellulose) and gel filtration chromatography (Sepharose, Sephadex). The various purification approaches used for microbial hyase purification are compiled in Table 16.5.

Bacillus sp. A50 Cryptococcus laurentii Penicillium purpurogenum Peptostreptococcus sp.

Propionibacterium acnes Pseudozyma aphidis Staphylococcus aureus

3. 4. 5.

7.

Streptococcus sp. group A Streptococcus suis

14.

15.

13.

12.

11.

Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus pneumoniae Streptococcus pyogenes

10.

8. 9.

6.

2.

Hyase-producing microorganism Arthrobacter globiformis Bacillus niacini

Sr. no. 1.

130

70 5.5

6.0

5.9

6.0

82

70

5.8–6.6

6.3

3.0 8.6

6.4

7.0

6.5 6.0 3.0

6.0

pH optima 6.0

125

116

130 84

85

160

120 120 30

120

MW (kDa) 73

37

40

45

37

37

40

37–45 37

40

46

44 37 43

45

Temp (°C) Optima 42

Table 16.3 Bio-physicochemical characteristics of microbial hyases

HA

HA

HA, Ch-S-A

HA

HA, Ch-S-A, dermatan-S HA, Ch

HA ND

HA, Ch-S-A, Ch-S-C HA, Ch-S-A, C

HA, Ch, Ch-S-A, Ch-S-C HA, Ch-S-A, C,D HA HA

Substrate specificity Ch-S-A, chitosan

ND

ND

3.8 × 10-4 M ND

ND

1610 μM/ min/mg ND

ND

ND ND

Allen et al. (2004)

Akhtar and Bhakuni (2003) Gerlach and Köhler (1972) Hill (1976)

Smirnou et al. (2015) Abramson and Friedman (1968) Ozegowski et al. (1994) Hamai et al. (1989)

Ingham et al. (1979)

Tam and Chan (1985)

Guo et al. (2014) Smirnou et al. (2015) Bakke et al. (2011)

0.27 min-1 ND ND 400 mM/ min/mg ND

Kurata et al. (2015)

References Zhu et al. (2017)

Vmax 4.76 μmol/ min/mL ND

ND

ND

2

8.17 × 10mg/mL 53 μM

ND ND

ND

0.14 mg/mL

0.02 mg/mL ND ND

0.011%

Km 0.11 mg/mL

484 S. P. Patil et al.

Streptomyces hyalurolytis

17.

33

54 6.0

6.0 37

45 HA

HA

7 × 10-2 mg/ mL ND ND

ND Suzuki et al. (2002)

Schaufuss et al. (1989)

Ch chondroitin, Ch-S-A chondroitin sulphate A, Ch-S-C chondroitin sulphate C, Dermatan-S dermatan sulphate, HA hyaluronic acid, ND not determined

Streptococcus uberis

16.

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Table 16.4 The preferred parameters for hyase production upon optimization by various researchers Sr. no. 1.

Organism Streptococcus equi

pH 5.5

Temperature 37 °C

C-source Dextrose

2.

Streptococcus mitis

5.8

37 °C

Sucrose

3.

Streptococcus pyogenes Streptococcus mitis MTCC 2695 Bacillus sp. LA_04

6.7

37 °C

Glucose

5.8

37 °C

Starch

6.5

37 °C

Maltose

Staphylococcus aureus

6.5

37 °C

Glucose

4. 5. 6.

N-source Ammonium sulphate Ammonium chloride Ammonium sulphate Ammonium chloride Ammonium chloride Ammonium chloride

Reference Sahoo et al. (2007) Sahoo et al. (2008) Lukyanenko (2011) Mahesh et al. (2012) Abed et al. (2012) Ahmed (2014)

16.6.1 Salt and Solvent Precipitation Upon fermentation, the biomass is separated either by centrifugation or filtration where the culture supernatant is then concentrated by salting out method (ammonium sulphate) (Hill 1976; Ingham et al. 1979). The use of ammonium sulphate for protein precipitation does not denature the protein even at higher concentrations. The method of solvent extraction by means of acetone and ethanol is also employed for the precipitation of enzyme hyase (Ahmed 2014). The salts and organic solvents lead to decrease the solubility of proteins in aqueous solutions and precipitate the proteins (Kumar and Takagi 1999). Several reports are available on the use of ammonium sulphate for the precipitation of enzyme hyase through salting out method. The 40% of salt concentration is reported for the precipitation of hyase from Streptococcus agalactiae (Ozegowski et al. 1994), 60% in case of hyase from Streptococcus uberis (Schaufuss et al. 1989), 80–90% for hyase from Staphylococcus aureus (Ahmed 2014) and 60% for hyase from Arthrobacter globiformis (Zhu et al. 2017). The use of 60–70% acetone and 60–70% ethanol for fractional precipitation of enzyme hyase from Staphylococcus aureus has been reported by Ahmed (2014).

16.6.2 Chromatographic Separations The chromatographic techniques like ion exchange, gel filtration and isoelectric focusing are used for the purification of the enzyme hyase with increased specific activities. Ion exchange chromatography is one of the frequently used protein purification methods. The surface residues of the protein and the buffer conditions are responsible for the net positive or negative charge on the protein molecule. The proteins have

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Table 16.5 Microbial hyase purification approaches Sr. no. 1.

Microorganism Staphylococcus aureus

2.

Streptococcal sp. group A

3.

Propionibacterium acnes

4.

Peptostreptococcus sp.

5.

Streptococcus uberis

6.

Streptococcus dysgalactiae

7.

Streptococcus dysgalactiae S. zooepidemicus Streptococcus equi

8.

Streptococcus agalactiae

9.

Penicillium purpurogenum

Separation strategies/methods Ammonium sulphate precipitation Sephadex G-100 chromatography Ammonium sulphate precipitation Sephadex G-200 chromatography DEAE Sephadex A-50 chromatography SDS-PAGE Ultrafiltration Ammonium sulphate precipitation Sephadex G-200 chromatography Isoelectric focusing Ethanol precipitation Sephacryl S-300 chromatography CM-Sepharose chromatography SDS-PAGE Ammonium sulphate precipitation DEAE cellulose chromatography Gel filtration: Ultragel ACA44 chromatography Isoelectric focusing ECTEOLA-cellulose column Phospho-cellulose column chromatography Sephacryl S-300 chromatography SDS-PAGE Ammonium sulphate precipitation DEAE-cellulose chromatography Isoelectric focusing Ammonium sulphate precipitation Phenyl Sepharose column chromatography Superdex G-200 chromatography Isoelectric focusing Ammonium sulphate precipitation

Reference Abramson and Friedman (1968)

Hill (1976)

Ingham et al. (1979)

Tam and Chan (1985)

Schaufuss et al. (1989)

Hamai et al. (1989)

Sting et al. (1990)

Ozegowski et al. (1994)

Bakke et al. (2011) (continued)

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Table 16.5 (continued) Sr. no.

Microorganism

10.

Bacillus sp. A50

11.

Bacillus niacini

12.

Pseudozyma aphidis Cryptococcus laurentii

13.

Arthrobacter globiformis A152

Separation strategies/methods Toyopearl-butyl 650 C column chromatography Q-Sepharose chromatography Ammonium sulphate precipitation Anion exchange: DEAE Sepharose chromatography Gel filtration: Superdex 200 chromatography Ultrafiltration Super Q-Toyopearl column chromatography Ammonium sulphate precipitation Butyl-Toyopearl column chromatography Membrane filtration DEAE-Sepharose chromatography Ultrafiltration Sephacryl S-200 chromatography Ammonium sulphate precipitation Ultrafiltration Q Sepharose chromatography Sephadex G-100 chromatography

Reference

Guo et al. (2014)

Kurata et al. (2015)

Smirnou et al. (2015)

Zhu et al. (2017)

negative charge at the physiological pH range (6–8) that can bind to positively charged resin molecules. The change in the pH of buffer can make the protein positively charged, which can bind to the negatively charged resin. The extensively used charged molecules in ion exchange chromatography are DEAE and CM, which are coupled with an inactive support material. The increase in the salt concentrations or changes in pH can elute the desired protein molecule from the column (Williams and Frasca 1999; Selkirk 2004). The ion exchange chromatography by using DEAE-Sepharose was recently reported by Smirnou et al. (2015) and Guo et al. (2014), Q-Sepharose by Bakke et al. (2011) and Zhu et al. (2017), DEAE-Cellulose by Sting et al. (1990) and CM-Sepharose by Tam and Chan (1985) for purification of enzyme hyase. In this method, the size of the protein is crucial for the purification. The column material comprises of a porous matrix for the diffusion of protein molecules. The smaller protein molecules get entangled into the porous matrix material, and hence their mobility is controlled, whereas the larger protein molecules do not get

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entangled into the matrix and passed through it. Hence, the larger molecules can be eluted first and the smallest molecules will be eluted later (Hagel 1998; Stanton 2004; Ó’Fágáin et al. 2011). The gel filtration chromatography by using Sephadex G-100 was recently employed by Zhu et al. (2017), Sephacryl S-200 by Smirnou et al. (2015), Superdex G-200 by Guo et al. (2014) and Ozegowski et al. (1994) and Sephacryl S-300 by Hamai et al. (1989) for purification of enzyme hyase.

16.7

Bio-physicochemical Characterization of Hyases

Several microbial hyases were purified and well characterized by researchers all over the world. The microbial hyases are differing from eukaryotic hyases in several aspects, namely, optima of pH, temperature, pH and thermal stability, substrate specificity and kinetic parameters. Table 16.3 summarizes several bio-physicochemical characteristics of purified microbial hyases.

16.7.1 Substrate Specificity of Hyases The hyases of prokaryotic and eukaryotic origins do not demonstrate the absolute substrate specificity towards substrate hyaluronic acid but can depolymerize other related GAG substrates like chondroitin, chondroitin sulphate, dermatan sulphate and chitosan along with the principal substrate HA (Patil et al. 2019). The reactions of hyases against these GAGs proceed more slowly as compared to HA (Rigden and Jedrzejas 2003), since natural binding affinity between hyase and other related GAGs does not exist (Turley and Roth 1980). The purified hyase of Bacillus niacini demonstrated the maximum activity against HA and also showed potential to degrade chondroitin, chondroitin sulphate-A and chondroitin sulphate-C at slower rate (Kurata et al. 2015). The hyase from Bacillus sp. A50 can depolymerize chondroitin sulphate-A, chondroitin sulphate-C and chondroitin sulphate-D along with HA (Guo et al. 2014). The hyase purified from coryneform bacterium Arthrobacter globiformis showed an ability to catalyse chondroitin sulphate-A and chitosan (Zhu et al. 2017) (Table 16.3).

16.7.2 Molecular Weight The gel filtration chromatography, SDS-PAGE and MALDI/TOF mass spectrometry have been employed by most researchers for determining the molecular weights of microbial hyases. The molecular weights of microbial hyases have been reported in the broad range of 33–160 kDa (Table 16.3) (Suzuki et al. 2002; Tam and Chan 1985; Smirnou et al. 2015; Guo et al. 2014; Kurata et al. 2015; Zhu et al. 2017; Akhtar and Bhakuni 2003). However, animal venom hyases are low molecular

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weight hyases with the molecular weight ranges from 33 to 82 kDa (da Silveira et al. 2007; Nagaraju et al. 2007; Sutti et al. 2014; Feng et al. 2008).

16.7.3 Optimum pH and Temperature The catalytic activity of microbial hyases has been greatly influenced by the pH, since change in pH of the reaction mixture may lead to the deactivation of enzyme. The microbial hyases catalyse their substrate with maximum possible rate in the acidic to near neutral range of pH, that is, 5.5–7.0 (Hamai et al. 1989; Schaufuss et al. 1989; Ozegowski et al. 1994; Suzuki et al. 2002; Allen et al. 2004; Kurata et al. 2015; Zhu et al. 2017) (Table 16.3). The temperature is another key physical factor in controlling the activity of microbial hyases. Most of the microorganisms that produce hyases are human pathogens. In pathogenesis, these organisms produce hyases to serve as their virulence factor to invade the host tissues. Hence, the microbial hyases were found to work optimally in the mesophilic range of temperature. The microbial hyases have been reported to have the optimum temperature which is in the range of 37–46 °C (Ingham et al. 1979; Tam and Chan 1985; Schaufuss et al. 1989; Akhtar and Bhakuni 2003; Suzuki et al. 2002; Allen et al. 2004; Bakke et al. 2011; Guo et al. 2014; Smirnou et al. 2015; Zhu et al. 2017).

16.8

Applications of Hyaluronidases

Hyaluronidases are extensively used in various fields, namely, medical, pharmaceutical, surgery, immunology, aesthetic medicine, oncology, ophthalmology, orthopaedics, internal medicine, gynaecology, dermatology, etc. (Patil and Chaudhari 2017). More specific applications of enzyme hyaluronidase are explained as follows.

16.8.1 Hyaluronidase Used in Cancer Therapeutics Hyases are used as the diagnostic indicators for the detection of bladder cancer. For example, the gene Hyal-1 is expressed in bladder cancer, and it serves as the perfect marker for high-risk bladder cancer. However, artificially increasing the hyase concentrations results in hyase functioning as a tumour suppressor. Hence, this enzyme can be used for developing the novel cancer therapeutics and diagnostics (De Maeyer and De Maeyer-Guignard 1992; Chang 1998). Hyases are also reported to have anticancer effects and able to suppress tumour development. Hyal-1 is a tumour suppressor gene which is inactivated in many tobacco-related lung tumours and also found to promote tumour cell cycling (St Croix et al. 1998).

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16.8.2 Hyaluronidases as Adjuvant Hyases are used as the adjuvants in clinical settings for the administration of local anaesthetics in the replacement fluids, corticosteroid, insulin and large molecules. Hyases have an inherent ability as diffusion-enhancing adjuvant for the infiltration analgesia of the skin (Wohlrab et al. 2012). Hyases by degrading the HA can reduce the viscosity of ECM that facilitates the proper diffusion of the anaesthetic agent. Hyases are also used in combination with lidocaine which can enlarge the anesthetized area, reduce the period of drug diffusion and improve the permeability of tissues enhancing the effect of anaesthesia (Pacella et al. 2010; Muchmore and Vaughn 2010).

16.8.3 Hyaluronidases in Ophthalmology The enzyme hyase has been used in the field of ophthalmology as an anaesthetic agent for various peribulbar, retrobulbar and sub-Tenon’s blocks. Hyases are also used as vitreous humour supplement or replacement during cataract surgery of the eye (Kim et al. 2011).

16.9

Commercial Hyases in the Market

In total, five different commercial formulations of enzyme hyase are observed in the market, namely, Hydase (manufactured by the Prima Pharm, Inc., USA), Amphadase (Amphastar Pharmaceuticals, Inc., Rancho Cucamonga, USA), Wydase (Wyeth-Ayerst Pharmaceuticals, Madison, USA), Vitrase (ISTA Pharmaceutical, Irvine, USA) and Hylenex (developed by the Halozyme Therapeutics, San Diego, USA). These commercial formulations of hyaluronidase can be injected intramuscularly or subcutaneously but not intravenously because this enzyme is quickly inactivated in the blood (Lee et al. 2010).

16.10 Conclusion Hyases are largely distributed enzymes in nature with exceptional substrate specificities, diverse catalytic mechanisms, a wide range of pH and temperature optima and a variety of functionalities. Owing to its characteristics, hyases are extensively utilized in many fields like ophthalmology, orthopaedics, surgery, oncology, gynaecology, internal medicine and dermatology. Much rigorous work have been done in the area of enzyme hyase; however, there is a huge scope for the work to be done on the three-dimensional structures of enzyme, site-directed mutagenesis, detail kinetics to develop the systematic understanding of substrate binding sites, recognition of more suitable substrates and accurate catalytic mechanisms. In

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order to improve the understanding of the interactions between enzyme and substrate, further detailed work on the substrate structure is also needed. Acknowledgements All authors acknowledge the kind help of R. C. Patel Arts, Commerce and Science College, Shirpur, MS, India, for providing infrastructural facilities. Corresponding author (BLC) is grateful to FIST grant of DST, New Delhi, for improving research facilities provided to the School of Life Sciences of KBCNMU.

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Strain Improvement Strategies of Industrially Important Microorganisms

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Sayani Ghosh, Pooja, and Supratim Datta

Abstract

Microorganisms are subjected to strain improvement techniques to improve the performance of metabolites produced and increase the efficiency of existing enzymes and pathways that produce compounds of industrial relevance. Strain improvement techniques are used to modify native pathways or to explore previously unknown properties of a non-model organism to synthesize the product of our interest. Recombinant DNA technology is a standard method for introducing desired mutations through plasmid transfer via techniques like transformation or electroporation into the host organism. These microorganisms are introduced with foreign properties by vital genetic manipulations like gene deletion, gene insertion, or overexpression to produce metabolites of industrial interest. Modern-day strategies like the CRISPR/Cas9 gene editing technique are excellent methods for performing genetic modifications in such cases. In this chapter, we elucidate the various strategies used to introduce gene modifications

Sayani Ghosh and Pooja contributed equally with all other contributors. S. Ghosh · Pooja Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India S. Datta (✉) Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Center for the Advanced Functional Materials, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India Center for the Climate and Environmental Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_17

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in these industrially relevant microorganisms toward the proper utilization of natural resources for the betterment of our society. Keywords

Industrial microorganisms · Mutagenesis · Epigenetic modification · Metabolic engineering · CRISPR/Cas9 in industrial biology

17.1

Introduction to Strain Improvement

Microbes are all around us. They are invasive in nature and spread quickly thus forming the biotic foundation for life in all environments. Without a competitor or antimicrobial agent, there is no environment in which a microbe cannot exist. From the beneficial microbes that impact our daily lives (such as the lactic acid bacterium that converts milk into curd) to the extremophilic bacteria that thrive in extreme environments like high salinity and high pH, microorganisms are well adapted to nature’s ways. Humans, being intelligent species, have studied microorganisms and their ways of thriving in nature for years. The process of harvesting the unusual properties of these organisms and putting them into solving our problems and making life better for us is one of the goals of modern industrial microbiology. It is the science of utilizing microorganisms to produce valuable products in mass quantities via microbial cell factories. The simple process of fermentation by bacteria holds the largest and most profitable industry of alcoholic beverages, along with some nonalcoholic fruit juices, which are fermented by various microbes. Antibiotic production is a significant part of modern health science. Large scale antibiotic production in industries (synthetic and semi-synthetic) are now being replaced by biosynthesizing, which has several advantages. Microbes also find extensive use in the biofuel industry to produce biofuels, biofertilizers, and biogas. Strain improvement is a crucial aspect of industrial microbiology. The microbes in their natural conditions often do not match the requirements of an industrial setup. They produce fewer metabolites and often require specific modifications to perform optimally. Strains are improved through the modification and selection of microorganisms with desired traits and the development of new strains through various techniques. With the availability of high-throughput instruments and sequencing technology, much more information can be derived about the genetic composition of a microorganism. Here we will discuss some of the key strategies used for strain improvement and shed light on the progress catalyzed by the discovery of the CRISPR-Cas system.

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17.2

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Classical Methods of Strain Improvement

The classical methods are still used to identify improved strains, wherein the cells are first exposed to mutagenic agents causing random mutations in the genome (Konar and Datta 2022). Repeated rounds of treatment lead to the survival of a few and the death of most strains. The surviving mutants that show the required trait are identified and isolated. Such methods are however time taking. To further understand the basis of the improvement or the preferred mutations, genetic studies are required. Today metabolic engineers often adopt a more targeted approach to strain improvement including revisiting the genomic mutations brought about by various mutagenic agents over time. Toward the target of overproducing β-lactams, the study of Penicillium rubens revealed that several mutations that occurred due to repeated rounds of mutagenesis over the years led to the inactivation of pathways that competed for resources with cysteine biosynthesis (Wu et al. 2020). Due to this the uptake of L-cysteine, one of the amino acids used for β-lactam production, was facilitated while tryptophan synthase, the enzyme converting L-serine into L-tryptophan, was inactivated by a mutation. Serine metabolism is an important pathway that connects to cysteine biosynthesis. Therefore understanding the genetic changes is critical in strain engineering. Two important classical methods, mutagenesis and recombinant DNA-based methods, are discussed next.

17.2.1 Mutation Mutagenesis is a technique that involves the use of physical or chemical agents to induce mutations in the DNA. A mutation is either spontaneous or induced. In both cases, the change caused is usually detrimental to the organism and eliminated over time, but some mutations are beneficial and can be preserved indefinitely. Due to these mutations, the resulting changes in the genotype result in a changed phenotype, which are then subjected to “strain selection” for an improved version of the wild type. Ionizing radiation is a classical physical method used for mutagenesis. This technique uses the property of X-rays and gamma rays to cause DNA damage by ionizing atoms and molecules. Microorganisms are subjected to low doses of radiation, specifically with gamma rays to induce mutations in microorganisms. Gamma rays are used industrially due to their high penetrability (short wavelength and highenergy electromagnetic radiation). This process, known as irradiation, has been utilized for the development of improved strains of bacteria in antibiotic production. The fungus Shiraia species (HA-producing strain ZZZ816) produces hypocrellin A, a natural pigment with antibiotic activity against a range of bacterial strains, also possessing antiviral and anticancer properties have been used to treat skin diseases (Liu et al. 2016). The spores from this fungus were treated with cobalt-60 gamma (60Coγ) to increase HA production by 414.9% compared to the original strain ZZZ816 (392 mg/L). The new strain yield was significantly higher than all the other industrial HA-producing strains. Alongside, the authors found a sweet spot

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between lethality (77%) and positive mutant frequency (35%) by varying the doses of radiation (Liu et al. 2016). Gibberellic acid (GA) is an essential plant hormone that has a role in flowering, plant growth, seed germination, etc. Industrial production of GA by an ascomycetous fungus, Gibberella fujikuroi, was limited by the quality of carbon source used for microbial growth. Treating the microbial cells with gamma rays led to overproduction of the product with the same amount of raw material, leading to reduced production costs and improvement in product quality (Meleigy and Khalaf 2009). Gamma radiation enhanced the activity of ligninolytic genes in the fungus Pleurotus sajor-cajo and Brachypodium distachyon, a model grass species from the family of wheat and barley that can be used as a substrate for biofuel production, leading to increased delignification of biomass (Abo-State et al. 2011; Kim et al. 2015). However, studies in microalgae showed negative impact of gamma irradiation on both biomass and lipid content (Abo-State et al. 2019). Ultraviolet light (200–400 nm) is part of the electromagnetic spectrum, emitted from the sun. UV-C (200–280 nm) with short wavelength and high energy emits in the range of wavelength absorbed by DNA (260 nm), causing maximum damage. Naturally, most of this dangerous radiation is blocked by the ozone layer. UV radiation results in the formation of pyrimidine dimers that halt the replication process, as these dimers cannot bind with other bases. The generation of singlet oxygen from irradiated oxygen leads to free radical production that oxidizes DNA bases. These oxidized bases do not pair correctly during replication, resulting in mutations (Cadet and Douki 2018). In an example of industrial strain improvement, UV radiation (physical mutagen) and NTG (chemical mutagen) enhanced lipase productivity (156% fold increase) in the wild-type strain of Rhizopus sp. BTS-24, while another mutant BTUV3 showed 164% higher lipase production than the parent strain (BTNS12) and 180% higher than the wild type (Bapiraju et al. 2004). Increases in lipase production has been reported in other studies too (Sandana Mala et al. 2001). Chemicals that can react or bind to DNA and cause mutations are chemical mutagens and include intercalating agents that bind or intercalate to DNA bases causing frameshift mutation during replication, base analogues which mimic DNA bases during replication and cause transition mutations, deaminating agents that remove the amine groups in DNA bases by oxidative depurination, and alkylating agents that introduce alkyl group causing ionization of nucleobases, resulting in base-pairing errors and eventual depurination or generation of gaps in the DNA polynucleotide. Common examples of chemical mutagens and mutations made by them are listed in Table 17.1. In a study based on two desert microalgae (Desmodesmus sp. S81 and G41), ethylmethane sulfonate (EMS) was used as a chemical mutagen to obtain two potential mutants, Desmodesmus sp. S5 and G3, where biomass and lipid production was increased by 48.41% and 46.01%, respectively (Zhang et al. 2016). The mutants showed reduced polyunsaturated fatty acid (PUFA) and glycol lipids, while monounsaturated fatty acids (MUFAs) were elevated with neutral lipid content. Hence, these mutants are appropriate for biodiesel production. UV and EMS treatment mutagenized a low fructosyltransferaseproducing strain, Aureobasidium pullulans NAC8, to increase the intracellular titers

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Table 17.1 Common mutagenic agents and their effects Mutagens X-rays

Gamma rays

Ultraviolet rays

N-MethylN′-nitro-Nnitrosoguanidine Ethyl methane sulfonate

Mutation induced Single or double-strand breakage of DNA Irradiation of spores

Overproduced compounds Streptomycin A

Pyrimidine dimerization and cross-links in DNA Methylation at high pH

6′-DO-BLM Z

Alkylation of bases C and A

Ethidium bromide

Intercalating between two base pairs

Acridine orange

Irradiation of spore Deamination of A, C, and G

Nitrous acid

Organisms Streptomyces fradiae

References Shier and Rinehart (1969)

Shiraia species and Fusarium moniliforme Streptomyces flavoviridis

Liu et al. (2016) and Meleigy and Khalaf (2009) Zhu et al. (2018)

Streptomycin A

Streptomyces fradiae

Shier and Rinehart (1969)

Lipid production enhancement of carotenoids and astaxanthin content Congo red biodegradation and overexpression of cellulases Citric acid production Lipase

Desmodesmus sp. and Haematococcus pluvialis

Zhang et al. (2016) and Sandesh Kamath et al. (2008)

Bacillus sp., and Trichoderma citrinoviride

Gopinath et al. (2009) and Chandra et al. (2009)

Aspergillus niger Aspergillus niger

Lotfy et al. (2007) Sandana Mala et al. (2001)

Hypocrellin A GA

by almost twofold and the extracellular titer by sixfold. A two-pronged statistical system was devised to determine important media parameters and optimize them according to the requirement (Ademakinwa et al. 2017). N-methyl-N′-nitro-Nnitrosoguanidine (NMG), known as nitrosomethyl guanidine, is an alkylating agent. It causes DNA mutations by introducing alkyl groups into the DNA bases. By treating the conidia from antibiotic nonproducer colonies of Streptomyces griseus strain 7-455F3 with NMG, mutants were identified that needed addition of streptidine into the media, to produce Streptomycin (Nagaoka and Demain 1975). Biosynthesis of new antibiotics, from supplementation of guanidinocyclitol, 2-deoxystreptidine, was achieved through this study, and it has been proven that the production of new antibiotics was possible by supplementing new metabolites to drive the secondary metabolite-generating pathway. Alteration of growth media to obtain biosynthetic replacements in antibiotics is already known. A mutant of Streptomyces fradiae 3535 forms four new antibiotics by utilizing aminocyclitols

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(streptamine and 2-epistreptamine) present in the media. These antibiotics were named hybrimycins (Shier and Rinehart 1969). This method of producing semisynthetic antibiotics was a new path forward for dealing with the evolving problem of antibiotic resistance. Ethidium bromide is another potent chemical mutagen used in industrial applications to introduce frame-shift mutations into the DNA. Biodegradation of Congo red, a toxic azo dye, utilizes a mutant Bacillus subtilis that efficiently degrades the dye by causing an overproduction of azoreductase enzyme. Mutagenesis using EtBr and UV led to improved mutant strain formation that offered better decolorization along with 12–30% reduction in time required for the complete degradation (Gopinath et al. 2009). The overproduction of enzymes, especially lipases, needs a special mention. UV irradiation and chemical mutagens (MNNG/NTG, NA, EtBr) have been used many times to overproduce lipases. A fungal strain Rhizopus oligosporus (var. microsporus) was mutagenized to improve the lipolytic potential by an overall 325% increase in activity (Iftikhar et al. 2010). Mutagenesis is a useful technique for improving the strain of an industrial microorganism, as it allows for the rapid generation of a large number of mutants, which can be screened for improved characteristics. For example, mutagenesis has improved the yield of various industrial products, such as enzymes, organic acids, and amino acids. In some cases, mutagenesis has been used to improve the tolerance of an industrial microorganism to environmental stressors, such as high temperatures and salinity or low pH. Effects of different concentrations of NaCl on biomass, photosynthetic pigments, chlorophyll fluorescence, and lipid accumulation were studied in S. obliquus XJ002. The salt-tolerant mutant showed decreasing biomass, chlorophyll, and carotenoid content, with increasing NaCl concentrations. Both the donor and acceptor sides of the PSII reaction centers were sensitive to NaCl stress. Additionally, the neutral lipid content of cells increased. This research provides insight into the physiological response to salt stress (Ji et al. 2018). This study displays a strategy to boost lipid content by adjusting salt levels in the growth medium. It is important to note that mutagenesis can also introduce unwanted mutations that may negatively affect the microorganism’s phenotype. Even the relevant mutations will show some detrimental effects, in growth or other attributes, but the focus remains on production of high yields of the required metabolite and requires a careful evaluation of the mutants.

17.2.2 Genetic Recombination (Recombinant DNA Technology) The naturally occurring process by which an organism exchanges DNA with another, to introduce genetic variability in its genome is termed as genetic recombination. The DNA is broken up and rearranged, resulting in alterations causing new combinations. This alters the offspring’s genetic makeup and differentiates it from the parent strains, thus creating a new strain. When the double-stranded structure of DNA was discovered, industrial microbiology underwent a rapid evolution with the incorporation of the recombinant DNA technology. Due to the low frequency of

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recombination, industrial reactions did not favor recombination. The focus was on mutagenesis and efficient methods of constructing mutant libraries. Only lately was it realized that ideal strain improvement is not excluding each other; instead both the methods can be used to complement one another, in a way that leads to improved functionality. Microbes such as single-celled fungi and bacteria show parasexual recombination, wherein DNA material exchange occurs without forming sexual parts. This is also known as horizontal gene transfer of bacteria allowing intact DNA transfer without variability. The three parasexual processes include conjugation, transduction, and transformation. Conjugation involves transfer of DNA between two bacteria via cell-to-cell contact, by forming a tube-like pili. Here, one bacteria consisting of a F-factor serves as the donor, while the other acts as receiver. In the environment, conjugation is a gene transfer method between bacterial communities, transferring advantageous metabolic properties, such as symbiotic lifestyle, virulence, or resistance to heavy metals and antimicrobials, and its role in microbial biofilm formation (Virolle et al. 2020). Transduction occurs in similar direction, that is, from host cell to recipient cell via mediation by bacteriophage. Here the virus acts as a vector for inserting the foreign DNA. Generalized transduction occurs randomly and more easily, while specialized transduction accounts for the location of the genes on the chromosome and the incorrect excision of the prophage (DNA transferred from the virus). The process of transformation involves uptake of naked DNA by competent bacterial cells, under the induction of some environmental stress conditions like starvation, cell density, etc., and chemicals like cold calcium chloride treatment, protoplasting, electroporation, and heat shock. Internally, genes can rearrange via translocatable DNA segments (insertion sequences or transposons) (Adrio and Demain 2010). The genes involved in daptomycin biosynthesis pathway were identified and cloned. A neutral insertion site was identified in Streptomyces fradiae, and an extra copy of the tylF gene was placed, leading to increased tylosin production (Shaikh et al. 2021). Hybridization: Hybridization involves the creation of new strains through the combination of two or more microorganisms. This can be achieved through techniques such as protoplast fusion or conjugation. Hybridization can be used to combine the desirable traits of different microorganisms, leading to the creation of new strains with improved performance. Protoplast fusion: Protoplast fusion also known as somatic fusion is a naturally occurring event in plant cells, where the protoplast of two distinct plant species fuses to generate a recombinant molecule. It is an important technique for generating recombinant DNA in plant species. Industrially, this technique has been used for recombination of genes involved in avermectin biosynthesis (Kohler and Darland 1988). Protoplast fusion as a means to produce novel strains and new products which were previously unobtainable by conventional methods has given rise to intraspecies, interspecies, and intergeneric hybrids for improving abilities of industrial microorganisms. Reports of industrial strain improvement through protoplast fusion have resulted in improvement in glucose-metabolizing activity in Brevibacterium, along with enhanced lignin degradation activity (Gokhale et al. 1993; Dahman et al.

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2019). Recent research has led to the discovery of an asymmetric protoplast fusion and screening method that opens the path for new cytoplasmic hybridization and the isolation of novel CMS lines of celeriac (Okamoto-Hosoya et al. 2003). Site-directed mutagenesis: Site-directed or site-specific mutagenesis is a gene editing technique, where the nucleotide sequence of a gene can be altered for studying structure function relations in proteins (Shaaban et al. 2010). Antibiotic production in Streptomyces lividans was activated by generating eight novel mutations via site-directed mutagenesis in a conserved region (Spadiut et al. 2014). Production and quality improvement of an anthelmintic group of drugs, known as avermectin, which is part of a gene cluster that has allowed the identification of the biosynthetic pathway in Streptomyces avermitilis. An avermectin analogue doramectin produced as a byproduct during the fermentation process is sold as an important commercial product, known as Dectomax™. The ratio of doramectin production in comparison to an undesirable analogue was increased by fourfold. This was brought about by a combination of site-directed and random mutagenesis of the aveC gene followed by integration of this engineered gene into the chromosome. This study is an important milestone toward engineering the biosynthetic pathway of a natural product in the production of a biopharmaceutical compound (Stutzman-Engwall et al. 2003). In a later attempt at rational engineering of this compound, the CHC-CoA biosynthetic gene cassette in the mutant strain S. avermitilis TG2002 was introduced to produce the precursor molecule for doramectin, and a 300-fold increase in production ratio was reported (Xia et al. 2020).

17.3

Epigenetic or Posttranslational Modifications (PTMs)

Epigenetics is an emerging strategy in the field of strain modification and improvement. Here changes in phenotype are observed with no changes in genotype, that is, no underlying change to the DNA. The manipulations are designed on the following levels of regulation: (i) DNA methylation and chromatin remodeling by histone modification, ii) transcription, and (iii) translational ribosome engineering and RNA interference.

17.3.1 Chromatin Remodeling Chromatin is the complex of tightly condensed DNA wrapped around histones to form the nucleosome, which consists of 146 base pairs of double-stranded DNA and eight histone proteins. This tightly packed chromatin, which is not accessible for transcription, is heterochromatin, whereas the loosely packed and transcriptionally accessible form is called euchromatin. These forms are interconvertible among themselves, and several transcription factors and other proteins bring about these changes and are targets for strain improvement (Aghcheh and Kubicek 2015). The H3 and H4 histones have particularly long tails that can undergo different types of

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modifications specific for each condition and are also known as the histone code. The primary toolkit comprises the methylation of lysine (K) and arginine (R) residues and acetylation of the lysine residues at various positions. Various histone-assisting proteins termed histone acetylases, protein arginine methyltransferases, etc. have been characterized in filamentous fungus. The transcriptional control brought about by these proteins are believed to play a role in the production of extracellular cellulase and the production of penicillin by interaction with chromatin remodeling complexes (Amann et al. 2019).

17.3.2 Ribosome Engineering Ribosome engineering involves the modification of the ribosome machinery in the cell to bring about changes to the production or synthesis of secondary metabolites, specifically the production of new antibiotics and a variety of structurally diverse bioactive compounds. Specific antibiotic resistant mutations were prepared through mutations in the ribosome, to enable the production of new compounds and the activation of cryptic gene clusters (Zhu et al. 2018). These clusters produce either at very low levels or not at all. Antibiotic-overproducing stains have been isolated from Streptomyces via this method. However, mutant screening from engineered ribosomes is laborious because the phenotypes generated from this mutation are previously uncharacterized (Yang et al. 2018). More studies have been reported targeting the rpsL gene loci in the ribosome, which keeps the translation accuracy in check. Some mutants showed the production of piperidamycin, a novel antibiotic not found in the host organism under normal conditions. Several point mutations in the rpsL genes enhanced antibiotic production by increasing protein synthesis and providing structural stability to the 70 s ribosome (Huccetogullari et al. 2019). These ribosomal mutants can be used in various applications, including the production of biopharmaceuticals, developing new enzymes for industrial processes, and optimizing protein expression for research. However, a careful consideration of the potential impacts of ribosome mutants on the function and stability of the ribosomes and the overall cell physiology is essential to obtain the best yields.

17.3.3 Engineering N-Glycosylation Sites With the range of model host organisms available for the production of biopharmaceuticals, posttranslational modifications (PTMs) of a protein and the host strain are necessary. The metabolites targeted during industrial overproduction often need to meet specific changes brought about posttranslationally, which are required to control the quality of the compound. Most eukaryotic proteins need other proteins and chaperones to bring about changes that control the abundance of the final active or isoform of any enzyme/secondary metabolite in the cell. For example, the production of monoclonal antibodies (mAbs) is difficult to maintain in human and other mammalian cell lines. The difference in the PTMs, like the glycosylation

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pattern of different host organisms, brings about stability issues, such as aggregation due to physical instability or deamidation or oxidation due to chemical instability. Glycosylation strongly impacts the production of recombinant mAb from the cell, as incompatible glycoforms can cause severe immunogenic reactions in patients. PTMs in S. cerevisiae are directed toward altering the intracellular protein trafficking reduction of proteolytic degradation by protease gene deletions and overexpressing the heat shock transcription factor (Hsf). The glycosylation machinery in S. cerevisiae is being engineered to adapt full-length mAb production. In case of E. coli, several strategies like the transport to periplasmic space and disulfide bridge formation are used to facilitate the proper folding of the heterologous proteins (Sheng et al. 2020). Glycosylation is not only species and cell specific but also affected by variable culture conditions and is the major reason for limited biopharmaceutical titers. The composition of sugar moieties impacts pharmacokinetic properties of antibodies displayed with a glycosylation site in the Fc region. Research in this field is still ongoing in an attempt to have an optimized production platform for biopharmaceuticals (Liu et al. 2022).

17.4

Genetic Engineering Strategies

Genetic engineering involves the modification of an organism’s genome through techniques such as gene insertion, deletion, or mutation by the targeted modification of specific genes and not allowing other accidental mutations to accumulate. Introducing new traits into an organism’s genome and improving existing ones have opened up new opportunities for industrial production of bio-products. Products of site-directed mutagenesis and other gene improvement techniques are incorporated into the genome-by-genetic integration, thus altering the overall genetic potential of the recombinant strain. The discovery of genetic recombination techniques and availability of genetic engineering toolbox in recent years have allowed the manipulation of extremely niche organisms in archaea and eukaryotes and have led to the emergence of newer “qualities” that were previously unknown. Genetic engineering or host engineering of organisms as a production platform to develop optimum quality and titer of a value-added product uses the following strategies: 1. The generation of inhibitory byproduct in the medium should be at minimum. 2. All the genes in the pathway should be functioning optimally. 3. Transcriptional and other global regulators of the major proteins need to be repressed or inactive. 4. Analysis of metabolic flux should be directed toward the pathway of interest. 5. A method to enable the extraction of the final metabolite from the engineered host is needed. Metabolic engineering of a host organism involves modification of an organism’s metabolic pathways to increase the production of a particular compound or enzyme.

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This can be achieved through the introduction of new genes or the manipulation of existing ones. For continuous and optimal production, a specific biosynthetic pathway needs to be the most energetically favored path for progression of a metabolite. Formation of inhibitory or feedback components during this progression needs to be channelized in some other pathways to not inhibit the forward pathway. Metabolic consumption of a precursor molecule (for the production of a compound) or carbon source, by an organism, is known as flux. Metabolic flux analysis (MFA) is one of the pillars of metabolic engineering where the regulatory networks or probable “sinks” of a compound are calculated. Accordingly, diverting the majority of the precursor source toward the pathway of interest is known as flux balance analysis. It is a constraint-based approach that paves the way for genome engineering strategies in the host organism. The biosynthetic pathway of 1,3-propanediol (1,3-PD) production encounters the production of an inhibitory intermediate product 3-HPA, which is toxic to bacteria. The two enzymes key to the bioprocess are susceptible to this accumulation and hence get inhibited. Even the production of exogenous 1,3-PD is toxic to the bacterial host, which leaves great scope for extensive genetic and metabolic regulatory network engineering (Jiang et al. 2013). Amino acid biosynthesis of L-alanine and other aromatic amino acids like L-tryptophan, L-tyrosine, and flavonoid production systems have been modified by metabolic engineering techniques (Jiang et al. 2015; Xu et al. 2015; Chuai et al. 2017).

17.5

CRISPR/Cas9 in Industrial Biology

CRISPR technology was developed by adapting a natural defense mechanism found in few bacteria and archaea to protect themselves against viruses and other pathogens. This defense mechanism is based on small RNA molecules called CRISPR RNA (crRNA), which are part of a larger system called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The CRISPR-Cas system works by using crRNA molecules to guide the Cas enzymes to specific sequences of DNA. When the Cas enzymes encounter a target sequence, they cut the DNA at that location. This allows the CRISPR-Cas system to defend the bacteria or archaea against invading viruses by destroying their DNA. This natural defense mechanism has applications in genetic engineering. The precise change of DNA of living cells can done with the help of CRISPR-Cas tool. This has led to many exciting applications in medicine, agriculture, and biotechnology. The most well-known CRISPR-Cas system is derived from Streptococcus pyogenes, but other systems have also been studied and used (Haft et al. 2005). The CRISPR system typically consists of a nuclease Cas9 or Cas12, CRISPR RNA (crRNA) for target recognition, and trans-activating crRNAs (tracrRNAs). CRISPR RNA (crRNA) is a type of RNA that recognizes and binds to a specific target sequence in DNA. A complex is formed by combining the precursor crRNA (pre-crRNA) with several tracrRNAs to form a complex called the dual RNA hybrid. Then RNase III recognizes this complex and cleaves the pre-crRNA and tracrRNA

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into mature crRNA and tracrRNA molecules. crRNA containing the targeting guide sequence fuses with a tracrRNA to construct single-guide RNA (sgRNA). This sgRNA can direct the Cas9 nuclease to a specific target site in the DNA, where it cuts both strands of the DNA helix to create a double-stranded break (DSB). After the DSB is created, the cell’s natural repair mechanisms (nonhomologous end joining and homologous recombination) can be used to alter the target gene by inserting, deleting, or replacing DNA at the site of the break. This methodology can be used to edit or regulate specific genes, depending on the desired outcome (Doudna and Charpentier 2014; Jinek et al. 2012; Cong et al. 2013). Nonhomologous end joining (NHEJ) is a major pathway for repairing DSBs. The broken ends of the DNA molecule are directly joined together, without the need for a homologous template for repair. NHEJ relies on recognition and processing of the broken DNA ends by specific proteins, which then facilitate the repair process. Homologous recombination (HR) is a DSBs’ repair process which requires a homologous template for repair. HR is a highly accurate and precise DNA repair process that helps maintain the genome’s integrity and prevent genetic abnormalities. The CRISPR/ Cas9 gene editing technique has revolutionized genetic engineering. There are a wide range of applications in industrial biotechnology for producing valuable chemicals, enzymes, vitamins, and other biochemicals through the expression of exogenous genes in microorganisms. For example, CRISPR/Cas9 has been used to delete or silence genes in microorganisms to improve the yield of enzymes or other chemicals or to alter their metabolic pathways to increase the production of chemicals. Bacteria such as Escherichia coli (E. coli) has been used in industrial biotechnology to produce various chemicals, drugs, and biofuels. Ao et al. inserted three heterologous genes in one round of recombination and selection to construct a recombinant E. coli to improve production of industrially useful chemical, 5-aminolevulinic acid (ALA) (Ao et al. 2018). In Clostridium ljungdahlii, butyric acid production was improved by introducing a butyric acid production pathway (Huang et al. 2019). Yeast has become a valuable biological system in industrial biology for production of pharmaceuticals, biocatalysts, food additives, fine chemicals, and renewable biofuels. Saccharomyces cerevisiae was the first yeast engineered by CRISPR-/ Cas9-based system to improve the efficiency of double-stranded break (DSB) repair; after that, HI-CRISPR system has further enhanced this efficiency (DiCarlo et al. 2013; Bao et al. 2015). In the lipolytic yeast Candida aaseri SH14, CRISPR-Cas9 system was used to delete six copies of acyl-CoA oxidase genes which shows that this genome engineering tool can be used to optimize yeast for industrial application (Hilmi Ibrahim et al. 2020). Filamentous fungi are a rich source of bioactive compounds, and they produce a wide variety of secondary metabolites. The CRISPR/Cas9 technology has increased genome editing efficiency in various filamentous fungi. CRISPR/Cas9 technology has also been used to study the function of specific genes and to construct strains with improved traits such as increased gibberellic acid production in F. fujikuroi (Shi et al. 2019). In Trichoderma reesei, Chai et al. increased the yield of three heterologous proteins (a bacterial xylanase XYL7, a fungal immunomodulatory protein LZ8, and the human serum albumin

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HAS) by using gene deletion approach (Chai et al. 2022). In M. thermophila, the protein production and cellulase activity has been increased by targeting nine genes which have role in the cellulase production pathway (Liu et al. 2019). The M. thermophila strain was engineered by CRISPR/cas for overexpression of GLT-1 (a glucose transporter) or CDT-1/CDT-2 (cellodextrin transport system) and for turning down the expression of pyc gene for ethanol production from glucose and cellobiose (Li et al. 2020). Microalgae are being extensively studied for their potential to produce a wide range of bioactive compounds, including nutraceuticals and pharmaceuticals, as well as other industrially important compounds such as carotenoids, omega-3 fatty acids, and polysaccharides that have nutritional and health benefits. CRISPR/cas9 tool has also been used in microalgae species, and C. reinhardtii was the first microalgae edited by codon-optimized Cas9 protein and four genes were successfully edited (Jiang et al. 2014). CRISPR-mediated genome editing in industrial important algae N. oceanica (Wang et al. 2016) was used to disrupt nitrate reductase gene, but the efficiency was very low at nearly 1%. The efficiency of CRISPR gene editing varies across microalgal species, and more research is needed to identify novel or optimized nucleases that can improve the efficiency of gene editing tool for microalgae.

17.6

Strategies for Improvement of Efficient CRISPR-/Cas-Based Genome Editing

17.6.1 Improvement in Repair Process There are two main pathways for repairing DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR) (Fig. 17.1). NHEJ is present in all eukaryotes but is less common in prokaryotes, which mainly rely on HR for DSB repair. Also, NHEJ is often the preferred pathway for repairing DNA breaks in industrial microorganisms because it is a faster and more efficient process than HR, which can be important in industries where rapid growth and high productivity are desired. To improve the homologous recombination efficiency, there are two main strategies: (1) coupling recombineering with Cas9 counterselection facilitates genome editing by homologous recombination and (2) non-recombineering-based homologous recombination (NrHR). There are two main types of recombineering: single-stranded DNA recombineering (SSDR) and double-stranded DNA recombineering (DSDR). ssDNA recombineering technique is used to introduce change in specific bases of the genome. In this recombineering method, an oligonucleotide is introduced into organisms with inducible expression plasmid of phagederived ssDNA binding protein. This lambda red recombineering coupled with CRISPR-Cas9 system leads to efficient recombination. The SSDR-CRISPR-Cas9based genome editing tool (Oh and van Pijkeren 2014) was first reported in E. coli. Similarly, in Lactobacillus reuteri ATCC PTA 6475, SSDR-CRISPR-Cas9 tool was used to remove the unedited cells (Oh and van Pijkeren 2014). DSDR double-

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Fig. 17.1 CRISPR/Cas9-mediated DSB repair mechanism

stranded DNA is used as a template strand and relies on inducible expression lambda red plasmid. This DSDR combined with CRISR-Cas tool was first used to edit E. coli and rescue low-efficiency native homology-directed repair systems. The application of the DSDR-CRISPR-based gene modification system was also used in T. citrea, an important host for production of the industrially relevant vitamin C precursor 2-keto-d-gluconic acid for metabolic engineering (Carter 1986). Singlenick-triggered homologous recombination (SNHR) is another approach for introducing genetic changes into the genome that does not involve recombineering. SNHR strategy can be used for precise deletion, insertion, or codon change to inactivate gene function and overcome the limitations of currently available genetic approaches for engineering industrial microorganisms. For example, C. cellulolyticum have minimal expression of nonhomologous end joining (NHEJ) components, and that is why cas9-induced double-strand breaks are lethal. Instead, it relies on the natural HR machinery of the cell to repair a DSB at a specific location in the genome. The single-nick-triggered HR strategy was reported to edit C. cellulolyticum in a single step with a high editing efficiency and precision (Carter 1986). HR requires the availability of a homologous template, which may not always be present in industrial microorganisms, hence posing as a limitation in this process. NHEJ is an error-prone process that can result in unwanted mutations. However, it can be useful in situations where HR is not possible, such as when a homologous template is not available.

17.6.2 Promoter Optimization for Expression of Cas9 and SgRNA It is important to carefully consider the choice of promoter when using the CRISPR/ Cas9 system, as the strength and specificity of the promoter can have significant impacts on the efficiency and specificity of gene editing. There are several different ways that the Cas9 enzyme and gRNA can be expressed. One approach is to use two separate plasmids, each containing a different expression cassette or a single plasmid containing two expression cassettes, one for the Cas9 enzyme and one for the gRNA. Cas9 should be expressed in a low-copy plasmid under a constitutive or induced promoter; in contrast the expression of gRNA should be in high-copy plasmids with a strong promoter. Cas9 expression by IPTG-inducible promoter has been used in E. coli (Li et al. 2015). Other inducible promoters like tetracycline derivatives

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(pTet), mannose, nisin, and arabinose have been used in several bacteria including E. coli, Bacillus subtilis, Clostridium acetobutylicum, and Lactococcus lactis (Reisch and Prather 2015; Altenbuchner 2016; Wasels et al. 2017; Berlec et al. 2018). Vector-dependent overexpression of key enzymes is a common strategy, but the ideal way is Cas9 or dCas9 inserted into chromosomes and subjected to induction expression, like the CRISPR-Cas9 toolkit for engineering Bacillus subtilis (Westbrook et al. 2016). The sgRNA flanked with two ribozyme sequences, 5′ end hammerhead (HH) and 3′ end hepatitis delta virus (HDV), to express under a strong RNA polymerase II promoter or synthesized hybrid promoters are better alternative to express sgRNA (Bao et al. 2015; Nødvig et al. 2015; Schwartz et al. 2016).

17.6.3 Optimization of Codon for Cas9 Optimizing the codons used to increase the expression and stability of Cas9 can potentially improve performance. Codon-optimized Cas9 is different for different organisms. For example in Saccharomyces cerevisiae, the natural spCas9 perform well, but there has been significantly higher cas9 expression in A. niger, Y. lipolytica, and K. phaffii after codon optimization (Bao et al. 2015; Nødvig et al. 2015; Schwartz et al. 2016; Cai et al. 2019). Human codon-optimized Cas9 gene also increases the expression in O. polymorpha to improve the target efficiency. There are also other aspects like designing of an efficient sgRNA to minimize the likelihood of off-target effects. Several online tools are available to predict and avoid potential off-target sites (Baltz et al. 1997). In some case, the targeted chromosomal region may escape the endonuclease activity of Cas9 enzyme, and prolonged incubation under selective pressure can be an effective solution for increasing efficiency.

17.7

Application of CRISPR/Cas in Synthetic Biology

CRISPR/Cas can be used to study the function of specific genes, to understand the interactions between different genetic elements, and to identify new genetic components that can be used to create novel genetic circuits. CRISPRi (interference) system (Fig. 17.2) uses a dCas9 protein fused to a transcriptional repression domain to bind to specific DNA sequences, thereby blocking the binding of RNA polymerase and repression of the target gene. CRISPR-mediated regulation of gene expression at the transcriptional level is an important aspect of synthetic biology to optimize the production of desired metabolites. Joseph et al. developed a dCas12abased CRISPR interference system for transcriptional gene repression in Clostridium pasteurianum and Clostridium acetobutylicum and showed a reduction in the mRNA profile of targeted gene (Joseph and Sandoval 2023). In Bacillus subtilis, CRISPRi tool was applied to downregulate the expression of pfkA or zwf resulting in improvements in hyaluronic acid production (Westbrook et al. 2018). In Bacillus subtilis, bioproduction of N-acetylglucosamine is improved by the reduction of

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Fig. 17.2 CRISPR-mediated regulation of gene expression at transcription level

expression of three genes (zwf, pfkA, glmM) which control the major competing reactions of GlcNAc synthesis (Wu et al. 2018). In the microalgae C. reinhardtii, CRISPRi was used to reduce the expression of PEPC1 to increase the lipid content (Kao and Ng 2017). CRISPRi tool based on Cas9 and Cas12a reduces the 85% transcript levels in N. oceanica (Naduthodi et al. 2021). CRISPRa (activation) system uses a dCas9 protein fused to a transcriptional activation domain to increase the expression of genes essential to produce the desired metabolite, thereby increasing the yield of that metabolite. To investigate CRISPRa, dCas9 fused with an omega (ω) protein to improve RNA polymerase activity and activate the transcription in E. coli, resulted in activation of reporter gene (Ferreira et al. 2019). Saccharomyces cerevisiae engineered for industrial-scale production of 3-hydroxypropionic acid (3-HP) using dCas9-based transcriptional regulation to target 168 genes successfully increased yields by 15–36% (Ferreira et al. 2019). Overall, CRISPR-based tools can provide a powerful means for controlling gene expression and optimizing the production of desired metabolites in synthetic biology.

17.8

Conclusions

Strain improvement of industrial microorganisms has been adapted to a more rational form and directed modification of strategies to fine-tune industrial bioprocesses. Ranging from targeted gene deletions, modifications of flux balances for obtaining increased titer of metabolite, glycosylational modifications of antibody production in yeast, to optimizing for the codon bias, CRISPR/cas have proved to be efficient strain improvement methods in industrial microorganisms. These reports hold a bright future for more specific host modifications that might bring new gene editing techniques and strategies to light.

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Acknowledgments This work was partly supported by MHRD-STARS/APR2019/CS/643/FS (SD). SG is supported by a Junior Research Fellowship from UGC, Government of India. Pooja is supported by a Senior Research Fellowship from CSIR, Government of India.

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Microbial Diversity for Agricultural Productivity

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Pompee Chanda, Bishal Pun, and S. R. Joshi

Abstract

The safety and security of the world’s food supply is at risk due to the present-day intensive farming methods. To fulfill the food demand of the population which is increasing at an alarming rate, agrochemicals are administered extensively to improve crop output. The improper use of chemical fertilizers and pesticides has caused several problems that pose a great danger to mankind. Moreover, the fertility of the cultivable fields is deteriorating exponentially. To combat the problem, researchers have come with a natural formulation known as biofertilizer that has the ability to promote plant growth. They are plantassociated microorganisms that hold great promise for resolving the problem. The benefits include enhanced nutrient availability, nitrogen fixation, phosphorus solubilization, phytohormone regulation, and they also act as a biocontrol agent. In addition to all the benefits, they are cost-effective. This chapter deals with the utilization of various strains of microorganisms as biofertilizers and the significant contributions made by these helpful bacteria in preserving the soil quality and boosting agricultural output. Keywords

biofertilizer · phytohormone · fertility · biomass · agrochemicals

P. Chanda · B. Pun · S. R. Joshi (✉) Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_18

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Introduction

Increasing food demand is directly correlated with increasing population. To address the increasing food demand due to the expanding world population which is likely to rise from 7.9 billion people to 10 billion in the next 50 years, the burden on production of food has increased. Other factors such as unavailability of fertile land, urbanization, and unpredictable weather occurrences linked to climate change, have put more pressure on the production of agriculture output (Glaser and Lehr 2019). Strategies such as the use of agrochemicals which contain N, P, or K pesticides, and insecticides, are applied to increase the crop yield and to guard against insect and pest attack during and after harvest (Liu et al. 2015). As the fertilizers have low application efficiency, less amount of nutrients around 30–40% of them are actually absorbed by the plants and the rest is lost to the soil and harms the environment (Kumar et al. 2022). Also harmful nonbiodegradable substances such as radionuclides and heavy metals are present in the fertilizer, which are hazardous to the environment. The other negative impacts comprise deterioration of plant roots, soil acidification (Wang et al. 2010), and eutrophication of ground water (Youssef and Eissa 2014). Nitrates, a type of nutrient seep into ground water and create “blue baby syndrome,” also known as “acquired methemoglobinemia,” (Kumar et al. 2022). As a result, it’s critical to consider environmentally friendly techniques, such as biofertilizers, which are essential to sustainable agriculture keeping in mind the safety of public health. Consequently, an innovative, safe, and sustainable strategy for increasing the quality of soil has emerged: employing advantageous microbiomes as biofertilizers in sustainable crop production. (Kumar et al. 2022). Biofertilizers are microscopic organisms that promote the host plant’s nutrient supply, hence promoting plant growth (Malusá and Vassilev 2014). They could be live or dormant cells added to crops to boost the absorption of nutrients from the soil (Fasusi et al. 2021). All terrestrial plants contain various microbial populations (Sasse et al. 2018). Epiphytic, endophytic, or rhizospheric processes are three ways that microbes can colonize plants (Rossmann et al. 2017). Different types of prokaryotic and eukaryotic species are found in the plant rhizosphere. Many of these microbes have numerous crucial roles in fostering the plant development that are crucial to agriculture as mentioned. The past twenty years have seen a surge in interest in the investigation of microbial communities that exist in varied habitats and their combined effects on plant development, growth, and defense (Bakker et al. 2012). Recent studies have shown that using biofertilizers is more cost-effective and environmentally friendly than using chemical-based fertilizers. Lately, there has been significant progress in the development of effective biofertilizers for many crops.

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Categories of Biofertilizers

On the basis of their role and mode of action, biofertilizers are categorized into different groups. These include nitrogen-fixers (N-fixers), potassium solubilizers (K-solubilizers), phosphorus solubilizers (P-solubilizer), and plant growthpromoting rhizobacteria (PGPR) (Nosheen et al. 2021).

18.2.1 Nitrogen-Fixing Biofertilizers (NFB) One of the main nutrients helpful for plant health is nitrogen. About 80% of the nitrogen in our environment is gaseous, yet green plants cannot absorb it straight from the air. 175 x 106 tonnes of nitrogen are fixed globally each year. Biological nitrogen fixation converts atmospheric nitrogen into ammonia with the help of rhizospheric bacteria. Some blue-green algae and soil bacteria have the capacity to convert atmospheric nitrogen into ammonia within their cells. Nitrogen-fixing bacteria transform organic compounds from inert air N2. Plants readily can take up ammonia produced during nitrogen fixation (Kumar et al. 2022). As a result, atmospheric nitrogen (N2)-fixing bacteria can provide considerable amounts of nitrogen for plant growth. Biological nitrogen fixation serves as a substitute or addition to mineral nitrogen fertilizers that are suitable to the environment. The availability of various crucial resources, including phosphate, molybdenum, and water, governs this process (Aasfar et al. 2021). Despite the fact that numerous N2-fixing bacterial species have been identified from the rhizosphere of different cereals, principally Azotobacter and Azospirillum members have been extensively studied to boost grain and legume yield in field settings. Several species of plants contain endophytic bacteria that fix nitrogen, which can provide up to 47% of the nitrogen from the atmosphere and foster plant growth. Bacterial ability to fix nitrogen can be studied using the total nitrogen difference method, the acetylene reduction test, the inspection of nitrogen solutes in the xylem and other plant components, and nitrogen labeling methods. Additionally, molecular techniques such as amplification, nif gene analysis, and qualitative and quantitative product estimation can be utilized to evaluate the bacteria’s capacity to fix nitrogen. They may also have one or more characteristics that can influence plant growth such as the synthesis of phytohormones, siderophores, the induction of systemic tolerance via the formation of 1-aminocyclopropane-1-carboxylase deaminase, and the induction of systemic resistance (Gupta et al. 2012).

18.2.2 Phosphate-Solubilizing Biofertilizer For plants to operate properly, they need the macronutrient phosphorus. Phosphorus deficit can hinder plant growth and development because it is essential to every element of plant growth and development (Daniel et al. 2022). Excessive conventional phosphorus fertilizer use has the capacity to reduce soil quality, pollute surface

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and ground water, eutrophize waterways, and accumulate toxic elements like high soil selenium and arsenic concentrations. This is done in an effort to boost agricultural productivity and meet the world’s growing food demand. Insoluble soil phosphate can be mineralized or solubilized by a variety of soil microorganisms, releasing soluble phosphate that is then available to plants. These bacteria help a number of crops thrive and produce more. Inoculating seeds, and soil with phosphate-soluble microbes is thus a possible method to increase global food supply without damaging the environment. Phosphorus-solubilizing microorganisms are extremely important for improving soil fertility, but they have not completely taken the place of traditional chemical fertilizers in commercial agriculture. It should be easier to use phosphate-solubilizing microorganisms as dependable components of sustainable agricultural systems if their functional variety, colonization capacity, mode of action, and prudent use are better understood (Alori et al. 2017). Bacterial strains and fungal strains are worth mentioning (Kalayu 2019). Phosphatesolubilizing microorganisms are present everywhere, and their numbers vary from soil to soil. The majority of phosphate-solubilizing microorganisms were discovered in the rhizosphere of different plants, where they are known to have higher metabolic activity (Khan et al. 2009; Walpola and Yoon 2012; Selvi et al. 2017).

18.2.3 Potassium-Mobilizing Biofertilizer An important requirement of all living cells as macronutrient is potassium. Because most of the potassium found in rocks and silicate minerals is insoluble, the amount of soluble potassium needed by plants in the soil is extremely low. Potassium is naturally present in soils in higher concentrations than any other nutrient, yet the majority of the potassium is inaccessible to plants. Some bacteria convert soluble potassium from inaccessible forms by means of a number of biological mechanisms. These potassium-mobilizing bacteria have the potential to boost soil potassium availability, which will be crucial for crop establishment in soils with low potassium levels (Patel et al. 2021). It has been discovered that soil bacteria living near/on the root surface and promoting plant growth in diverse ways are present in the paddy rhizosphere. The ability of two isolates, Bacillus pumilus and Pseudomanas pseudoalcaligenes, to solubilize potassium to aid plants in promoting growth in greenhouse conditions was tested. In addition to being examined for several growthrelated aspects, including the buildup of carbohydrates as an osmoprotectant under saline stress, selected bacteria were also examined for their capacity to solubilize potassium on various sources. Potassium-solubilizing bacteria improve growthrelated physiology in plants, such as stomatal conductance, electrolyte leakage, and lipid peroxidation, to protect them against salinity damage. The kind and quantity of soluble carbohydrates accumulated by plants infected with potassiummobilizing bacteria promote the plant to combat osmatic stress (Jha 2017).

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18.2.4 Sulfur-Oxidizing Biofertilizer Plants also require sulfur as a micronutrient. According to some reports, sulfur is essential for improving certain characteristics of soil. Sulfur is renowned for protecting soil from high pH levels. Sulfur is said to boost the effectiveness of fertilizers that contain nitrogen and phosphorus as well as the capacity of crops to absorb micronutrients (El-Halfawi et al. 2010). These biofertilizers function by oxidizing sulfur to sulfates, which can be absorbed by plants. Thiobacillus thioxidans can oxidize sulfur to plant-usable sulfates (Riaz et al. 2020; Vidyalakshmi et al. 2009). According to studies, there are two main methods by which sulfur is oxidized (Dasgupta et al. 2021): • The S4 intermediate process, which involves the direct conversion of thiosulfate to sulfate through the synthesis and oxidation of tetrathionate or polythionate, • The paracoccus sulfur oxidation pathway.

18.2.5 Zn Solubilizer Zinc is an important element required by plants. Potential substitutes for zinc supplements are zinc-solubilizing bacteria (Kamran et al. 2017). Divalent cation of zinc is readily usable by plants which, however, is present in small amounts (KabataPendias 2000). The remaining majority of zinc is found in forms not usable by the plants. To encourage zinc uptake by plants, crop rotation, multiple cropping, conventional breeding, and transgenic strategies have been applied in a number of contexts (Gunes et al. 2007; Gustin et al. 2009; Mhatre et al. 2011; Tan et al. 2015). These methods, however, are pricy and time-consuming. Application of zinc-solubilizing rhizobacteria is a superior approach to overcome the limitations of already available methods. Some of the most effective zinc-solubilizing strains include Pseudomonas, Bacillus, Acinetobacter, Gluconacetobacter, Thiobacillus, and Rhizobium. This will increase the amount of soluble zinc in the soil, which will improve plant development and yield. Many plant growth-promoting rhizobacteria (PGPR) are good zinc solubilizers and make zinc more readily available to plants (Kamran et al. 2017). They increase the solubility of zinc by production of organic acids to acidy the soil (Alexander 1978), utilization of chelating agents (Jones and Darrah 1994), and generation of siderophores (Chang et al. 2005; Saravanan et al. 2011; Wakatsuki 1995).

18.3

Symbiotic Nitrogen-Fixing Bacteria

Numerous types of nitrogen fixers are connected to the root nodules of leguminous plants (Kawaka 2022). A crucial limiting ingredient for overall plant growth is nitrogen. Although nitrogen is abundantly present in the atmosphere, it can be utilized by plants only in its reduced form. Biological nitrogen fixation is a process

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performed by a certain subset of prokaryotes. They convert the atmospheric nitrogen (N2) to ammonia conversion using the enzyme nitrogenase (NH3). In order to create the aforementioned nitrogenous proteins, plants can easily ingest NH3 as seen in the symbiotic relationship between the water fern Azolla and the cyanobacterium Anabaena azollae where the cyanobacteria fix a sizable amount of nitrogen in specialized cells known as heterocysts. In Southeast Asian wetland paddies, this symbiosis has been utilized as a biofertilizer for at least 1000 years. Another illustration is the relationship between actinomycete Frankia and actinorhizal plants and shrubs like Alder (Alnus spp.). These plants, which are native to North America, do well in conditions with little nitrogen.They are frequently the founding species in successional plant communities and are frequently the most prevalent nonlegume nitrogen fixers. The interactions between legumes and the bacteria Rhizobium and Bradyrhizobium are important nitrogen-fixing symbiotic partnerships in the global ecology of nitrogen fixation.

18.3.1 Rhizobium The well-known bacterial symbiont of legume plants’ root nodules that fixes nitrogen is known as rhizobium. Recent research has demonstrated that this soil microbe also forms organic, close, and occasionally advantageous endophytic relationships with different cereal crops including rice and wheat. These microorganisms in the form of biofertilizers can be used in sustainable agriculture and reduce the need for chemical fertilizer applications to achieve maximum grain yield (Schaechter 2009). Rhizobia are Gram-negative bacteria (van Berkum & van Berkum and Eardly 1998). They have the ability to cause their legume hosts to develop root nodules, which are specialized structures for fixing atmospheric dinitrogen to ammonia. The plant receives access to ammonia, which in turn provides the bacteria with carbon sources (Gage 2004; Jones and Darrah 1994). Rhizobial symbiosis with legumes is typically a specialized relationship because both partners have specific hosts. The success of the interaction depends on a complex signal system. Only the right combination results in an effective symbiosis (Broughton et al. 2000). Rhizospheric bacteria sense chemotactic substances released by plant roots, such as flavonoids, phenolics, sugars, dicarboxylic acids, and amino acids (Brencic and Winans 2005). Rhizobia then move and cling to populate the root surface. Expression of the nodulation genes is induced by flavonoids, and polycyclic aromatics produced by the host (nod, noe, nol, and others). The resultant proteins produce and export a class of compounds known as lipochito-oligosaccharides or Nod factors. Other phytohormones produced include indole acetic acid, lumichrome, and riboflavin. They consist of a conserved backbone that is surrounded by accessory groups that are organized in various ways. The first rhizobial determinant of host specificity is a strain that can only nodulate a certain legume host when certain types and combinations of Nod factors are present (Spaink 2000). Calcium spikes and modifications to the cytoskeleton of root hair are two early responses to Nod factors. Due to the localized presence of Nod factor molecules, the root hairs curl. Root cortex cells are induced to restart mitosis

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concurrently, resulting in the development of nodule primordia (Gage 2004). The associated bacteria become trapped within the distortion as a result of root hair curling. Following local lysis of the root hair cell wall, the plant cell membrane invades. The infection thread is an invaginated tube-like structure where the rhizobia then enter. The infected thread advances inward toward the nodule primordium’s developing cells and the base of the root hair. Rhizobial surface polysaccharides, including cyclic glucans, exopolysaccharides, lipopolysaccharides, and capsular polysaccharides, interact with the host plant during the production of infection threads. Their proper composition is necessary for successful symbiosis (Gage 2004). The nodule, a brand-new plant organ, is created as the nodule primordium divides further. Once the bacteria have left the infection thread, they can fix nitrogen in the nodule interior’s microaerobic environment by differentiating into bacteroids. In addition to the surface polysaccharides and Nod factors previously mentioned as nonproteinaceous host specificity determinants, secreted proteins make up another group of rhizobial signals that influence symbiosis. Rhizobia can produce and release a variety of phytohormones such as IAA, lumichrome, riboflavin, lipochito-oligosaccharide NOD factors, gibberelins, jasmonates to name a few. While these characteristics may help plants adapt to various edapho-climatic challenges, such as the lack of certain nutrients needed to promote plant growth, maximizing their effectiveness requires an understanding of the mechanisms underlying their action.

18.3.2 Free-Living Nitrogen-Fixing Bacteria Symbiotic connections with plants are not necessary for free-living nitrogen-fixing bacteria to exist and multiply. This is crucial since many plants, including corn, do not develop symbiotic connections with bacteria that fix nitrogen. Free-living nitrogen bacteria can be aerobic such as Azotobacter and Azospirrillum and anerobic such as Clostridium pasteurianum. Free-living oxygen-dependent nitrogen fixers have developed a specialized metabolic route to maintain extremely low oxygen concentrations inside their cells (Vadakattu and Paterson 2006). Even though they make only a minor portion of the soil’s microbial ecosystem, these natural microorganisms are there. Other examples of these kind of naturally occurring organisms that fix nitrogen in soil include Firmicutes, Cyanobacteria, Proteobacteria, Archaea, and others.

18.3.2.1 Azotobacter Azotobacter are very varied, widely distributed, nonsymbiotic nitrogen-fixing bacteria found in soils all over the world. In habitats lacking symbiotic nitrogen fixing, this bacterial community may serve as the primary natural source of nitrogen (Choudhury and Kennedy 2004; Das and Saha 2007). Additionally, the prevalence of Azotobacter species in the soil may increase the availability of phosphorus as well as nitrogen through the biological nitrogen fixation processes (Din et al. 2019; Velmourougane et al. 2019). Azotobacter species can directly influence plant growth

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in addition to biological nitrogen fixation by synthesizing phytohormones, avoiding various stresses, degrading pesticides and oil globules, and metabolizing heavy metals. These hormones can indirectly shield host plants from phytopathogens and encourage other advantageous rhizosphere bacteria in addition to promoting plant growth and nutrient uptake (Arora et al. 2018; Sahoo et al. 2014). Furthermore, some commercially relevant cereal and pulse crops showed considerable yield improvements (up to 40%) when Azotobacter strains were applied as bioinoculants, with positive impacts on plant development and crop production (Choudhury and Kennedy 2004; Kannan and Ponmurugan 2010; Ritika and Utpal 2014; Yanni and ABD EL-Fatiah 1999). The potential for environmentally engineering Azotobacter species with high nitrogen inputs while minimizing reliance on N-containing fertilizers like urea is presented by these favorable characteristics (Bageshwar et al. 2017). Azotobacter species have certain distinctive characteristics, such as the ability to create cysts that provide resilience to environmental stressors (Sadoff 1975). These characteristics are examined in light of the potential for creating unique formulations based on Azotobacter cysts.

18.3.2.2 Azospirillum Azospirillum is a gram-negative, aerophilic rod-shaped bacteria. These biofertilizers are necessary for the cultivation of crops. More nitrogen from the atmosphere can be fixed by azolla bacteria than rice plants have absorbed in their root zones. The organism can thrive in both anaerobic and oxygen-consuming environments; however, it favors micro aerophilic environments for development. Numerous plant species, many of which are important in terms of agronomy and ecology, are affected in terms of growth and yield (Pandey et al. 2009). Auxins, cytokinins, and gibberellins, among other phytohormones secreted by Azospirillum, alter the architecture of plant roots and encourage the creation of adventitious roots and root hairs in their host plants, which is advantageous because it stimulates root growth (Pandey et al. 2009). Agriculture has made extensive use of PGPR inoculation (dos Santos and Maranho 2018) and one of the most investigated genera is Azospirillum (Bashan & de-Bashan, 2010; Kaushal and Wani 2016). The growth promotion of Azospirillum, which involves nitrogen fixation and the generation of phytohormones, polyamines, and trehalose (Bashan and de-Bashan 2010) is the most widely recognized idea about the mechanism of action of this organism. Azospirillum has several different methods of action (Bashan and de-Bashan 2010). These mechanisms eventually result in plants that are bigger and frequently more productive (Cassán et al. 2015; García et al. 2017). Wheat, corn, and sugar cane crop yields have all increased thanks to Azospirillum (Bashan and de-Bashan 2010). Cacti, fruit trees, and chili peppers have all benefited from its application (Bashan and de-Bashan 2010). Many regions of the world, including those with tropical, subtropical, and temperate climates, as well as standing crops produced in a variety of soil types, are home to Azospirillum spp., which has been discovered to fix atmospheric nitrogen, has been detected in the rhizospheres of numerous tropical and subtropical nonleguminous plants. Pulsed-field gel electrophoresis strains were used to investigate the genomes of Azospirillum species. When first isolated, the soil

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bacterium Azospirillum was known as Spirillum lipoferum. Azospirillum strains have been used in pollution degradation procedures, according to reports (Bashan and de-Bashan 2010). One study that assessed the degradation of phenol and benzoate is one example. The genus Azospirillum (Beijerinck 1925) contains the bacterial group that can either be endophytically connected if the intercellular spaces of the roots are colonized (Van Dommelen and Vanderleyden 2007) or rhizospheric colonized. Azospirillum is the best-studied genus of PGPR and 19 of its species have been described (Zeffa et al. 2019). Azospirillum brasilense, Azospirillum lipoferum, Azospirillum halopraeferens, and Azospirillum oryzae are among the major species of the genus and are frequently employed as biofertilizers, particularly for grain crops.

18.3.2.3 Cyanobacteria Since the past three billion years, Cyanobacteria (also known as blue-green algae) have helped produce oxygen in the planet’s atmosphere. They are the main photosynthetic microorganisms that can be spread in diverse habitats, including rivers, seas, soil, and bare rock. These microbes can be found as single cells, colonies, or filaments. Cyanobacteria are minuscule in size, yet they can still be seen when they form colonies in the form of crusts or flowers (Catherine et al. 2013). Cyanobacterial biomass is the best source of biofertilizer for recovering the physico-chemical characteristics of damaged soils, including its capacity to hold water and the condition of its mineral nutrients. Because of their extensive distribution, quick reproduction, and ability to fix atmospheric nitrogen, Cyanobacteria are unique. Some of the prominent species include Anabaena, Nostoc, and Plectonema. There is growing interest in using Cyanobacteria as bioinoculants to increase soil fertility and environmental quality. Due to their enhanced organic acid synthesis, Cyanobacteria can raise soil phosphate levels. Around 1 billion hectares of soils are impacted by salinization worldwide, yet these soils can be restored utilizing biological, physical, and chemical remediation techniques. Cyanobacteria can assist enhance soil biomass, inhibit weed growth, and reduce soil salinity (Saadatnia and Riahi 2009). In a recent study, it was found that Spirulina platensis filtrates increased the amount of elemental components and chlorophyll in radish seedlings. These results demonstrated the possibility of the application of algal extracts in horticulture and agriculture in the future. Products made from Spirulina species promise assurance that there will be enough food for an expanding population. Cyanobacteria are significant members of the soil microbial community and contribute biologically to the biological restoration of the effect of soil deterioration (Singh et al. 2016). The water-binding ability and nutritional makeup of depleted soils are improved by applying cyanobacterial and microalgal biomass to soils as fertilizer. Overall, biofertilizers like Cyanobacteria are an economically and environmentally viable alternative to synthetic fertilizers like urea for use in agriculture since they require less capital and energy. Other than use as biofertilizer, new potential for the environmentally and financially sustainable production of biofuels has been made possible by the development of genetically altered Cyanobacteria with special genes

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for creating a variety of biofuels, including biodiesel, biohydrogen, biomethane, and syngas (Singh et al. 2016).

18.4

Phosphorus-Solubilizing Microorganisms

In an effort to increase agricultural productivity and satisfy the world’s growing food demand, excessive conventional phosphorus fertilizer use has the potential to deplete soil fertility, pollute surface and ground water, eutrophize waterways, and accumulate toxic elements like high soil concentrations of selenium and arsenic. Numerous soil microbes have the ability to mineralize or solubilize insoluble soil phosphate in order to release soluble phosphorus and make it available to plants. These bacteria help a number of crops thrive and produce more. Thus, introducing phosphatesolubilizing microorganisms to seeds, crops, and soil is a promising method for increasing global food supply without endangering the environment. Phosphorus, which makes up around 0.2% of a plant’s dry weight, is one of the important minerals required for plant development and growth. When it comes to mineral nutrients that most frequently prevent crops from growing, it is only second to nitrogen (Azziz et al. 2012). Phosphorus-solubilizing microorganisms are extremely important for improving soil fertility, but they haven’t completely taken the place of traditional chemical fertilizers in commercial agriculture. Pseudomonas spp., Agrobacterium spp., and Bacillus circulans are examples of soil bacteria that have been shown to mobilize phosphorus that is poorly accessible by solubilization and mineralization. Some phosphorus-solubilizing microorganisms show biocontrol agent role against phytopathogens in addition to solubilizing phosphorus. Phosphorus-solubilizing microorganisms produce antifungal chemicals (including phenolics, and flavonoids), which help to limit phytopathogens. The use of phosphate-soluble microorganisms in saline-alkaline soil improves its fertility and suitability for agricultural use with no environmental or health risks (Alori et al. 2017; Babalola and Glick 2012; Jahan et al. 2013; Kumar et al. 2022; Mamta et al. 2010). Here we will discuss few of the phosphorus-solubilizing bacteria.

18.4.1 Bacillus A major phenomenon is the solubilization of natural phosphate by different microorganisms (Collavino et al. 2010). The majority of naturally occurring bacteria break down mineral phosphates (Hayat et al. 2010), and may be used as a replacement for inorganic phosphate fertilizers (Gupta et al. 2012). A kind of bacteria called rhizobacteria aid in the growth of plants. It encompasses numerous genera, including Paenibacillus, and Bacillus (Marciano Marra et al. 2012). They are important as biofertilizers and have pivotal role in biogeochemical cycle of phosphorus (Das et al. 2007). One study revealed the isolation of Bacillus subtilis from cow dung. This strain has been shown to be thermotolerant (50 °C) and to play a part in the

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biotransformations of soil minerals like phosphorus and sulfur (Swain and Ray 2009).

18.4.2 Pseudomonas Rhizosphere colonization is necessary for plant PGPR to be applied in the field. Interaction of plants and microorganisms occurs through intricate signal exchanges and mutual recognition. Here, wheat germ agglutinin was used to separate the phosphate-solubilizing bacterium Pseudomonas spp. P34 from a wheat rhizosphere. This strain belongs to PGPR family and has an affinity for wheat (Liu et al. 2019). A study has shown that psychrotolerant Pseudomonas spp., isolated from high altitudes of Indian Himalayas to solubilize phosphorus at low temperature(Adhikari et al. 2021). Due to their numerous biofertilizing actions, which include enhancing soil fertility, secreting phytohormones, and suppressing soil-borne infections, Pseudomonas species have been noted as among the most effective phosphatesolubilizing bacteria and as a significant bioinoculant (Parani and Saha 2012).

18.5

Potassium-Solubilizing Microbes

The rhizospheric potassium-solubilizing microorganisms convert insoluble forms of potassium to soluble forms of potassium for plant uptake. Potassium is the third most important macronutrient for plants and is the seventh most plentiful element in the universe. It is crucial for metabolism, enzyme activation, osmoregulation, charge balance, minimizing needless water loss, and controlling plant stomatal movement. Its absence causes poor growth and development, which have a direct impact on agricultural yield and disease resistance. Although potassium is fixed in soils very quickly, it is released slowly. Numerous bacterial, strains, fungal strains, and some arbuscular mycorrhizal fungi are involved in the potassium-solubilization process. Different organic acids secreted by microbes cause potassium to be liberated from minerals and become accessible to plants. The availability of potassium content in soils is decreased by potassium fixation, plant uptake, and leaching. By transforming the potassium into an accessible form, the use of microbial inoculants enhances growth and yield. In agriculture, potassium-solubilizing microorganisms act as biofertilizers to increase productivity, increase nutrient availability, and decrease the usage of agrochemicals in a sustainable, cost-effective manner (Pandey et al. 2009).

18.6

Mycorrhiza

Arbuscular mycorrhizal fungi (AMF) are essential bridge between plants and the mineral nutrients in the soil. The crucial mutualistic relationship between the two kingdoms of plants and fungi is known as mycorrhiza. More than 90% of land plants

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contain arbuscular mycorrhizae, the common endotrophic symbiont, which are taxonomically and functionally varied. They belong to the monophyletic phylum Glomeromycota (Schüβler et al. 2001). Mycorrhizae can be classified into seven different categories: Arbuscular, Ecto, Arbutoid, Ectendo, Ericoid, Monotropoid, and Orchidaceous mycorrhizae. The most prevalent and widespread of them are Ectomycorrhizae and Arbuscular (Dwivedi and Ram 2015). They exchange water and mineral nutrients for photosynthetic byproducts from the host plant (Smith and Read 2008). AMF mycelium can have access to portions of the soil where plant roots cannot get to obtain nutrition (Smith et al. 2000; Allen 2011). Then, at the point where the plant and the fungus meet, nutrients are shared within the roots. Arbuscules, which are thought to be the functional location of nutrition exchange, are highly sensitive. As a result, AMF can reduce the restriction in plant development brought on by an insufficient nitrogen supply (Nouri et al. 2014). AMF interactions help plants by increasing their nutritional supply as well as their ability to withstand drought, salt, and disease (Berruti et al. 2016). According to several studies AMF is known to reduce heavy metal toxicity in the host plants (Berruti et al. 2016). Additionally, AMF play important ecological roles by enhancing soil structure and aggregation, driving plant community structure, and increasing productivity (Berruti et al. 2016). Recently, researchers have looked into how AMF symbiosis affects greenhouse gas emissions (Lazcano et al. 2014). AMF could control N2O emissions by increasing plant uptake and assimilation of nitrogen (Berruti et al. 2016). AFM exhibit a wide spectrum of abiotic factor tolerance. The ability of mycorrhizal inoculation with various AFM species to plants under low- or hightemperature stress and varying nitrogen levels to minimize temperature stress and enhance phosphorus content in comparison to non-AFM plants has been well proven. Plants that have AFM symbiosis are more resilient to the effects of alkalinity stressors as well as to the low-pH acidic soil that inhibits plant growth (Muthukumar et al. 2014). Under drought, they encourage osmotic correction (Augé et al. 2015). Additionally, the symbiosis boosts phenolics, carotenoids, chlorophyll, etc.

18.6.1 Ectomycorrhiza Fungus associated with the feeder roots of higher plants form a symbiotic relationship known as Ectomycorrhiza (ECM). The vast majority of fungi that can synthesize ECM are classified as Basidiomycetes, Ascomycetes, and a very few Zymomycetes which produce fruiting bodies such mushrooms, puffballs, coral fungi, toadstools, truffles, etc. Even while ectomycorrhizal (ECM) fungi have a number of benefits, they are very vulnerable. The vegetative mycelium must be handled carefully and with the appropriate instruments because it is a sensitive material (Lazcano et al. 2014). A crucial part of the nutrient cycle in terrestrial ecosystems, particularly in forest systems, is played by ECM fungi. The Hartig network serves as the interface for metabolic communication between the fungus and the root in these symbiotic systems. The mobilization, absorption, and transport of soil nutrients and water to the roots are all directly facilitated by the mycorrhizal

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mantle, which is linked to fungal filaments that extend into the soil (extraradical mycelium). ECM fungus expands the area of land that is available to the plants by increasing the water and nutrient supply to plant. It aids in phosphorus and nitrogen absorption. A root’s ECM colonization may offer defense against soil infections. Forest habitats are being degraded by humans on a worldwide scale. The commodities and services they offer are being badly impacted by this process, which is diminishing biodiversity. EMF have a potentially crucial function in restoration and management actions in these ecosystems because of the prevalence of this symbiotic relationship (Policelli et al. 2020).

18.6.2 Endomycorrhiza Roots of plants serve as a source of carbohydrates for fungi, and fungi deliver nutrients and water to the plant roots through a relationship known as endomycorrhizae. (Nagpal et al. 2021). The fungus develops structures inside the cortical cells and also spreads between them. There are several different kinds of endomycorrhiza, with arbuscular mycorrhiza (AM, known as vesicular-arbuscular mycorrhiza (VAM)), ericoid, and orchid mycorrhiza being the most well-known. The endomycorrhiza with the largest diversity is arbuscular mycorrhiza (Smith and Read 2008). Today, the AM fungus are categorized under a different phylum called Glomeromycota. The four genera Acaulospora, Gigaspora, Glomus, and Sclerocystis are where they primarily belong. The production of intracellular structures (such as arbuscules or hyphal coils) within the cortex cells, intercellular hyphae in the cortex, and a mycelium that penetrates deeply into the soil are all characteristics of the AM. Regarding the structures created in the cortical cells, it is now understood that there are two varieties of AM: Paris type mycorrhiza, which creates hyphal coils, and Arum type mycorrhiza, which is characterized by arbuscules (Smith and Read 2008). It’s interesting to note that a particular AM fungus can produce either hyphal coils or arbuscules depending on the host plant. The primary sites of solute exchange with the host are the arbuscules and coils; however, these are transient and only remain active for about 7 days. Ericoid mycorrhiza is found in the Ericales; they produce individual hyphae that extend into the soil and create coils of hyphae within rhizodermal (epidermal) cells, as in the case of AM. Orchid mycorrhiza is fungi that grow on plants of the Orchidaceae family (Marschner and Marschner 2012). AM can help with nutrient deficiencyrelated plant growth restrictions. AM interactions benefit plants in other ways as well, such as by increasing their ability to withstand disease and salt as well as drought. AMF have a reputation for reducing the harmful effects of heavy metals on their host plants.

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Action Mechanism of Biofertilizer

Through a number of methods, including atmospheric nitrogen fixation, phosphate solubilization and mobilization, composting, phytohormone synthesis, and disease suppression, biofertilizers improve plant growth and productivity. Plant nutrients are supplied through the first four methods. Phytohormones are essential for the growth and differentiation of various plant tissues. They also promote the uptake and transfer of minerals, particularly iron. Through the production of antibiotics, hydrolytic enzymes (such as 1,3-glucanase and chitinase), siderophores, HCN, and ammonia, plant growth-promoting microorganisms inhibit the growth of plant diseases while increasing plant defenses and competition for nutrients and habitats (Singh et al. 2021) Table 18.1.

Table 18.1 Types of Biofertilizer and its action mechanism Type of fertilizer Nitrogen fixation Phosphorus mobilizing

Potassium solubilizing

Potassium mobilizing

Phosphorus solubilizing

Nutrient uptake Plant growth promoting

Action mechanism Fix atmospheric nitrogen and make available Capable of hydrolyzing insoluble phosphorus compounds, both organic and inorganic, into soluble P forms that are simple for plants to absorb Release potassium into plantavailable forms from soil or minerals may be a sustainable alternative. By increasing the plant’s growth-related physiology, such as stomatal conductance, electrolyte leakage, and lipid peroxidation, it protects from salinity damage. Production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil. Zinc solubilizing

Production of hormones for plant growth

Organisms Azotobacter Bacillus spp, Pseudomonas spp, and Rhizobium spp, Penicillium spp and Aspergillus spp

Reference Din et al. (2019) Kalayu (2019)

Bacillus mucilaginosus, B. edaphicus

Maurya et al. (2016)

Pseudomonas spp, Burkholderia spp, Enterobacter spp, Acidothiobacillus ferrooxidans

Jha (2017) Chaudhary et al. (2019)

Pseudomonas spp, Bacillus spp and Rhizobium spp

Alori et al. (2017)

Pseudomonas spp., Mycorrhiza spp, and Bacillus spp. Agrobacterium spp, Pseudomonas fluorescens, Arthrobacter spp, Erwinia spp, Bacillus spp, Rhizobium spp

Kamran et al. (2017) Nosheen et al. (2021)

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18.7.1 Nitrogen Fixation Numerous biomolecules need nitrogen (N), an important element, in order to function and support life (Ferguson 1998). This element is widely distributed in the Earth’s atmosphere as the gas dinitrogen (N2), but because N2 is largely inert, most organisms are unable to utilize it. Instead, the majority of species must receive their N in “fixed” forms like nitrate or ammonia (NH3) (NO3–). Life can only be sustained through the conversion of N2 to NH3. Thisprocedure, which is often referred to as N2 fixation, is crucial for the biogeochemical cycle of nitrogen. N2 fixation can take place in three different ways: (i) biologically, through the action of the enzyme nitrogenase, which is only found in a few number of microorganisms (Burk 1934; Burk et al. 1934); (ii) by geological phenomena, such as lightning; (iii) industrially, through the Haber-Bosch process (Haber 1922, 1923; Smil 2004). The Haber-Bosch process became widely in use in the 1950s (Smil 2004; Thamdrup 2012). The Haber-Bosch process today accounts for the majority of the reduced nitrogen used in agriculture, as the world’s population becomes more and more dependent on agriculture to sustain itself. Around 380 teragrams of nitrogen are fixed annually through all the different processes combined. However, it is unclear that using reduced nitrogen generated with fossil fuels will be sustainable. The agents of biological nitrogen fixation are diazotrophic bacteria that can fix atmospheric nitrogen via the nitrogenase enzyme. This is largely achieved through the symbiotic interaction between specific diazotrophic bacteria and plants (Geddes and Oresnik 2016). The idea that microbes may “fix” N2 was originally put forth by Jodin over 150 years ago (Hoffman et al. 2014), and the first N2-fixing bacteria were only isolated in the early 1900s. Burk introduced the word “nitrogenase” in 1934 and launched the first thorough investigations of nitrogenase in living cells. Nitrogenase is an enzyme that catalyzes the conversion of N2 to a bioaccessible form of nitrogen. The most significant biological nitrogen-fixing biosystem is known to be the symbiotic relationship between rhizobia and legumes (Aasfar et al. 2021). Due to the high energy cost of biological nitrogen fixation—16 ATP molecules are required to break down a single Ns molecule—nitrogen fixing is a costly process. For the digestion and transport of NH4+, a total of 12 more ATP molecules are needed. To partially benefit 1 g N, the nodulating plants must give their bacterial partners 12 g of glucose (Buscot and Varma 2005). The Haber-Bosch process, created in 1913, is still more expensive in terms of energy than this method. The Haber-Bosch process needs a temperature of 400–500 °C and a pressure of between 200–250 bars to produce the same amount of nitrogen. Nitrogenase, which is found in the majority of nitrogen-fixing bacteria, catalyzes N2 fixation. Dinitrogenase MoFe (molybdenum-iron protein), which serves as the catalytic component, and dinitrogenase reductase make up the enzyme complex known as nitrogenase (Fe protein). The nif genes, namely the nifD and nifK genes for MoFedinitrogenase and the nifH gene for Fe dinitrogenase reductase, are responsible for encoding these two metal components (Soumare et al. 2020). Nif genes encode various regulatory proteins involved in nitrogen fixation in addition to nitrogenase. There are three types of nitrogenase, based on whether it needs molybdenum (Mo), vanadium (V), or iron (Fe). For the reduction of the substrate, each

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Fig 18.1 The nitrogen cycle. (Figure taken from app.biorender.com)

nitrogenase has an active site that is made up of a complex metal group called the FeV-cofactor, FeFe-cofactor, and FeMo-cofactor, for the respective nitrogenases V-nitrogenase, Fe-nitrogenase, and Mo-nitrogenase. Nitrogenase, irrespective of type, is inactivated in an aerobic environment due to its high O2 sensitivity. In fact, oxygen inhibits nitrogen fixation and assimilation processes, inactivates nitrogenase, and destroys nitrogenase While Mo-nitrogenase is only somewhat less sensitive to oxygen, Fe-nitrogenase and V-nitrogenase are highly sensitive (Soumare et al. 2020). Figure 18.1 depicts the nitrogen fixation using various pathways.

18.7.2 Phosphorus Solubilization and Mobilization Nutrients are necessary for the healthy growth and development of plants. Phosphorus is the second important element that plants need after nitrogen. Although phosphorus is often used in the form of H2PO4, it is also commonly found in the form of polyprotic phosphoric acid (H3PO4). Along with adsorption and desorption, the entire phosphorus conversion processes include mineralization and immobilization, weathering, and precipitation. Three kinds of enzymes in the soil environment are responsible for the release of organic phosphate, while microbial species are responsible for the mineralization of inorganic phosphate. For access to phosphorus (P) in a variety of forms, plants are dependent on the growth and activity of soil microbes. By (1) accelerating the dissolution of P-containing minerals through soil acidification or by releasing metal chelating ligands like organic acids with low molecular weights, (2) secreting extracellular enzymes like acidic phosphatases like

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phytase to mineralize organic P; and (3) mobilizing P that is inaccessible to plants, these organisms are able to mineralize and mobilize P into orthophosphate.

18.7.3 Potassium Solubilization Agricultural output is tremendously influenced by the solubilization of essential macronutrients; potassium being one of them. Dissolution of silicate minerals into soluble forms of potassium takes place through weathering process. But, application of potassium-solubilizing microorganisms can contribute to the availability of soluble forms of potassium to plants. These potassium solubilizers carry out this conversion process through acidification of the soil, chelating agents, and other biological mechanisms and promote plant growth to increase agricultural yield (Masood and Bano 2016).

18.7.4 Intake of Micronutrients Microbes play a key role in the cycling of nutrients in soil. The composition of the soil nutrient pool and the organization and functioning of the soil microbiome are both influenced by environmental factors. Microbes play a critical role in mobilizing and absorbing nutrients. They carry out numerous activities that promote plant growth. Some of these microorganisms, such as symbiotic bacteria that fix nitrogen or mycorrhizal fungi that improve mineral nutrition, have long been known to play significant roles in plant performance. Root exudates, a collection of chemicals that is particularly promising for luring advantageous bacteria to the rhizosphere, are one of the crucial elements. It is widely known that in legumes, the flavonoid pathway plays a significant role in luring rhizobia bacteria to the roots and activating NOD gene expression. Additionally, flavonoids are essential for hyphal branching, which encourages mycorrhizal contact. Both of these interactions promote the intake of nutrients by plants; mycorrhiza and root nodules, respectively, increase phosphorus and nitrogen uptake. Furthermore, phosphate and sulfate solubilization, plant growth promotion, siderophore production, and denitrification are some other well-known microbial-mediated plant growth-promoting pathways (Prakash et al. 2015). For nutrition, growth, and development, plants need inorganic minerals which comprise both macronutrients and micronutrients. Plant tissues contain very little concentrations of micronutrients. Different types of these nutrients can be found in nature, and microbial inoculants absorb them and make sure that they are bioavailable to plants. Microorganisms participate in a variety of processes that help plants assimilate nutrients and promote plant growth (Maitra et al. 2021). By improving nutrient absorption and availability through the processes of N fixation, P solubilization and mobilization, K mobilization, and other micronutrient mobilization, bioinoculants thriving close to roots improve plant health (Maitra et al. 2021). Plants can’t carry out all necessary physiological processes in unfertile soil since there isn’t enough micronutrients present (Bouguyon et al. 2015). It is feasible to capture

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atmospheric N biologically. Diverse microorganisms play a key role in the process called biological nitrogen fixation (BNF). N is captured from the atmosphere by certain bacteria and methanogenic archaea, which then provide it to plant roots in a usable form as already discussed. The siderophore released by Pseudomonas spp. has the ability to make Fe accessible. Another micronutrient that contributes to numerous physiological and metabolic processes but is required in lower concentrations is zinc (Zn). Zn aids in the absorption and movement of water as well as the mitigation of stressors like heat and salinity. In soil, Zn solubilizers include Pseudomonas fragi, Pantoeadispersa, Pantoeaagglomerans, and Sedum alfredii. Another essential element for plant growth is manganese (Mn), which has a variety of roles in physiologic and metabolic processes. It is a crucial component in the structure of the proteins and enzymes that are involved in photosynthesis. AMF and other microbes can make Mn available to plants (Maitra et al. 2021).

18.7.5 Production of Plant Hormones A plant’s metabolism results in the production of a class of chemicals known as plant hormones. They clearly affect plant growth physiologically at very low doses. Auxins, gibberellins (GAs), cytokinins, abscisic acid (ABA), and ethylene are the five main categories of plant hormones salicylates and brassinosteroids are two unusual plant hormones that have been found and named as study has advanced. Plant extraction, chemical synthesis, and microbial fermentation are the three main ways to get plant hormone compounds. Bacteria produce biologically active substances such as phytohormones, antifungal substances, enzymes, and suitable solutes. Microbes’ capacity to synthesize phytohormones in the rhizosphere or root tissue is one of the ways they help plants grow and cope with stress (Etesami et al. 2015). Isolated endophytic Klebsiella and Enterobacter bacteria from sugar cane produce IAA (de Santi Ferrara et al. 2012). In Cd-contaminated soil, Bacillus, Klebsiella, Leifsonia, and Enterobacter bacterial isolates from the rhizosphere of a vegetable (bitter gourd) were able to create IAA and enhance maize growth (Ahmad et al. 2011). Arthrobacter, Bacillus, Azospirillum, and Pseudomonas were among the cytokinin-producing bacteria that were observed to enhance plant root development. Additionally, ABA was found in root-associated bacteria from different plants. As ABA-producing bacteria, Proteus mirabilis, Phaseolus vulgaris, and Klebsiella pneumoniae have all been identified (Karadeniz et al. 2006) (Fig. 18.2).

18.7.6 Disease Control The biological control of plant diseases is the eradication of plant pathogen by biological means. Through a variety of mechanisms, microbial agents shield crops against disease damage. Competition for resources and space is another indirect contact with pathogens. Microbial biological control agents may also engage in hyperparasitism or antibiosis with the phytopathogen (Köhl et al. 2019).

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Fig 18.2 Different types of phytohormones

18.8

Application of Microbial Fertilizers Toward Sustainable Agriculture

The use of beneficial microorganisms in sustainable crop production will receive a lot of attention in the upcoming decades (Nina et al. 2014). These microorganisms are a consortium of naturally occurring bacteria, whose introduction into the soil ecosystem improves the soil’s physicochemical qualities and microbial (Sahoo et al. 2014). Plant growth-promoting rhizobacteria, N2-fixers, and helpful bacteria that prevent plant diseases are among the microbial communities that are valuable for agriculture (Singh et al. 2011). Biofertilizers are an important element that can significantly help in sustainable crop productions (Sahoo et al. 2014). Some of the PGPRs include Azotobacter, Azospirillum, Rhizobium, Cyanobacteria, phosphorus and potassium-solubilizing microorganisms, and mycorrhizae (Kemal et al. 2011). Azotobacter has a variety of metabolic processes, which contribute to its major involvement in the nitrogen cycle in nature (Aasfar et al. 2021). In addition to its role in nitrogen fixation, Azotobacter can also create plant hormones as well as vitamins (Revillas et al. 2000). Azotobacter promotes seed germination, advances root architecture, and inhibits pathogenic microbes near agricultural plant roots to boost plant growth (Gholami et al. 2009). Another free-living, mobile, gram-

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Fig. 18.3 Applications of biofertlizer

variable, aerobic bacteria that can flourish in flooded areas is called Azospirillum (Bhardwaj et al. 2014). It supports a number of elements of plant growth and development. It’s interesting to note that Azospirillum inoculation has been shown to alter root shape by creating chemicals that control plant development via siderophore synthesis. Additionally, it boosts the growth of root hairs and lateral roots, increasing the amount of root surface area available for adequate nutrient absorption (Fig. 18.3).

18.8.1 Role of Microbes as Biosensors in Agricultural Activities A biosensor is an analytical tool that converts biological reaction into an electrical signal. In order to transform the response to analytes into a quantifiable signal that is proportional to the analytes’ concentration, an analytical device known as a biosensor combines a biological recognition element with a signal transducer. A microbial biosensor is a type of biosensor that uses microbes as the bioelements, which are made up of multiple enzymes. Living cell enzymes are able to respond specifically and selectively to the analytes. Different categories of biosensors comprise tissue-based, enzyme-based, DNA, immuno, piezoelectric, thermal, DNA-based biosensors, magnetic biosensors, optical biosensors, and FRET-based biosensors (Mehrotra 2016). The following are the application of biosensors in agriculture.

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• A diagnostic instrument for evaluating illness and soil quality: The Research Center of Advanced Bionics in Japan asserted that it had created the world’s first biosensor for diagnosing soil with the goal of quantifying soil qualities based on soil bacteria. The fundamental idea is based on quantitative measurements of the relative activity of “good microorganisms” and “bad microbes” in the soil or differential oxygen consumption in respiration. In order to evaluate the simultaneous microbial respiration of two separate strains immobilized on two different transducer systems, a dual sensor system was created. This system’s measurements are proportional to the drop in dissolved oxygen when it is submerged in soil extract. Finally, a correlation matrix can be created by estimating disease symptoms using ratio response. The results are based on numerical databases with a focus on quick illness outbreak prediction rather than long-term experimentation findings (Banik 2021). • Pesticide residue determination: High-performance liquid chromatography (HPLC) and gas chromatography (GC) examinations of pesticides necessitate time-consuming extraction and cleanup procedures that raise the possibility of analytical and human error. However, biosensors are an affordable alternative for pesticide residue determination. In AchE-based biosensors, pH-sensitive transducers can be utilized to connect the amount of choline produced to the enzymatic activity based on the inhibitory effects of carbamate and organophosphorus pesticides. Pesticides propoxur and carbaryl are determined using fiber optic-based biosensors(Banik 2021). • Application in different physiological processes of plants. Understanding diverse Ca++ dependent signaling processes as well as cellular ionic equilibrium at distinct cell and tissue types requires monitoring real-time dynamics of calcium imaging. Identifying main metabolites produced by distinct metabolic processes in animal and plant systems to ensure optimal development and growth under varied environmental circumstances To identify the crops that are stress-resistant, researchers analyze reactive oxygen species and redox changes under biotic and abiotic stress situations. To find the hormonal distribution in multicellular tissues that is associated with development, growth, and stress tolerance (Mehrotra 2016)

18.9

Conclusion: Limitations and Future Prospects

A biological strategy for the sustainable intensification of agriculture is the use of biofertilizers. However, there are a number of restrictions on how they can be used to boost agricultural production. Biofertilizers frequently respond differently in the field than in the lab or greenhouse because they are more subject to biotic and abiotic stress. There are many different environmental factors that affect how crops are cultivated, including temperature ranges, rainfall patterns, soil types, soil

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biodiversity, and crop varieties. Because of these variances, the effectiveness of the biofertilizers varies. In addition, biofertilizers take longer to work than synthetic fertilizers since it takes time for the inoculum to colonize the root and build up its concentration. To overcome these limitations, proper and systematic field trials considering all the essential parameters should be done before the development of biofertilizers for commercial applications (Zambrano-Mendoza et al. 2021). With the increase in the human population at an alarming rate, there is an increasing pressure to meet their food demand. However, biofertilizers can aid to increase the agricultural outputs and ease the existing burden present in the food production. Moreover, over-dependence on chemical fertilizers is not a sustainable approach in the long run because of the costs associated with establishing fertilizer factories and maintaining output, both in terms of domestic resources and foreign cash. Once farmers and producers have access to sufficient knowledge, biofertilizers are a class of goods that are likely to hold commercial promise in the long run. In addition to having an effect on the economic growth of sustainable agriculture, the use of biofertilizers will benefit the planet’s ecosystem sustainability and human wellness in general (Daniel et al. 2022). It is anticipated that the market for biofertilizers would grow from US$2.3 billion in 2020 to US$3.9 billion in 2025. In accordance with Development Sustainable Goal 12 established by FAO: “promote sustainable consumption and production patterns,” the increase in market value will be supported by government agencies and businesses to educate farmers and consumers about the advantages of using biofertilizers (Zambrano-Mendoza et al. 2021). Research on the use of metagenomics to comprehend the microbial communities connected to the rhizosphere is expanding and will continue to do so. Numerous microorganisms invade the rhizosphere of plants, and as a result, there are many microbial genes that interact with plant genes. The relationship between meta transcriptomic and meta proteomic data and plant growth, for example, requires more in-depth study (Jha and Kumar 2021). For better agronomic techniques and innovative biofertilizer formulations, the omics data must be applied in the field. Genetic modification is an alternate method for enhancing the characteristics of rhizobacteria that promote growth. This makes it likely that PGPR genes will be recognized by their unique benefits to plants and applied in gene-editing or transgenic techniques (Bruto et al. 2014). On non-susceptible plant hosts, for example, certain tumor-inducing Agrobacterium strains may be able to stimulate plant growth (Walker et al. 2013). Additionally, bacterial genes directly conferring plantbeneficial properties, like nif (nitrogen fixation) or phl (phloroglucinol synthesis), have been identified. Nano fertilizers are increasingly being used in agriculture. Nano fertilizers reduce production costs, boost nutrient use efficiency, and are nontoxic. The conjugation of gold, aluminum, and silver nanoparticles in the encapsulation of nano biofertilizers will help to prolong the release of PGPR to the target cell (Gupta et al. 2015).

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Role of Microbes in Bioremediation

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Devargya Ganguly, K. L. V. Prasanna, Swaroopa Neelapu, and Gargi Goswami

Abstract

Biodegradation is a comprehensive term that encompasses practically any biologically induced substrate change, and the process of minimizing pollutant complexity through biodegradation is known as bioremediation. Microorganisms play a pivotal role in bioremediation, which can be accomplished when microbes interact within their niche under optimal conditions. Recent technological advances in microbial bioremediation include biostimulation, bioaugmentation, bioaccumulation, biosorption, and use of biofilms. A variety of microbes which include bacteria, yeast, fungi, and microalgae can biotransform or decompose or biodegrade several pollutants. However, control and optimization of bioremediation is a complex process depending on both biotic and abiotic factors. The current chapter provides an overview on the different types of bioremediation, types of microbes associated with bioremediation, and factors influencing the remediation process. A detailed account of the application of microbes toward bioremediation of contaminated soil, pesticides, heavy metals, hydrocarbons, antibiotics, dyes, radioactive wastes, etc. has been discussed from the perspective of sustainable environment management. Keywords

Bioremediation · Biodegradation · Microorganisms · Waste management · Sustainable environment

D. Ganguly · K. L. V. Prasanna · S. Neelapu · G. Goswami (✉) Department of Biotechnology, GITAM School of Science, Gandhi Institute of Technology and Management (Deemed to be University), Visakhapatnam, Andhra Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_19

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Abbreviations ARB ARHDs EPS G-HCH HGT HM LFL LPS MICP OP PAC PAH PGPR ROS

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Acid Red B Aromatic ring hydroxylating dioxygenases Extracellular polymeric substances Gamma-hexachlorocyclohexane Horizontal gene transfer Heavy metal Landfill leachate Lipopolysaccharides Microbial-induced calcium carbonate precipitation Organophosphorus Powdered activated carbon Polycyclic aromatic hydrocarbons Plant growth-promoting rhizobacteria Reactive oxygen species

Introduction

Chemical fertilizers, pesticides, and herbicides have enhanced agricultural productivity and crop yield, but they have also added harmful amounts of phosphorus and nitrogen to the soil and consequently to the terrestrial ecosystem. Besides, the contamination of natural resources by hazardous pollutants released from diverse man-made sources has resulted in the shortage of potable water and decline in soil fertility (Chen et al. 2013; Kumari et al. 2013). The rapid increase in the anticipated number of polluted locations is posing a global threat. The traditional methods for treating these polluted sites include excavation and disposal of the contaminated soil into a landfill or capping and subduing the contaminated parts of a site. The excavation method includes handling, and transport of hazardous materials that may pose serious dangers and merely shifts the contamination to another location. Finding new landfill locations for the eventual disposal of these materials is also extremely challenging and getting more and more expensive. The cap and contain method is another temporary fix because the contamination is still present on the scene, necessitating ongoing monitoring and upkeep of the isolation barriers with all the expenses and risks that entails. One alternative is bioremediation, which makes use of natural biological processes to potentially eliminate or render harmless a variety of contaminants. According to van Dillewijn et al. (2007), “bioremediation” can be described as the process by which environmental contaminants are converted into relatively less toxic forms by using a variety of biological agents, primarily microorganisms. Tang et al. (2007) have described bioremediation as the process by which selective microbes convert hazardous toxins to produce energy and biomass while

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immobilizing or converting organic or inorganic wastes to a nontoxic state or to concentrations below acceptable levels. Bioremediation technique utilizes microorganisms to reduce, eliminate, contain, or transform harmful chemicals present in water, soil, and sediment. The detoxification procedure aims to remove hazardous substances by mineralizing, transforming, or altering the molecules (Shannon and Unterman 1993). Energy is generated during the bioremediation process in microbial cells through redox reactions. These redox reactions involve different biological processes which are required for the reproduction and maintenance of cells. Bioremediation is only efficacious in environments that encourage microbial growth and activity causing contaminants to break down rapidly. According to El Fantroussi and Agathos (2005), microorganisms used for remediation can either be indigenous to the polluted site or be isolated from a different environment and introduced to the contaminated location. High-temperature burning and different types of chemical breakdown mechanisms are among technologies that have been utilized to completely destroy contaminants, or at least turn them into harmless compounds (Vidali 2001). However, in spite of significant efficiency, these processes have a number of shortcomings which notably include technological complexities and high cost incurred for small-scale operations. Besides, incineration enhances the exposure of the site employees and surrounding inhabitants to toxins and lacks public acceptance. On the contrary, bioremediation employs biological activity, relies on natural attenuation, and is typically carried out on-site. It is also relatively low-cost, low-tech, and has a high level of public acceptance (Vidali 2001). This book chapter succinctly covers the bioremediation techniques currently in use, varieties of microorganisms employed—their applications—and how they function in relation to remediation of various types of contaminants.

19.2

Types of Bioremediation

Based on the methodologies used, bioremediation is classified into: In-Situ Bioremediation and Ex-Situ Bioremediation.

19.2.1 In-Situ Bioremediation In-situ bioremediation is an onsite method employing biological therapy to get rid of dangerous toxins in the natural environment. The aerobic breakdown is encouraged. The methods are less expensive than ex situ bioremediation methods since there are no additional expenses for excavation activities. However, sophisticated equipment needs to be built and set up on-site to promote microbial activity during bioremediation, which in turn incurs significant expenses. Also, the depth of soil requiring efficient remediation may cause constraints in in-situ treatment. The two forms of in-situ bioremediation include:

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1. Natural attenuation 2. Enhanced methods (a) Bioventing (b) Biosparging (c) Bioaugmentation (d) Biostimulation

19.2.1.1 Natural Attenuation This is a form of in-situ bioremediation where contaminants are controlled naturally without the use of chemicals, machinery, or other human intervention. Instead, the moisture content, suitable nutrients, temperature, and oxygen levels of the ground can all be controlled naturally. Engineering biotechnology principles are applied to immobilize or transform pollutants into less hazardous or bioavailable forms through the process of natural attenuation, which uses soil and groundwater systems as natural bioreactors (Banwart et al. 2007). Using a gadget, it would be possible to determine whether or not the pollutant concentrations on the locations had been decreased to acceptable levels. Native environmental microorganisms selfproliferate and lower the concentration of contaminants in the suitable environment. No movement of the contaminants, as observed through the monitoring equipment, implies that the contaminants are being biodegraded. Natural attenuation is a timeconsuming approach for removing harmful contaminants because only around 10% of all the soil microorganisms have been reported to degrade toxins (Dzionek et al. 2016). 19.2.1.2 Enhanced Methods These are techniques used to clean up soils and groundwater, contaminated by fuels and other organic pollutants, through breakdown by local organisms. Under aerobic conditions, organic contaminants are converted into water, carbon dioxide, and enhanced microbial cell mass. Uncontaminated water when enriched with nutrients and saturated with dissolved oxygen and percolated or injected enhances soil bioremediation. Bioventing Native microorganisms innately present in the contaminated soil are stimulated when air and nutrients are supplied through wells. In this process, the unsaturated soil zone eventually attains saturation through the movement of air, particularly oxygen. Most fuels polluting the natural environment are oxidized by oxygen since it is an electron acceptor. The vented oxygen encourages the growth of any bacteria or fungi that have been added or are present organically. The supplied oxygen is used by the microorganisms for their enzyme activity, further contributing to the breakdown process. The adsorbed fuel residues are biodegraded, the volatile components decay into vapors and move very slowly through biologically active soil (Azubuike et al. 2016). In passive technology, the effect of atmospheric pressure is used to execute the gas exchange through vent wells; however, in active technology, a blower or pump is used to assist the process.

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Biosparging Remediation through biosparging involves either physical methods or biological methods or a combination of both. Volatile pollutants are transported from the aqueous phase to the gaseous phase with the help of an injected air stream and subsequently removed through physical process. Increased oxygen delivery is used in biological processes to promote aerobic respiration (Weymann 1995). In this method, nutrients are pushed into the contaminated zone, under pressure, in order to promote aerobic biodegradation. Due to low degree of microbial bioavailability, the heavier contaminants require a lengthier process of treatment, whereas the lighter ones are cleaned out with ease. This technique aims to treat the remaining contaminants in contamination zone by degrading chemicals including xylene, naphthalene, mineral oils, toluene, benzene, and ethylbenzene that can be decomposed under aerobic conditions (Kao et al. 2008). The main objective of biosparging is to reduce the load of organic compounds and maximize pollution biodegradation. Most typically, biosparging is suggested in areas with pollution from mid-weight petroleum hydrocarbons, which include jet fuel and diesel. Gasoline is a lighter pollutant which can be physically removed from the saturated zone. For heavier contaminants, such as oils, longer carbon chains limit microbial bioavailability, thereby necessitating longer restorative periods (Sharma 2019). Bioaugmentation It is a technique wherein microbial culture populations are added, and such populations have the capacity to break down pollutants in ground water and particular types of soil. Using this method, strains that are natural or genetically modified can be added to the polluted site to hasten biodegradation. This method is mostly used in the waste water treatment processes. Microbes from the contaminated area are collected, cultivated in vitro, genetically altered, and then released back onto the site. They constitute the population of in-situ microorganisms necessary for removing and transforming pollutants into non-toxic forms. When highly hazardous chlorinated ethenes, for instance, contaminate soil or groundwater, in-situ microorganisms break down these noxious compounds into ethylene and chloride. Due to the low efficiency of native species in degrading some harmful pollutants, organisms are genetically altered through DNA manipulation to aid the degradation process. These genetically modified microbes exhibit improved resistance to a variety of chemical and physical contaminants and are considered to be promising alternatives toward the bioremediation of soil, groundwater, and sludge (Azubuike et al. 2016). Biostimulation It is a process of promoting the growth of beneficial microbes, already present in the contaminated environment, by providing them with essential nutrients (Sharma 2019) and electron acceptors like nitrogen, oxygen, and carbon. As a result, their growth and population rise, initially increasing the rate of bioremediation. Biostimulation can accelerate the rate of disintegration by injecting one or more biosurfactants or manipulating the concentration of nutrients systemically. Both

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open systems (like vast water bodies) and closed systems (like soil and storage tanks) can use biostimulation processes. Indigenous microorganisms can be stimulated either through provision of environmental parameters like temperature, pH, and oxygen to boost their rate of metabolism, or by dispensing fertilizers, growth factors, and trace minerals. Pollutants operate as stimulants that can activate the operons for bioremediation enzymes with just a miniscule amount.

19.2.2 Ex-Situ Bioremediation Ex-situ bioremediation entails excavating the pollutants from the source and moving them to a different place for treatment. This method is often based on the cost, the depth of the contaminants, the level of pollution, the location, and the type of pollutant. High efficiency and predictability of ex-situ bioremediation techniques are crucial for effectively reducing risks to both human and environmental health. The presence of severe contamination of highly hazardous compounds essentially employs ex-situ techniques, helpful, when a risk-free and effective intervention is required. The selection of ex-situ techniques is presented in accordance with the criteria.

19.2.2.1 Biopile In order to enhance bioremediation by augmenting microbial activity, the excavated soil is piled up together with modified nutrients and, if necessary, aeration. When air is injected or vacuumed into the sediments of the vadose zone to serve as an oxygen supply and to promote microbial oxidation of the hydrocarbons, it is known as active bioventing. The biopile technique is quite similar to this method. The contaminated material is excavated and then blended with other materials, such as fertilizers, sand, sawdust, wood chips, compost, or other comparable bulking agents, as needed, to increase permeability and moisture retention (Hazen et al. 2003). It is then put onto a specially designed structure, which fosters and promotes the biological processes, necessary for hydrocarbon oxidation. Typically, this is a composting procedure involving forced air injection or vacuum extraction, moisture management, fertilizer input, and environmental monitoring utilizing commercially available tools such as vacuum pumps, blowers, leachate pumps, moisture probes, and thermocouple temperature probes (Hazen et al. 2003). Cost effectiveness is a major advantage of this technique. Nutrients, leachate collection systems, aeration, irrigation, and treatment bed are the essential elements of these procedures. This method can be used to efficiently remediate in environments with harsh conditions, such as in cold climates. 19.2.2.2 Windrows Windrows is an ex-situ bioremediation method which relies on the routine rotating of polluted soil to accelerate the activities of native and hydrocarbonoclastic bacteria already present in the soil. The windrows are usually built parallel to the slope of the site and perpendicular to the prevalent wind direction. The benefit of this technique is that windrow composting concedes the handling of a considerable amount of

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material. For the biosolids to blend and become homogeneous, a bulking agent typically wood chips is added. The mixture is then organized into one or more than one windrows, which are lengthy piles, exposed to the air and built to be mechanically spun by the appropriate gear (Dentel and Qi 2014). Frequent rotation of the composting material is necessary to ensure an adequate supply of oxygen (Dentel and Qi 2014). This soil turning up combined with the addition of water promotes aeration, nutrients, microbial degradative activity, and uniform distribution of contaminants, which in turn accelerates the bioremediation process. Windrows have a higher rate of hydrocarbon removal when compared to biopiles. However, the rate of biodegradation depends on the type of soil.

19.2.2.3 Bioreactor Raw materials are transformed into biological reaction products in a bioreactor. The bioreactor can operate in a variety of ways, but the operation chosen will solely depend on economy and expenditure. Technologies based on bioreactors may offer effective solutions to discard various contaminants from the air, groundwater, and soil. According to Tekere (2019), “the bioreactor type selected for each application should be easy to operate and maintain for the selected purpose and application” (Tekere 2019). The bioreactor imitates the natural culture conditions of the microbes and promotes growth under ideal process parameters. This technology has the key advantage of perfect control of variables including pH, temperature, substrates, aeration rate, agitation, and inoculum concentrations. However, change of intrinsic and extrinsic process variables can influence the rate of bioremediation and an optimal combination of the process parameters can eventually lead to enhanced bioremediation.

19.3

Types of Microbes Associated with Bioremediation

19.3.1 Bacteria Bacteria constitute a varied group of organisms frequently used in biodegradation and subsequently bioremediation. They are robust microbes which can survive harsh climatic conditions and have the ability to reproduce under regulated circumstances. They play a significant role as biosorbents to clean up the contaminated areas. Gramnegative bacteria use enzymes, glycoproteins, lipopolysaccharides, lipoproteins, and phospholipids on their cell walls as active sites for binding heavy metals, whereas gram-positive bacteria use peptidoglycan layer, composed of glutamic acid, alanine, and teichoic acid, as the active site for heavy metal binding. Thus, heavy metals are removed from the contaminated environment. A gram-negative soil bacterium called Pseudomonas putida is used in toluene bioremediation (Harding et al. 2003). Coates et al. (2001) have reported a gram-negative soil bacterium called Dechloromonas aromatica, which can oxidize aromatic compounds like benzoate, chlorobenzoate, and toluene. Another study has reported a rod-shaped bacteria called Alcanivorax borkumensis which consumes fuel-derived hydrocarbons and generates carbon

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dioxide (Kadri et al. 2022). In recent past, Jariyal et al. (2020) found that consortia of Pseudomonas sp. can remove dye from contaminated wastewater more efficiently than pure cultures.

19.3.2 Rhizobacteria Studies have reported the bioremediation of heavy metal-polluted soil by the plant growth-promoting rhizobacteria (PGPR) (Sayyed et al. 2013). Rhizobacters are mostly employed to help plants absorb nutrients from their surroundings or to combat plant diseases. However, they colonize the plant roots and accelerate plant growth, even in soils contaminated with high concentrations of metals, through reduction of metal toxicity. Three objectives are feasible with metal bioremediation employing rhizobacteria: (a) lowering metal uptake; (b) metal hyperaccumulation in plants; and (c) in-situ stability of the metals as organocomplexes. Malathion, an extensively used pesticide, is an organophosphorus compound, that has been reported to be broken down by the free-living, nitrogen-fixing rhizobacteria Azospirillum lipoferum (Romeh and Hendawi 2014). According to Jariyal et al. (2020), rhizoremediation employs a combination of microbial bioremediation and phytoremediation, making it an effective technique for eliminating toxins from sites with potential harm.

19.3.3 Fungi Fungi are essential for environmental bioremediation as they break down both inorganic and organic pollutants and promote cycling of elements. Compared to bacteria, this group of microorganisms have superior environmental and metabolic adaptability. They can survive in difficult environmental conditions including low moisture and high concentrations of pollutants and are capable of oxidizing a wide range of contaminants. Some fungal species even have the enzymatic functional capacity to digest complex compounds like lignin. A white rot fungus, Phanerochaete chrysosporium, can metabolize a variety of important environmental pollutants (Cameron et al. 2000). Numerous fungal genera, including those from the families Cladosporium, Aspergillus, Cunninghamella, Penicillium, Fusarium, and Mucor, have been reported to degrade aliphatic hydrocarbons and the more challenging-to-degrade aromatic hydrocarbons (Dell’Anno et al. 2021).

19.3.4 Yeast Yeast can withstand unfavorable and extreme environmental conditions. They constitute a potential group of microorganisms for bioremediation. Their negativecharged cell walls can interact electrostatically to bind with heavy metal ions. Yeast detoxify through mobilization, immobilization, or conversion of contaminants. The

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biosorption process, in which the metals interact with the cell membrane through adsorption, precipitation, and crystallization, is one way through which heavy metals are immobilized. Bioaccumulation involves uptake of toxic metal compounds inside the cells. And biotransformation is a process of converting harmful compounds into less hazardous forms. The mobilization mechanism includes bioleaching through excretion, and production of acids which interact with the metals to form an insoluble complex (Csutak et al. 2010). In Wang and Chen 2006, Wang & Chen reported that Saccharomyces cerevisiae can be widely employed for the biosorption of metals, namely, chromium, lead, zinc, mercury, cadmium, and nickel.

19.3.5 Algae Algae are considered to be one of the most effective organisms for the breakdown of xenobiotics and bioaccumulation of metals. In aquatic environments, algae are used for biomonitoring and managing organic pollutants. These include bioremediation of polychlorinated biphenyls, petroleum hydrocarbons, and polycyclic aromatic hydrocarbons. Phycoremediation, a biological process for wastewater treatment, uses algae. It has the capability to retain radioactive elements within its own cells. As the primary producer in water systems, algae thrive in water contaminated with various organic pollutants, through detoxifying mechanisms. Algal degradation can occur either intracellularly, extracellularly, or through a combination of both. Algae adsorb metals through their surface, which are then gradually transported into the cytoplasm, through the process of chemisorption. The freshwater filamentous algae Spirogyra hatillensis has been reported to absorb Ni, Cr, Fe, and Mn through biosorption; while Cladophora glomerata and Oedogonium rivulare temporarily remove Cu, Pb, Cd, and Co from aqueous solutions (Dwivedi 2012).

19.3.6 Protozoa The biodegradation of organic contaminants, such as petroleum hydrocarbons, is accomplished through the use of protozoa in both in-situ and bioreactor bioremediation methods. To improve and facilitate bioremediation, protozoa can be introduced either alone or in combination with bacteria to the pollution site. Protozoa excrete mineral nutrients which is utilized by bacteria for efficient use of nutrients resulting in enhanced growth and metabolism (Ratsak et al. 1996). Nutrients released by these microorganisms help to maintain soil fertility. Additionally, protozoa are crucial in the treatment of sewage and industrial waste waters.

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Factors Associated to Microbial Bioremediation

19.4.1 Biotic Factors Bioremediation, a process involving environmental biodegradation, uses microbes to address and eliminate concerns related to various toxins. The rate at which pollutants degrade depends on (i) microbial load capable of metabolizing the pollutants and (ii) the rate of expression of particular enzymes by the cells. The key biological factors influencing the bioremediation process include population size and composition, horizontal gene transfer, interaction, mutation, and enzyme activity (Sayler and Ripp 2000; Ikuma and Gunsch 2012). In the prokaryotic kingdom, horizontal gene transfer (HGT) naturally occurs, wherever and whenever microorganisms need genetic adaptation to survive. HGT might be used as a tool, even in harsh environments, to modify microbial communities and facilitate the degradation of hazardous contaminants. In bacteria, plasmid conjugation transfer is a well-known and well-studied method of HGT, which enables the transmission of characteristics like tolerance to toxicants like metals and antibiotics as well as the ability to degrade complex chemical compounds (Ikuma and Gunsch 2012).

19.4.2 Abiotic Factors 19.4.2.1 Temperature Microorganisms exhibit maximum growth and metabolism when the environment temperature is at its optimal. Growth is hindered if the temperature is either higher or lower than the optimum. Further, temperature also affects the activity of the biological enzymes engaged in the degradation process. The microbial growth rate and metabolism increases with the increase in temperature, and attains the maximum value at the optimal temperature. With further rise or fall in temperature, a sudden decline is observed both in growth and enzyme activity. To summarize, temperature has a profound impact on the degradative activities that bacteria are principally responsible for, as well as on the pace of bacterial growth, enzyme activity, chemical composition of contaminants, variety, and physiology of the bacterial community. In other words, a suitable temperature range is essential for the viable bacteria species to complete the breakdown of dangerous contaminants (Singh and Chandra 2014). Studies have shown that with the increase in temperature, microbial membranes become more permeable to harmful pollutants yet the rate of degradation decreases at higher temperatures (40 °C). A possible explanation can be lowering of metabolic activity and oxygen solubility in case of aerobic bacteria. On the other hand, at lower temperatures, low molecular weight hazardous compounds become less volatile, their solubility decreases, all of which delay the microbial biodegradation process. Thus, in frigid regions like the Arctic, natural oil breakdown processes are very slow (McFarlin et al. 2014). Warm climates, ranging between 30 °C and 40 °C, have been

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reported to induce the highest rate of microbial biodegradation (Børresen and Rike 2007).

19.4.2.2 pH pH significantly affects both the rate of bacterial growth and bioremediation (neutral, acidic, or alkaline). In polluted soils, microbial activity—nutrient availability— bioavailability—solubility, as well as pH, are all impacted by the production and accumulation of bacterial wastes. Although dangerous pollutants can be partially degraded in the alkaline pH range of 7.5–10, the efficient degradation process takes place at a pH range of about 5.2–7.0 (Sonawdekar 2012). Maintaining an optimal pH is essential for regulating the microbial growth and enzyme activity, which in turn promotes biodegradation (Maliji et al. 2013). 19.4.2.3 Availability of Nutrients The inorganic nutrients’ load affects the population of biodegrading microorganisms in the environment. The process of biostimulation is accelerated when macronutrients like nitrogen (N), phosphorus (P), and potassium (K) are added to the contaminated soil. In order to support growth and enzymatic activity of soil microorganisms, organic matter in the soil supplies nutrients and enhances various soil properties. However, the breakdown of pollutants produces toxic intermediates, which makes the soil hazardous, especially when there is a surplus amount of organic nutrients present. In contaminated areas, where there is a considerable accumulation of organic carbon, quick depletion of trace elements such as calcium, sulphur, magnesium, iron, and manganese takes place due to the rapid bacterial metabolism which in turn slows down the biodegradation process (Kuppusamy et al. 2016). A deficiency in oxygen may result from high levels of harmful pollutants changing the NPK (nitrogen, phosphorus, potassium) ratio, and the growth of the biodegrading bacteria of the soil may be inhibited by the lack of mineral nutrients (Sihag et al. 2014). 19.4.2.4 Concentration of Oxygen In addition to serving as an electron acceptor and chemical reactant in the enzymatic oxygenase reaction for the biterminal, subterminal, and terminal oxidation and ring breakage of aromatic pollutant compounds, oxygen also serves as a limiting factor for the aerobic bioremediation process (Koshlaf and Ball 2017). It has been established that compared to anaerobic metabolism, aerobic catabolism results in a higher level of biodegradation. The rate of pollutant breakdown by microorganisms is accelerated by increasing the concentration of the electron acceptor, where oxygen acts as the main electron acceptor for the aerobic bioremediation process (Clarkson and Abubakar 2015). 19.4.2.5 Toxic Compounds Bacterial metabolism and degradation may be influenced by the chemical nature of the contaminants, including their components, molecular weight, and structures, as well as their physical properties, including weight, viscosity, occurrence, and

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diffusion rate. Besides, the concentration, toxicity, and structural characteristics of the pollutants, present in the contaminated environment, also affect the bacterial population significantly. As compared to unbranched alkanes (intermediate length: C10–C25) and lighter PAHs (two or three rings: naphthalene, phenanthrene, and anthracene), polycyclic aromatic hydrocarbons with high molecular weight (four or more rings: pyrene, chrysenes, fluoranthene, and coronenes), and highly condensed cycloalkane compounds are more resistant to microbial degradation (Varjani 2017).

19.4.2.6 Moisture Content The water film in the soil acts as a conduit for both the transportation of soil nutrients and the elimination of metabolic wastes of the bacterial population. The moisture content of soil also affects the pH, aeration, gaseous transfer, diffusion process, nature and concentration of soluble compounds, osmotic pressure, bioavailability of hydrocarbons, and soil toxicity Kebede et al. (2021). The presence of hydrocarbon contaminants reduces the soil porosity and water-holding capacity (Huang et al. 2019). Studies have shown that microbial and soil water activities are completely correlated. This implies that the rate of microbial activity decreases with the decline in the moisture content of the soil and vice versa. According to Varjani and Upasani (2019), the efficiency of the bioremediation process depends on an adequate moisture content; however, extreme high moisture levels prevent microbial growth and metabolism. 19.4.2.7 The Soil The regular influx of organic materials from plants and animals together with the adequate supply of oxygen to the topsoil (surface soil and the unsaturated zone of soil) is primarily responsible for supporting a large bacterial population (Kebede et al. 2021). The amount of oxygen and organic matter available to the soil microbes decreases with the increase in soil depth, thereby resulting in a low bacterial population in the sediments or saturated zone of the soil (Macaulay 2015). The size of soil particles affects the soil permeability and in turn, the rate of degradation of hydrocarbons (Macaulay 2015). According to Kebede et al. (2021), “fine soil particles like clay soil, having small interstitial spaces, can retain the hydrocarbon at the soil surface and reduce the availability of nutrients and oxygen; whereas, coarse soil particles like sand soil, with large interstitial spaces, drain the hydrocarbon pollutants through the soil to the unsaturated zone.” The bioavailability of the contaminant and the metabolism and proliferation of potential indigenous bacteria are both enhanced by drained (porous) soil particles (Huang et al. 2019).

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Applications of Microbial Bioremediation

19.5.1 Bioremediation of Pesticides Pesticides constitute a wide variety of chemical compounds that are used in various chemical formulations. Pesticide usage and application rates have increased as a result of rising agro-product demand and shifting regional climate (Shetty et al. 2008). Intensive and perennial use of these harmful chemicals coupled with their delayed and ineffective degradation have resulted in persistent presence in the ecosystem. Pesticide degradation in soil via physiochemical processes is constrained by their low solubility and highly stable structure, and biochemical degradation is constrained by their restricted bioavailability to plants and microbes. In the past, methods such as recycling, pyrolysis, and landfilling were used to get rid of them from the environment, but these methods are harmful to the environment and produce toxic intermediates (Paul et al. 2005). The eco-friendly, cost-effective, and adaptable bioremediation technique is a promising one that makes use of the capacity of microorganisms to clean up environmental toxins (Finley et al. 2010). Comprehension of metabolic and co-metabolic pathways involved in pesticide biotransformation in microorganisms is essential for developing bioremediation strategies in contaminated soil. The biotransformation process includes oxidative, reductive, hydrolytic, and conjugation pathways. However, the metabolic pathways differ based on the properties of pesticide, the environment, and the type of microbes. The process starts with oxidative transformation involving oxidative enzymes such as cytochrome p450, peroxidases, and polyphenol oxidases. In the second step, hydrolytic transformation is catalyzed by hydrolases. These hydrolytic enzymes introduce hydrogen or hydroxyl group from water molecules thereby resulting in the release of substrate bonds. The third step, i.e., anion removal by reduction is accomplished by reductive transformation which involves the activity of nitroreductase. Finally, the conjugation reaction is initiated. This chemical reaction promotes mineralization through the addition of an exogenous or endogenous natural compound to the pesticide. This is a co-metabolic process because it uses already-existing enzymes. Xyloxylation, alkylation, acylation, and nitrosylation are all parts of this process. The final process is reductive dehalogenation, carried out by the enzyme reductive dehydrohalogenase. Organohalides serve as the final electron acceptor in the ATP synthesis process. Pesticide degradation by microbes is influenced by a number of factors, including temperature, pH, and nutrients, in addition to the enzyme system. The presence of anionic species in some pesticides renders them recalcitrant while others can be easily degraded. Pesticide-degrading bacteria are members of genus Flavobacterium, Arthobacter, Aztobacter, Burkholderia, and Pseudomonas (Glazer and Nikaido 2007). Recently, it was discovered that Raoultella sp. also degrades pesticides (Uqab et al. 2016). As a result of extensive research on organophosphorus (OP) compounds, there is a wealth of literature available that describes the enzymes degrading OP. Flavobacterium sp. ATCC 27551, isolated from soil samples in the Philippines in 1973, is the first report of a bacterium capable to break down OP

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compounds (Uqab et al. 2016). Since then, it has been discovered that OP compounds can be used as source of carbon, nitrogen, or phosphorus by a number of bacteria, several species of fungi, and cyanobacteria (Uqab et al. 2016). The Pseudomonas species also sequester neonicotinoids in addition to OP compounds. Pesticides are typically biotransformed by fungi, which undergo minor structural changes and render them non-toxic; these non-toxic forms, produced as a result of biotransformation, are subsequently discharged into the soil, where they can be further broken down by bacteria (Gianfreda and Rao 2004). In particular, oxidoreductases, laccases, and peroxidases from fungi have significant applications in the remediation of polycyclic aromatic hydrocarbons (PAHs), which are significant contaminants present in terrestrial, marine, and freshwater environments (Balaji et al. 2014). These enzymes are essential for the biodegradation of xenobiotic compounds. A variety of pesticides are broken down by the fungus Phanerochaete chrysoporium. White-rot fungi is reported to degrade various classes of pesticides to varying degrees, including lindane, atrazine, diuron, terbuthylazine, metalaxyl, DDT, gamma-hexachlorocyclohexane (g-HCH), dieldrin, aldrin, heptachlor, chlordane, lindane, and mirex (Kennedy et al. 1990; Hickey et al. 1994; Mougin et al. 1994; Singh and Kuhad 1999; Bending et al. 2002; Quintero et al. 2007). A number of fungi, including Agrocybe semiorbicularis, Auricularia auricula, Coriolus versicolor, Dichomitus squalens, Flammulina velupites, Hypholoma fasciculare, Pleurotus ostreatus, Stereum hirsutum, and Avatha discolor, have demonstrated their capacity to break down different pesticide groups, including phenylamide, triazine, phenylurea, dicarboximide, chlorinated, and organophosphorus compounds (Bending et al. 2002).

19.5.2 Bioremediation of Heavy Metals Rapid population growth and increasing necessity for industrial facilities to suffice the needs of human population have led to issues like overuse and exploitation of the resources available and increased air-water-land pollution (Medfu Tarekegn et al. 2020). Heavy metals are economically significant for industrial usage and have gradually become a serious global environmental menace (Siddiquee et al. 2015; Igiri et al. 2018). Excessive use of fertilizers, pesticides, and mulch has led to the soil being contaminated with heavy metals, which in turn has negatively impacted the crop production and food quality (Su et al. 2014). Contrary to organic pollutants, metals are non-degradable, persist in the environment for a longer time period, and have the potential of disrupting plant metabolism, when present at high concentrations (Ferraz et al. 2012). Studies have reported that heavy metals can effect toxicity through (i) disruption of important enzymatic functions, (ii) reacting as redox catalysts in the production of reactive oxygen species (ROS), (iii) disrupting ion regulation, and (iv) directly influencing DNA and protein synthesis (Gauthier et al. 2014). These harmful effects of heavy metal ions have thus necessitated the development of novel technologies for their eradication from soil, water bodies, and

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wastewater. According to Ahirwar et al. (2016), microbial removal of heavy metals from soil and water is an effective and prudent prospective. Extracellular polymeric substances (EPS), present on the bacterial cell walls, are reactive compounds and have been reported to have a significant impact with respect to metal adsorption (Comte et al. 2008). EPS exhibit strong metal binding properties toward complex heavy metals using (i) the concept of proton exchange and (ii) micro-precipitation of metals (Comte et al. 2008; Fang et al. 2010). When the rate at which a contaminant is being adsorbed is higher than its loss, the compound accumulates inside the microorganism. This process of containment of the contaminant(s) is called bioaccumulation (Chojnacka 2010; Medfu Tarekegn et al. 2020). Candidate organisms for bioaccumulation should be tolerant to a wide range of contaminants and concentrations. Additionally, they might exhibit enhanced biotransformational abilities, converting the hazardous compound into a nontoxic form, and enabling the microorganism to lessen the toxicity of the pollutant while keeping it contained (Mishra and Malik 2013). Bioaccumulation of metals might be useful and economically profitable, if it results in significant metal concentrations. It is a characteristic of many environmental bacterial species to accumulate significant amounts of metals inside of cells, on the surface of the cell wall, or in areas surrounded by the cytoplasm (Medfu Tarekegn et al. 2020). Considering the soil or the aquatic environment, the amount of this deposit may even amount to 6% of the dry cell mass, resulting in lowering the concentrations of heavy metal ions in the environment temporarily (Medfu Tarekegn et al. 2020). According to Kisielowska et al. (2010), such phenomenon “allows living for other organisms to be as of ecocommunion, including humans.” Heavy metals undergo oxidation, reduction, methylation, and demethylation through enzymatic reactions in microbes. For example, Kisielowska et al. (2010) have reported that Gram-positive bacteria, principally tannery sewer isolates, reduced highly toxic chromium (VI) to chromium (III), which is less toxic, and can be removed from the environment thus making the process practically useful. Microbial activity can thus precipitate or crystallize heavy metal compounds, resulting in modification of the metal and reduction of its toxicity. Certain precipitation and biocrystallization processes, participating in biogeochemical cycles, include microfossil formation, deposition of iron and manganese, and mineralization of silver and manganese. Metal precipitation on the cell wall or in the cytoplasm of the microbial cells can be the consequence of direct microbial enzyme activity or galactosis of secondary metabolites (Sklodowska 2000). Owing to their abundance, size, ability to grow in controlled environments, and resistance to environmental conditions, bacteria are considered to be significant biosorbents (Srivastava et al. 2015). A number of heavy metals have been tested using different bacterial genera like Flavobacterium, Pseudomonas, Enterobacter, Bacillus, and Micrococcus. High surface-to-volume ratios and the presence of teichoic acid (which function as active chemisorption site) on the cell wall may be the possible reasons for their potential biosorption capabilities (Sannasi et al. 2006; Mosa et al. 2016). Consortia of cultures are better suited for field applications as mixed cultures allow bacteria to thrive and remain stable for a longer period of time, besides possessing superior metabolic capabilities for metal biosorption (Kader et al.

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2007). Chromium was reported to be reduced by 78% by a bacterial consortium of Acinetobacter sp. and Arthrobacter sp. at a metal ion concentration of 16 mg/L (De et al. 2008). Similarly, Abbas et al. (2014) reported the effective removal of heavy metals, namely, chromium, zinc, cadmium, lead, copper, and cobalt by a bacterial consortium, roughly at the rate of 75–85% in less than 2 h of contact time. Pb was removed in significant amounts from a synthetic medium using Micrococcus luteus (Puyen et al. 2012). The study reported the ability of Micrococcus luteus to eliminate Pb was found to be 1965 mg/g under ideal conditions. In 2018, Abioye and his colleagues studied the biosorption of Pb, Cr, and Cd in tannery effluent using Bacillus subtilis, Bacillus megaterium, Aspergillus niger, and Penicillium sp. B. megaterium was recorded to reduce Pb at the rate of 2.13–0.03 mg/L, seconded by B. subtilis (2.13–0.04 mg/L). In the same study, A. niger demonstrated the greatest ability to decrease Cr concentration (1.38–0.08 mg/L), followed by Penicillium sp. (1.38–0.13 mg/L) after a span of 20 days; while B. subtilis demonstrated the greatest ability to decrease Cd concentration (0.4–0.03 mg/L), followed by that of B. megaterium (0.04–0.06 mg/L) (Abioye et al. 2018). Kim and coauthors immobilized Desulfovibrio desulfuricans, on zeolite and designed a batch system that could effectively remove 99.8% Cr+6, 98.2% Cu, and 90.1% Ni (Kim et al. 2015). Fungi have demonstrated exceptional ability to uptake and recover metals making them popular biosorption candidates for the remediation of toxic metals (Fu et al. 2012). Majority of studies have revealed that fungal cells, both living and dead, are crucial for adhering of inorganic chemicals (Tiwari et al. 2013). Srivastava and Thakur (2006) have reported on the effectiveness of Aspergillus sp. in removing chromium from tannery wastewater. While Aspergillus sp. could remove 85% of the chromium from a synthetic medium at pH 6 in a bioreactor system. However, the same strain could remove only 65% of chromium from the tannery effluent. A possible explanation might be the inhibition of growth by organic pollutants present in the tannery effluent. Coprinopsis atramentaria was reported to bioaccumulate 76% of Cd2+ at a concentration of 1 mg/L and 94.7% of Pb2+ at a concentration of 800 mg/L, thereby demonstrating successful mycoremediation through the bioaccumulation of heavy metal ions Cd2+ and Pb2+ (Lakkireddy and Kües 2017). According to a study by Park and coauthors, the dead fungal biomass of Aspergillus niger, Rhizopus oryzae, Saccharomyces cerevisiae, and Penicillium chrysogenum could convert toxic Cr (VI) into less toxic or nontoxic Cr (III) (Park et al. 2005). According to Chabukdhara et al. (2017), ‘Phycoremediation’ can be described as the remediation process including either degradation and assimilation, or removal, using different species of algae and cyanobacteria. Algae are autotrophic, require comparatively less nutrients while producing significant quantity of biomass, in comparison with other microbial biosorbents, and have been extensively exploited for the removal of heavy metals (Abbas et al. 2014). Functional groups, namely, hydroxyl, carboxyl, phosphate, and amide moieties, present on the algal surface, act as metal-binding sites (Abbas et al. 2014). Goher et al. (2016) could successfully use dead cells of Chlorella vulgaris to remove Cd2+, Cu2+, and Pb2+ ions from aqueous solutions under varying conditions of pH and treatment time. The results were

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encouraging and implied that Chlorella vulgaris biomass can be effectively used as a biosorbent to remove 95.5% of Cd2+, 97.7% of Cu2+, and 99.4% of Pb2+ from a mixed solution of a 50 mg/dm3 of each metal ion.

19.5.3 Bioremediation of Hydrocarbons One of the most significant and most pervasive environmental hazards include hydrocarbons (Heider et al. 1998). Oil spills came to the attention of the world in 1967 after the Torrey Canyon supertanker released 1,20,000 tons of crude oil into the English Channel. It is frequently thought that the isolation of hydrocarbon degraders was a remarkable discovery because the capacity of living organisms to use them as a substrate is typically regarded as a distinguishing quality. Microorganisms which have the ability to decompose hydrocarbons and utilize them as a substrate can be efficiently used for the process of bioremediation. The cytotoxic and mutagenic properties of hydrocarbons are responsible for the deep-routed pollution aftermath (Fuentes et al. 2014). According to Fuentes et al. (2014), “a more bioavailable toxic compound not only shows increased noxious effects but also has higher accessibility for biodegradation. In contrast, strongly adsorbed fraction is less toxic but more recalcitrant.” Petroleum hydrocarbons have a tendency to tightly adsorb to these matrices and hence the preceding concept(s) is pertinent for developing biological cleanup methods of contaminated soils or sediments (Baboshin and Golovleva 2012). Hydrocarbon compounds can be activated and oxidized by microbes. One or two hydroxyl groups are added to the hydrocarbon skeleton as a part of the initial stage in the aerobic degradation of hydrocarbons (Koshlaf and Ball 2017). Oxygenases constitute the key enzymes used in hydrocarbon degradation pathways (Rojo 2009). Monooxygenases add one oxygen atom to the hydrocarbon, whereas the addition of two hydroxyl groups is catalyzed by dioxygenases (Rojo 2009). Microorganisms use alkane hydroxylases (such as AlkB), which are soluble or integral-membrane non-haem iron monooxygenases, to break down aliphatic hydrocarbons (n-alkanes) through substrate hydroxylation (Rojo 2009). In essence, primary alcohol is produced by the oxidation of the terminal methyl group, which in turn initiates the aerobic degradation of alkanes. Aldehyde dehydrogenases and alcohol further oxidize this product to obtain aldehyde. Finally, the aldehyde undergoes oxidation and is transformed to fatty acid. Acetyl-CoA is formed when fatty acid and CoA is coupled together, and is then transported into the ß-oxidation pathway (Koshlaf and Ball 2017). Degradation of long-chain alkanes occurs through both terminal and sub-terminal oxidation pathways (Kotani et al. 2007). The produced secondary alcohols are converted, through the process of sub-terminal oxidation, into the corresponding ketone. In the subsequent reaction, a Baeyer–Villiger monooxygenase oxidizes the ketone into an ester. An esterase enzyme finally hydrolyzes the ester to produce a fatty acid and an alcohol (Koshlaf and Ball 2017). Studies have reported that Pseudomonas sp., Rhodococcus sp., Sphingobium sp., and Sphingomonas sp. can break down aromatic hydrocarbons. Elliot et al.

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(2010) have stated that “the rate-limiting step of these catabolic mechanisms is the oxidation of the aromatic ring, followed by the systematic breakdown of the compound to PAH metabolites and/or carbon dioxide.” The reaction is fundamentally catalyzed by aromatic hydrocarbon ring hydroxylating dioxygenases (ARHDs), which results in the formation of cis-dihydrodiols (Koshlaf and Ball 2017). The cytochrome P450 monooxygenase is used by a small number of bacteria to oxidize PAHs, producing trans-dihydrodiols (Bamforth and Singleton 2005). ARHD activity is determined by the specific PAH. PAH dihydroxy derivatives are produced by the oxidation of dihydrodiols, catalyzed by dehydrogenases. According to Koshlaf and Ball (2017), these PAH dihydroxy derivatives are further subjected to the enzymatic action of ring-cleaving dioxygenases. In this reaction, .a pair of oxygen atoms are integrated into the dihydroxy derivative molecule coupled with the cleavage of the aromatic ring (Koshlaf and Ball 2017). Dihydroxylated intermediates can then be cleaved via the ortho or meta cleavage pathways, producing catechols, which are then broken down by the TCA cycle into carbon dioxide and water (Koshlaf and Ball 2017; Baboshin and Golovleva 2012). Two distinct metabolic pathways are involved in the fungal mineralization of PAH. PAHs are transformed into arene oxides by non-ligninolytic fungi. These group of microbes catalyze the incorporation of one oxygen atom into the ring of the substrate through the cytochrome P450 monooxygenase pathway. On the other hand, ligninolytic fungi, such as white-rot fungi, mineralize PAHs using soluble extracellular ligninolytic enzymes like laccases, lignin peroxidase, and manganese peroxidase (Koshlaf and Ball 2017). The most effective agents for degrading petroleum are bacteria, which primarily degrade different target molecules found in soil, water, and sludge (Brooijmans et al. 2009). Numerous genera of organisms have been described as hydrocarbonoclastic, capable of degrading diverse petrogenic hydrocarbon components. Common bacterial species include Mycobacterium spp., Arthrobacter spp., Marinobacter spp., Achromobacter spp., Alcaligenes spp., Corynebacterium spp., Flavobacter spp., Micrococcus spp., Nocardia spp., and Pseudomonas spp. (Germida et al. 2002). In recent times, researchers have discovered that the genera Bacillus, Dietzia, Gordonia, Halomonas, Cellulomonas, Rhodococcus, and halotolerant Alcanivorax spp. have the capacity of oxidizing and degrading a variety of crude oil compounds (n-alkanes and aromatic hydrocarbons) (Borzenkov et al. 2006; Wang et al. 2007; Mnif et al. 2009; Dastgheib et al. 2011). Numerous studies have reported that a variety of fungi, including those from the genera Aspergillus, Penicillium, Cunninghamella, Fusarium, Saccharomyces, Amorphotheca, Syncephalastrum, Neosartorya, Phanerochaete, Paecilomyces, Talaromyces, and Graphium, can mineralize petroleum hydrocarbons with different degradation rates (Germida et al. 2002; Chaillan et al. 2004). Studies have documented that there are a number of filamentous fungi and white-rot fungi which can oxidize and dissipate a variety of PAHs into safer metabolites. Benzo[a] pyrene, benz[a]anthracene, and 9,10-dihydrobenzo[a]pyrene, for example, have been transformed and degraded by the filamentous non-ligninolytic soil isolate fungus Cunninghamella elegans (Cerniglia et al. 1985). Psilocybe spp., Cyclothyrium spp., and Penicillium simplicissimum constitute other examples of

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filamentous fungi with hydrocarbonoclastic activity against different PAHs (Tortella et al. 2005). Photosynthetic unicellular prokaryotes or eukaryotes including cyanobacteria, green, brown, and red algae are thought to be potential candidates for bioremediation applications because of their high growth efficiency and biomass generation (Duan et al. 2019). It was first reported, almost 50 years ago, that it might be possible to use microalgae to degrade aromatic compounds like naphthalene (Cerniglia et al. 1980). In more recent studies, pyrene degradation using green microalgae (Chlamydomonas, Chlorella, Scenedesmus, and Selenastrum) or cyanophytes (Synechocystis) was reported to range from 34% to 100% over the course of seven days (Lei et al. 2002). Similar to this, another study reported that Skeletonema costatum and Nitzschia sp. could effectively degrade phenanthrene and fluoranthene (Hong et al. 2008). The green microalga, Chlorella vulgaris, was reported to demonstrate significant bioremediation of crude oil-contaminated waters, the bioremediation efficiency ranging from 88% to 94% (Xaaldi Kalhor et al. 2017). In Al-Hussieny et al. 2020, in a study conducted by Al-Hussieny et al., five cyanophytes, namely, Westiellopsis prolifica, Anabaena variabilis, Oscillatoria princeps, Phormidium mucicola, and Lyngbya digueti were reported to lower the concentrations of several hydrocarbon compounds, found in waste waters from oil refineries, by 24% to 92%. It has been reported that marine cyanobacteria, namely, Agmenellum quadruplicatum, Microcoleus chthonoplastes, and Phormidium corium can remove phenanthrene (Ghasemi et al. 2011). In addition, the eustitgmatophyte, Nannochloropsis oculata, and the haptophyte, Isochrysis galbana, have been acknowledged as potential candidates for removing hydrocarbons from tainted seawater (Ammar et al. 2018).

19.5.4 Bioremediation of Mined Wasteland and Landfill Leachates The land of mining areas is significantly impacted by activities such as drilling, blasting, building various auxiliary facilities, and storing excess waste materials that are imposed (Sharma et al. 2013). Groundwater alteration or contamination due to metal toxicity, effluent discharges, and acid drainage are some examples of other environmental problems brought on by increased land degradation and a decline in the ratio of people to land. Some microorganisms can be considered to play a significant role in the bioremediation of mined lands owing to the cost-intensive physicochemical remediation methods. When Mwandira et al. (2017) used Pararhodobacter sp. to bioremediate a Pb-contaminated mining site, they reported the complete removal of 1036 mg/L of Pb2+. This was accomplished using microbial-induced calcium carbonate precipitation (MICP) (Mwandira et al. 2017). When introduced into the ground, Pararhodobacter sp. is buoyant due to its high urease activity, and it retains this enzyme activity for an extended period of time to immobilize the toxic ions (Danjo and Kawasaki 2016; Li et al. 2016). Landfilling is considered to be one of the most popular and widely accepted methods for the disposal of waste. The process of landfilling results in the production

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of leachate. Infiltration and percolation of wastewater through decomposing solid waste is known as landfill leachate (LFL). LFL poses a major threat to both surface and groundwater. Since bioremediation has a lower operating cost than physicochemical methods, it can be considered a viable alternative strategy. LFL can be bioremediated by bacteria, fungi, and algae. In their experiment, Morris et al. (2018) reported the isolation of bacterial strains belonging to the phyla Firmicutes, Actinobacteria, and Proteobacteria from LFL. These isolates were able to remove 90% of ammonia and 60% of both nitrate and phosphate. In Paskuliakova et al. 2018, Paskuliakova and co-researchers used the Chlamydomonas sp. algal strain SW15aRL and observed a significant reduction in ammonia nitrogen between 70% and 100% (Paskuliakova et al. 2018). In an altogether different study, Spina et al. reported detoxification of the contaminants through evaluation of the bioremediation potential of two indigenous fungi, Pseudallescheria boydii MUT 721 and Phanerochaete sanguinea MUT 1284 (Spina et al. 2018).

19.5.5 Bioremediation of Dyes Dye is a colored compound that imparts color to a material through chemical bonding with that particular material. The chromophore group present in a dye is responsible for the color of the dye. There are many different kinds of dyes, but they all have at least one N = N bond (azo groups). Azo complexes have gained recognition as one of the most significant and adaptable groups of artificial colorants, and are frequently used in a variety of industries, including food, paper, textiles, and cosmetics (Pandey et al. 2007). Extremely high dye concentrations in the wastewater from the textile industry impede the photosynthetic ability of the aquatic life. Additionally, it has harmful, toxic, and carcinogenic effects on the living organisms on land. Typically, different bacteria have the potential to decolorize azo dyes under conventional cultivation conditions, i.e., aerobic, anaerobic, and facultative anaerobic conditions. Aerobic decolorization of colorants, especially sulfonated azo dyes, is difficult to sequester even in the presence of aerobic bacteria (McMullan et al. 2001; Pearce et al. 2003; Lin et al. 2010). The anaerobic microbial method is successful at removing color, primarily through azo reductases that cut the N=N azo dye linkages, but it also produces lethal oncogenic as well as mutagenic, colorless aromatic amines (Popli and Patel 2015). Since azo dyes are resistant to extreme aerobic conditions and produce and accumulate environmentally toxic amines under anaerobic conditions for mineralization, sequential anaerobic–aerobic biological techniques have been identified as the most efficient and cost-effective approaches (Ajaz et al. 2019). It has been determined, in one of the studies, that enzymatically the azo colorant is converted into a variety of end products (Ajaz et al. 2019). These metabolites are utilized through a variety of pathways; for instance, 4-guanidinobutyric acid is used as a substrate during amino acid metabolism. Catabolism of the amino acid catabolism may result in the formation of pyruvate. The pyruvate is converted into acetyl coenzyme A which goes through the TCA

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cycle to produce reduced molecules. A different product, a phthalate derivative, can be converted into various fatty acids and aldehydes. These molecules can then move directly or indirectly into fatty acid ß-oxidation reactions, producing both NADH2 and FADH2. The reduced molecules can be applied for ATP synthesis. Azo dyes can be broken down by the enzymes: (i) azoreductase, used by bacteria; and (ii) peroxidases, used by fungi. In-depth studies on dye degradation, using single bacterial cultures of Proteus mirabilis and Pseudomonas luteola, have demonstrated key promising results in anoxic conditions (Chang et al. 2001; Yu et al. 2001; Kalyani et al. 2009). Pseudomonas aeruginosa decolorized Navitan Fast Blue S5R, a commercial colorant, when glucose was added under aerobic conditions (Nachiyar and Rajakumar 2005). The biodegradation of reactive red-BLI dye, under static anaerobic conditions, was studied by Kalyani and co-workers using the facultative anaerobic bacterium Pseudomonas sp. SUK1, isolated from dye-contaminated soil (Kalyani et al. 2009). When the initial dye concentration was 50 mg/L, researchers reported the isolate with a decolorization efficacy of up to 99.28% after 1 h of incubation. This bacterial strain breaks down poisonous dye into harmless products. Bacillus sp. YZU1, an isolate from dye-polluted soil, has been used in static conditions to study reactive black-5 dye decolorization. The researchers discovered that the azoreductase enzyme of the strain catalyzed the degradation of dye, resulting in a 95% degradation rate of 100 mg of colorant in 120 h (Wang et al. 2013). The Enterococcus faecalis YZ 66 strain demonstrated the capability to degrade different synthetic dyes, most notably the Direct Red 81 diazo dye, which it can completely degrade after 1.5 h of incubation under static anoxic conditions (Sahasrabudhe et al. 2014). In 2014, Imran and colleagues used Shewanella sp. IFN4 to reductively decolorize azo dyes (acid red-88, direct red-88, and reactive black-5) from synthetic wastewater with an efficiency of 90% under anaerobic–aerobic system (Imran et al. 2014). Under optimal static conditions, the bacterial consortium of Bacillus pumilus HKG212 and Zobellella taiwanensis AT1–3 could effectively remove the reactive green-19 dye. However, the removal efficiency improved up to 97%, within 24 h of incubation, when yeast extract was added to the cultivation medium. This significant bioremediation efficiency was observed even at 100 mg/L initial concentration of the colorant (Das and Mishra 2017). Reports suggest that fungi produce extracellular ligninolytic enzymes including lignin peroxidase, laccase, and manganese peroxidase which help degrade complex organic compounds (He et al. 2018; Guo et al. 2019; Mani et al. 2019; Zahran et al. 2019). Besides the white rot fungus, Phanerochaete chrysosporium, other species, including Aspergillus ochraceus, Trametes versicolor, different species of Pleurotus and Phlebia have been extensively studied with reference to azo dye degradation and decolorization; (Pointing and Vrijmoed 2000; Saratale et al. 2006; Kunjadia et al. 2012; Sen et al. 2016). However, an extended growth cycle and the prerequisite for N2 restraining conditions are few drawbacks associated with the removal of dye from textile wastewater using white rot fungus. Another key concern to take into account is the upkeep of fungi in bioreactors (Stolz 2001). But since wastewaters do not

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naturally contain white rot fungus, the production of enzymes for dye degradation is an independent phenomenon (Robinson et al. 2001). The bioremediation potential of yeasts through enzymatic biodegradation and decolorization of various azo dyes have been assessed using Candida tropicalis, Candida zeylanoides, Debaryomyces polymorphus (Yang et al. 2003), and Issatchenkia occidentalis (Ramalho et al. 2004). Recent studies have reported that Saccharomyces cerevisiae MTCC-463 has the potential to decolorize Malachite Green and Methyl Red (Jadhav and Govindwar 2006; Jadhav et al. 2007). Galactomyces geotrichum MTCC 1360 has been reported to degrade reactive textile colorants, triphenyl methane, and azo (Jadhav et al. 2008). Navy Blue HER decolorization has been studied using an enzymatic mechanism inherently present in Trichosporon beigelii NCIM-3326 (Saratale et al. 2009). According to Lucas et al. (2007), Candida oleophila caused the decolorization of Reactive black 5, an azo dye, in an assay using Fenton’s reagent. Under ideal circumstances, Cyberlindnera samutprakarnensis was observed to decolorize Acid Red B (ARB) with a 97% efficiency after 18 h of incubation (Song et al. 2018). Algae and cyanobacteria are common photosynthetic organisms in nature. It has been hypothesized that by using their azoreductases, algae are adept at degrading azo dyes (Hafez Omar 2008; Vijayaraghavan and Yun 2008). Algae can remove azo dyes using three different mechanisms of assimilative chromophores-utilization processes, i.e., (i) production of algal biomass; (ii) changing colored into colorless molecules by H2O and CO2 alterations; and (iii) adsorption of chromophores on algal biomass (Acuner and Dilek 2004; Mahalakshmi et al. 2015). In Yan and Pan 2004, Yan & Pan have reported the complete degradation of more than 30 azo complexes and basic aromatic amines by Chlorella vulgaris, Chlorella pyrenoidosa, and Oscillateria sp.

19.5.6 Bioremediation of Radioactive Wastes An acute health response to radionuclide or radiation exposure starts with headache, nausea, and vomiting (Prakash et al. 2013). With enhanced exposure, a person may also experience low blood pressure, dizziness, nausea, hair loss, fatigue, weakness, fever, diarrhea, blood in the stool, and eventual death (Prakash et al. 2013). The effects of radiation damage at cellular level in fetuses can result in the formation of smaller heads or brains, poor formation of eyes, mental retardation, and irregular development (Nussbaum 2007; Al-Zoughool and Krewski 2009; Bogutskaya et al. 2011). Presently, soil contaminated with radionuclides is excavated and shipped to a far-off waste disposal facility (Prakash et al. 2013). In Lloyd and Renshaw 2005, Lloyd & Renshaw have reported that, an amount of over a trillion dollars in the USA and 50 billion pounds sterling in the UK had been assessed to be spent on cleaning up these sites. Owing to the high cost of the physiochemical approaches, microorganisms and plants have gained the focus of the scientific community toward detoxification of sediments and waters impacted by nuclear waste (Lloyd and Renshaw 2005; Kumar et al. 2007).

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Desulfuromonas ferrireducens and Rhodanobacter sp. have been found to interact with such contaminants (Amachi et al. 2010; Green et al. 2012). Studies have suggested that radionuclides, such as uranium (U), technetium (Tc), and chromium (Cr), can be detoxified enzymatically by microbes. The high solubility of the oxidized forms of U, Tc, and Cr, in aqueous media, is the responsible factor for their mobility in ground water. On the contrary, their reduced forms are insoluble and frequently precipitable into the solution (Amachi et al. 2010). It has already been documented that the microbial isolates Desulfovibrio desulfuricans, Geothrix fermentans, Deltaproteobacteria, and Clostridium precipitate metals and radionuclides extracellularly (Suzuki et al. 2003; Brodie et al. 2006). Understanding the processes that lead to the reduction of Fe(III), U(VI), and Tc(VII) by Geobacter sulfurreducens has made substantial progress (Lloyd 2003). In two different studies, it was found that surface bound c-type cytochrome (9.6 kDa), associated with the periplasmic surface of Geobacter sulfurreducens, initiated U(VI) reduction; however, G. sulfurreducens mutant which lacked the c-type cytochrome could not detoxify U(VI) (Lloyd 2003; Lloyd et al. 2003). In De Luca et al. 2001, De Luca et al. reported the detoxification of Tc(VII) by Desulfovibrio fructosovorans, through a periplasmic Ni/Fe containing hydrogenase enzyme. The Ni/Fe containing hydrogenase enzyme catalyzed the reduction of the metal using hydrogen as the electron donor. One of the main risk-inducing contaminants is technetium 99, which primarily exists in an oxidized form in soil and groundwater before changing into an anionic pertechnetate form. Direct reduction of pertechnetate (Tc(VII)O4) by metalreducing bacteria results in the formation of the immobile solid [Tc(IV)O2(s)] (Burke et al. 2005). Cr(IV)- reducing microorganisms, such as Arthrobacter aurescens, Pseudomonas aeruginosa, Pantoea agglomerans, and Desulfovibrio vulgaris, have been isolated from chromate-contaminated waters, oils, and sediments (Ganguli and Tripathi 2002; Arias and Tebo 2003; Goulhen et al. 2006; Horton et al. 2006). The chromate ion is highly soluble and is reduced to form Cr (III), which eventually precipitates in the form of Cr(OH)3. During the process of bioaccumulation, solutes are transported into the cytoplasm through the cellular membrane. Different microbes, including Micrococcus luteus, Arthrobacter nicotianae, Bacillus megaterium, and Citrobacter sp. N14, have been reported to be used in the process of bioremediation of radioactive waste materials (Macaskie et al. 2000; Tsuruta 2003). Biosorption is referred to as the association of soluble materials with the cell surface. Kumar et al. (2007) have stated that “This reactivity arises from the presence of a wide array of ionizable groups, such as carboxylate and phosphate, present in the lipopolysaccharides (LPS) of a Gramnegative, and the peptidoglycan, teichuronic acids, and teichoic acids of a Grampositive, bacterial cell wall.” A number of surface structures, which are composed mainly of carbohydrate polymers (capsules) or proteinaceous surface layers (S-layers), may overlayer the bacterial cell wall and interact with metal ions (Douglas and Beveridge 1998). Merroun et al. (2005) have reported that Bacillus sphaericus is capable of accumulating uranium in cells with an S layer. In a study conducted by Khani and colleagues in 2005, the brown marine alga Cystoseira indica was described to efficiently adsorb the radionuclide U(VI) (Hassan Khani

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et al. 2006). The same study stated that the efficiency of radionuclide adsorptions can be enhanced by pre-treating the alga with calcium (Hassan Khani et al. 2006). Firmicutes and Citrobacter freundii have been identified as radionuclide biosorbents among other microorganisms (Haferburg et al. 2007; N’Guessan et al. 2008; Xie et al. 2008). The addition of nutrients increases the quantity or activity of native microflora available for bioremediation. This process is referred to as “biostimulation” (North et al. 2004). The most prevalent radionuclide contaminant at nuclear complex sites is Uranium U(VI). Biostimulation can effectively cause immobilization of U(VI), involving reduction from UO22- to insoluble U(IV) oxide, and is considered to be an effective method for in-situ remediation of U(VI) (Vrionis et al. 2005). The Geobacteraceae family of dissimilatory metal-reducing microorganisms are effectively stimulated when acetate is added to a uranium-contaminated aquifer, thereby speeding up the in-situ immobilization process of uranium remediation (OrtizBernad et al. 2004). Typically, biofilms are described as one or more populations of microorganisms that have formed an attachment to a surface, either abiotic or biotic, using extracellular polymeric substances (Kumar et al. 2007). Biofilms consisting of the sulfatereducing bacterium Desulfovibrio desulfuricans have been used to immobilize hexavalent uranium U(VI) (Beyenal et al. 2004). According to scientific studies, the accumulation of radionuclides, particularly 60Co from contaminated water, is directly caused by the biofilm population found in spent nuclear fuel (Sarró et al. 2005). The soil microbes cannot absorb iron as a free ion because it is mostly present in aerobic soil in the form of Fe(III), which has a poor water solubility. Siderophores are low-molecular-weight chelating compounds that are produced by microbes. They bind to iron and facilitate in the energy-dependent process of transporting iron into the cell (Pierwola et al. 2004). Thorium, uranium, neptunium, and plutonium are examples of actinides that siderophores effectively bind to (Renshaw et al. 2003). Microbacterium flavescens, the common soil bacterium, was found to be bound with plutonium Pu(IV), iron Fe(III), or uranium U(VI) when incubated with the siderophore desferrioxamine (John et al. 2001). The rate at which the microbial cells were able to consume the Pu-siderophore complex was much slower than the rate at which they degraded the Fe-siderophore complex by using transport proteins. The U-siderophore complex, however, was incapable of biodegradation. The mutual inhibition of uptake between Pu(IV)-DF and Fe(III)-DF, the two complexes, suggests that they might be competing for the same binding sites or transport mechanisms in the microbe (Kumar et al. 2007). Since Pu(IV)-DF and Fe(III)-DF complexes have comparable structures, it is likely that the bacterial uptake system can recognize both of these compounds (Kumar et al. 2007). These reports might have a significant impact on future bioremediation efforts and provide reliable predictions of the behavior of plutonium and other actinides in the environment (Kumar et al. 2007).

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Advantages and Disadvantages of Bioremediation

19.6.1 Advantages 1. Bioremediation, a natural process where microbes multiply and simultaneously degrade the contaminants, results in the production of cell biomass and releases water and carbon dioxide as bi-products. Typically, the biodegradative intermediates decreases with the removal of pollutants (Singh et al. 2022). 2. The bioremediation process is less tedious and may frequently be completed on-site, often without significantly disrupting daily operations. The necessity to move significant amounts of waste off-site is minimized, as are the associated risks to the environment and public health. 3. Pollutants are completely degraded and most of the hazardous substances are converted into harmless products thus eliminating the danger associated with future accountability linked to the treatment and disposal of contaminated material. 4. As compared to other traditional methods for hazardous waste cleanup, it is a more affordable approach. This is a crucial technique employed for cleaning up oil-contaminated areas (Montagnolli et al. 2014).

19.6.2 Disadvantages 1. The process of bioremediation is applicable only to biodegradable substances and includes the possibility that the byproducts of biodegradation might be more hazardous or enduring than the original chemicals. 2. Biological processes are frequently very specific. Important site criteria for success include the presence of metabolically competent microbial populations, acceptable environmental growth conditions, and appropriate quantities of nutrients and pollutants. 3. There is a need for extensive study to develop bioremediation techniques that are suitable for sites with complex combinations of pollutants unevenly distributed in the environment. 4. The technology is time-intensive and the scale-up from bench and pilot-scale research to large-scale field applications is challenging.

19.7

Microbial Bioremediation and Sustainable Environment Management

Unprecedented urbanization and associated anthropogenic activities have resulted in alarming pollution levels across the globe. Industrialization is a ‘necessary evil’ since on one hand, it is essential for emerging countries to modernize, evolve, and adapt more quickly; whereas on the other hand, industrialization results in environmental pollution, without sustainable control. Thus, industrialization has a negative

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impact on millions of individuals, the number only increasing exponentially with time, leading to numerous illnesses and fatalities. Environmental pollution involves the conscious release of toxic compounds into the ecosystem owing to human activity. These harmful substances exert a toxic effect and renders the environment unsustainable. Contaminated locations can be cleansed using many standard techniques. These methods include concealing polluted locations; moving contaminated soil to a landfill; excavation, processing, and transportation of hazardous materials; regardless of the fact that these strategies could pose considerable dangers and that finding additional landfills for the disposal of materials is an expensive and demanding process. Other approaches like hightemperature incineration and chemical degradation, involving base-catalyzed dechlorination and UV oxidation, are also employed. However, though these procedures have the potential to significantly reduce levels of an extensive range of contaminants, yet they have a number of drawbacks, including technical shortcomings and greater exposure to contaminants for site employees and adjacent households (Vidali 2001). It has been observed that a number of alternative physical and chemical processes have resulted in high removal efficiencies for phenolics. These include coagulation with alum or lime followed by adsorption on powdered activated carbon (PAC). However, these strategies produce a significant amount of toxic sludge without complete degradation of the contaminants (Mehta and Chavan 2009). A more preferable approach involves either complete elimination of the pollutants or conversion into less toxic forms which are naturally degradable. This strategy can be accomplished through bioremediation. Bioremediation is an interdisciplinary technology encompassing microbiology, engineering, ecology, and chemistry, and is realized to be safer, cleaner, more economical, and environmentally friendly, besides the prevalent fact that it can be applied to any site of operation. Microorganisms are crucial for the upkeep and sustainability of any ecosystem because they can adapt more quickly to environmental changes and assist in the degradation of the pollutants. The use of microbes in biodegradation is a helpful strategy for cleaning up, managing, and recovering the environment from pollution. They produce different metabolic enzymes, which can either directly destroy the chemical or transform the toxins into less dangerous or toxic intermediates (Dash and Das 2012). Numerous microbe species with various mechanisms are used in bioremediation, depending on the environment and the types of pollutants present (Boopathy 2000) either to eliminate the harmful pollutants from the environment or to break them down. Thus, microorganisms offer a beneficial platform to be employed for an improved bioremediation model owing to their versatility. Microbial bioremediation is successful when microbes interact with the environmental contaminants under the most optimum circumstances and within their niche (Riccardi et al. 2005). Organic carbon and an energy source are essential for these life forms to function irrespective of their varying cultivation requirements. Hence, in addition to common places like soil, water, and sediments, microorganisms also inhabit areas with high chemical contaminants. The effectiveness of undesirable chemical breakdown is influenced by the presence of competing biological agents, the availability of critical nutrients, and

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unfavorable external abiotic factors such as aeration, moisture, pH, temperature, and pollutant bioavailability. These characteristics may sometimes make biodegradation difficult under natural circumstances. However, bioremediation is effective when environmental factors promote microbial growth and activity. Bioremediation is far less expensive compared to other technologies that are often employed to clean up hazardous wastes. However, the process of bioremediation is still regarded to be at its nascent stage. Future anthropogenic activity-related pollution control can be made viable and sustainable through thorough investigation and development of more comprehensive methods of using microorganisms, in their niche, to degrade pollutants. Acknowledgement Devargya Ganguly is thankful to his mentor, Dr. Gargi Goswami, for her constant guidance and support. K.L.V. Prasanna and N. Swaroopa are thankful to Dr. Gargi Goswami for entrusting them with the responsibility and opportunity to write this chapter. Author Contributions Conceptualization and designing of the contents of the book chapter— Gargi Goswami. Drafting of the book chapter –Devargya Ganguly, K.L.V. Prasanna, Swaroopa Neelapu, and Gargi Goswami. Critical revision of the book chapter—Gargi Goswami and Devargya Ganguly. All the authors have read and approved the final draft of this chapter. Competing Interests The authors state that the work was carried out without the existence of any financial or commercial associations that could be interpreted as a probable conflict of interest.

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Uqab B, Mudasir S, Nazir R (2016) Review on bioremediation of pesticides. J Bioremed Biodegr 7: 1–5 van Dillewijn P, Caballero A, Paz JA, González-Pérez MM, Oliva JM, Ramos JL (2007) Bioremediation of 2,4,6-trinitrotoluene under field conditions. Environ Sci Technol 41:1378–1383 Varjani SJ (2017) Microbial degradation of petroleum hydrocarbons. Bioresour Technol 223:277– 286 Varjani S, Upasani VN (2019) Influence of abiotic factors, natural attenuation, bioaugmentation and nutrient supplementation on bioremediation of petroleum crude contaminated agricultural soil. J Environ Manag 245:358–366 Vidali M (2001) Bioremediation. An overview. Pure Appl Chem 73:1163–1172 Vijayaraghavan K, Yun Y-S (2008) Biosorption of C.I. reactive black 5 from aqueous solution using acid-treated biomass of brown seaweed Laminaria sp. Dyes Pigments 76:726–732 Vrionis HA, Anderson RT, Ortiz-Bernad I, O’Neill KR, Resch CT, Peacock AD, Dayvault R, White DC, Long PE, Lovley DR (2005) Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site. Appl Environ Microbiol 71:6308–6318 Wang J, Chen C (2006) Biosorption of heavy metals by Saccharomyces cerevisiae: A review. Biotechnol Adv 24:427–451 Wang YN, Cai H, Chi CQ, Lu AH, Lin XG, Jiang ZF, Wu XL (2007) Halomonas shengliensis sp. nov., a moderately halophilic, denitrifying, crude-oil-utilizing bacterium. Int J Syst Evol Microbiol 57:1222–1226 Wang ZW, Liang JS, Liang Y (2013) Decolorization of reactive black 5 by a newly isolated bacterium Bacillus sp. YZU1. Int Biodeterior Biodegrad Geomicrobial Ecotoxicol 76:41–48 Weymann DF (1995) Biosparging used in aquifer remediation. Pollut Eng 27 Xaaldi Kalhor A, Movafeghi A, Mohammadi-Nassab AD, Abedi E, Bahrami A (2017) Potential of the green alga Chlorella vulgaris for biodegradation of crude oil hydrocarbons. Mar Pollut Bull 123:286–290 Xie S, Yang J, Chen C, Zhang X, Wang Q, Zhang C (2008) Study on biosorption kinetics and thermodynamics of uranium by Citrobacter freudii. J Environ Radioact 99:126–133 Yan H, Pan G (2004) Increase in biodegradation of dimethyl phthalate by Closterium lunula using inorganic carbon. Chemosphere 55:1281–1285 Yang Q, Yang M, Pritsch K, Yediler A, Hagn A, Schloter M, Kettrup A (2003) Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates. Biotechnol Lett 25:709–713 Yu J, Wang X, Yue PL (2001) Optimal decolorization and kinetic modeling of synthetic dyes by pseudomonas strains. Water Res 35:3579–3586 Zahran SA, Ali-Tammam M, Hashem AM, Aziz RK, Ali AE (2019) Azoreductase activity of dye-decolorizing bacteria isolated from the human gut microbiota. Sci Rep 9:5508

Reuterin: A Broad Spectrum Antimicrobial Agent and Its Applications

20

Kiran S. Dalal, Sandip P. Patil, Girish B. Pendharkar, Dipak S. Dalal, and Bhushan L. Chaudhari

Abstract

Probiotic bacteria play a vital role in living animals including human health by helping in the digestion of foods and boosting the immune system. Hence, microbes with better properties are of interest. Some probiotic bacteria produce specific substances that can inhibit or inactivate other organisms which compete for nutrients and space. The β-hydroxypropionaldehyde (3-HPA) known as reuterin is produced by Lactobacillus reuteri. It has broad antimicrobial activity against potentially harmful microorganisms without adversely affecting the beneficial gut flora. Its production occurs under the anaerobic condition where the enzyme glycerol dehydratase facilitates the conversion of glycerol to reuterin by removing water molecules. The structure of reuterin contains both hydroxy and aldehyde functional groups. Reuterin is a low molecular weight water-soluble compound resilient to proteolytic and lipolytic enzymes, and remains active at a varied pH and hence finds applications in industries. This chapter focuses on reuterin production, stability, antimicrobial activity, and its applications.

K. S. Dalal · B. L. Chaudhari (✉) School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India e-mail: [email protected] S. P. Patil Department of Microbiology and Biotechnology, R. C. Patel Arts, Commerce and Science College, Shirpur, Maharashtra, India G. B. Pendharkar Department of Microbiology, Sadguru Gadage Maharaj College, Karad, Maharashtra, India D. S. Dalal School of Chemical Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_20

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Keywords

Reuterin · Lactobacillus reuteri · 3-hydroxypropionaldehyde · Antimicrobial activity · Probiotics

Abbreviations 1,3-PDO 3-HP 3-HPA FT-IR GRAS HCA HHP HPLC IC-PAD MRS RP-HPLC TVC

20.1

1,3-propanediol 3-hydroxypropionic acid 3-Hydroxypropionaldehyde Fourier-transform infrared spectroscopy Generally regarded as safe Hydroxycinnamic acid High hydrostatic pressure High-performance liquid chromatography Ion chromatography-pulsed amperometric detection De Man, Rogosa and Sharpe Medium Reverse phase high-performance liquid chromatography Total viable counts

Introduction

The intestinal microbiome has a vital role to play in the health of living organisms especially humans and animals by helping in the digestion of foods and boosting the immune system. Generally, short-chained fatty acids from nondigestible carbohydrates are synthesized by the bacteria present in the gastrointestinal tract which also synthesize secondary bile acids that modulate immune system in intestine (Louis et al. 2014). Several microorganisms present in food get entry into the gastrointestinal tract while a few of them could survive under these harsh conditions. The microflora present in the gastrointestinal tract get affected by food properties, nutritional changes, pharmaceutical drugs, and diseases like inflammatory bowel disease, obesity, cancer, and type II diabetes (Zhang et al. 2015). Hence, microbes with better properties having GRAS (Generally Regarded as Safe) status are of choice to be introduced with the food materials. The microbes with GRAS status are always nonpathogenic and can also help in healing diseased conditions (Anadón et al. 2014). These organisms are also called beneficial microbes and being supportive to life are referred to as probiotic microorganisms which can adhere to mucosa and intestinal cell lining through competition with pathogenic microbes. Probiotic microbes are also known to modulate activities related to enzymes in the intestinal tract and enhance immunomodulatory effects that can be used to manage dysbiosis (Plaza-Diaz et al. 2019).

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Some intestinal bacteria are known to produce specific substances that can inhibit or inactivate other organisms which compete for nutrition and space (Guarner and Malagelada 2003). These compounds are generally the bacteriocin type of compounds and organic acids like lactic acid, acetic acid, etc. Owing to the antimicrobial effect of these compounds, these are good agents to treat dysbiosis in diseased persons and among immunocompromised patients. Lactobacillus reuteri is known to produce reuterin having low molecular weight that exerts antimicrobial effect (Axelsson et al. 1989) which could be nonhydratedeuter, hydrated, or dimeric in nature. Reuterin has a broad antimicrobial activity against potentially harmful microorganisms. Reuterin can inhibit the growth of yeast, fungi, protozoa, and various harmful bacteria without adversely affecting the beneficial lactic acid bacteria of the gut flora. It is generally resistant to enzymes that have proteolytic nature, stable at a wide range of pH (El-Ziney et al. 1999), and active against Gram-positive as well as Gram-negative bacteria and other organisms like fungi and protozoa (Avila et al. 2014). It is also more stable throughout its refrigerated shelf life. The reuterin is more sensitive to Gram-negative microorganisms and resistant to Gram-positive microorganisms (Ortiz-Rivera et al. 2017; Cleusix et al. 2007). Reuterin exerts its antimicrobial effect through oxidative stress induced by thiol groups present in proteins which can also inhibit DNA synthesis (Schaefer et al. 2010; Cleusix et al. 2007). Interestingly, reuterin exerts its effect in cooperation with the other peptides having antimicrobial action, and hence reuterin can replace antibiotics and win against antibiotic-resistant pathogens (Hanchi et al. 2017). In a few reports, reuterin (8 AU/mL) in combination with nisin (100 IU/mL) proved to be effective against L. monocytogenes in milk (Arqués et al. 2004). The inhibitory activity of combining three natural preservatives such as nisin, reuterin, and the lactoperoxidase system was effective against the L. monocytogenes and S. aureus in cuajada (Arqués et al. 2008a). The analysis of reuterin system can be done by colorimetric methods, HPLC, LC-MS, IC-PAD, etc. The recent literature highlights the potential antimicrobial agents, including those derived from lactobacilli for the treatment of dental diseases (Yang et al. 2021). Reuterin (HOCH2CH2CHO) consists of both hydroxy and aldehyde functional groups can find its usage for the synthesis of various economically important chemical compounds like acrolein, 3-hydroxypropionic acid, 1,3-propanediol, or polymeric materials like acrylamide, and acrylic acid, and metabolites like malonic acid (Bauer et al. 2010a, b; Dishisha et al. 2015; Vollenweider and Lacroix 2004). Talarico et al. (1988) reported the production of reuterin during fermentation of glycerol from Lactobacillus reuteri. Reuterin synthesis is also catalyzed by B12dependent enzyme, glycerol dehydratase which converts glycerol to reuterin (Sriramulu et al. 2008; Daniel et al. 1998). Most of the reuterin is produced by Lactobacillus reuteri is the only lactic acid bacterium capable of producing a large amount of reuterin. However it can be also produced by other microorganisms like Enterobacter agglomerans, Klebsiella pneumoniae, Citrobacter freundii, Aerobacter aerogenes, and Clostridium butyricum. Sauvageot et al. (2000) showed reuterin synthesis by other strains like Lactobacillus collinoides obtained from French cider. Martín et al. (2005), found a new strain Lactobacillus coryniformis

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capable of producing reuterin isolated from goat’s milk cheese. The role of reuterin from L. reuteri in gastrointestinal immunity is not yet fully elucidated. The detailed mechanistic studies are essential for knowing the role of reuterin to a healthy intestinal environment. The mechanism of action of probiotics includes increased adhesion to the intestinal mucosa and epithelial cells, suppression of the growth of pathogenic microorganisms, synthesis of natural antimicrobial compounds, and modulation of immune function (Bermudez-Brito et al. 2012). One of the most studied organisms that produce reuterin is Lactobacillus reuteri, which can harbor the colon of mammals. In humans, L. reuteri is found in different body sites, including the digestive and urinary tract, skin, and breast milk (Mu et al. 2018). Obligate heterofermentative L. reuteri is found in gastrointestinal tract (Oh et al. 2010; Walter 2008a, b) which has also been reported as advantageous for conferring immunemodulatory effect (Greifova et al. 2017), reducing body cholesterol (Jones et al. 2012), improving gut health by acting as a barrier (Li et al. 2019), anti-inflammatory effect (Hsieh et al. 2021), producing antimicrobial substances (Axelsson et al. 1989), etc. They have gained an ability to survive under the stress of digestive enzymes like proteases, lipases, etc., which can harbor and grow in the digestive tract. Apart from that they may produce many important antimicrobial substances that control the growth, binding, and invasion of pathogenic microorganisms. The Lactobacillus strains support epithelial cells and growth helping inhibition of proinflammatory cytokines such as TNF-alpha, and increase protective cytokines such as IL-10 and TGF-β levels (Patel and Pyrsopoulos 2019). Several strains of Lactobacillus reuteri can produce reuterin (Vollenweider and Lacroix 2004). However, L. reuterin is a main reuterin-producing strain having major applications as a remedial culture for better health of animals and human beings. Kandler et al. (1980), proposed to classify the Lactobacillus fermentum biotype II strains into Lactobacillus reuteri as the new species identified by Reuter and Lerche; the German scientists who had worked on these organisms. The L. reuteri generally dwells in the gastrointestinal and urogenital tracts of humans and other animal species (Molin et al. 1992; Naito et al. 1995; Rodriguez et al. 2003). The strain was also often isolated from fermented foods and milk products (Vollenweider and Lacroix 2004; Langa et al. 2013). L. reuteri received the status of “generally regarded as safe” (GRAS) by the United States’ Food and Drug Administration in 2008 (FDA 2008). This species is predominantly found in the intestines of animals and humans (Casas and Dobrogosz 2000). Based upon its dominant nature of conferring health benefits for humans and animals, the scientific community thought of finding out more advantageous effects of L. reuteri related to intestinal health. Ratcliffe et al. (1986) reported that fermentation of yogurt and milk by using L. reuteri could reduce the coliform count and increase the Lactobacillus count throughout the gut. After that Talarico et al. (1988), reported the advantages of reuterin which was a by-product of glycerol fermentation (Doleyres et al. 2005). A recent experiment showed that reuterin system can increase the shelf life of raw milk which is an alternative method for the preservation of raw milk (Kumar et al. 2020). In this experiment reuterin alone or in combination with bacteriocins, effectively

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Fig. 20.1 Reuterin system (3-HPA) biosynthesis from glycerol and its different components formed by in situ in the aqueous solution

regulate the microbial load in milk and prolong its shelf life. A complex reaction pathway in which reuterin system formed by starting glycerol plays a central role in the formation of different components in the aqueous solution Fig. 20.1.

20.2

Synthesis and Composition of Reuterin

Lactobacillus reuteri strain is a probiotic strain that can convert glycerol to reuterin where glycerol as the electron acceptor, is the main ingredient or intermediate required for the synthesis of reuterin. The glycerol dehydratase enzyme along with co-enzyme B12 is responsible to convert glycerol to reuterin under anaerobic condition (Toraya 2000; Zeng et al. 1993). In presence of water, reuterin combines with water forming HPA hydrate and by the process of dehydration, it forms acrolein (Talarico and Dobrogosz 1989). Physico-chemical factors like pH, oxygen and glucose concentration, temperature, incubation time, growth stage of cells, and biomass concentration play a crucial role in reuterin biosynthesis. However, if the excess glucose concentration was supplied during the conversion of glycerol to reuterin production was inhibited because of the NAD+ dependent oxidoreductase produced during the conversion of glycerol to reuterin. The enzyme propanediol dehydrogenase converts reuterin to 1,3-propanediol based on the availability of NAD+. Subsequently, reuterin gets transformed into 3-hydroxypropionic acid by the enzyme aldehyde dehydrogenase.

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Morita et al. (2008), compared genome analysis of two strains of reuterinproducing lactobacilli viz. L. reuteri from human feces, and L. fermentum from fermented plant material. It was observed that pdu-cbi-cob-hem gene cluster could be responsible for the production of reuterin and cobalamin. In the same investigation, the glycerol dehydratase enzyme encoded by gupCDE in L. reuteri could be associated with the synthesis of reuterin. Santos et al. (2011), provided the first experimental evidence of having genes responsible for glycerol utilization and vitamin B12 synthesis present in Lactobacillus reuteri, where a regulatory protein or gene PocR (lreu_1750) modulates glycerol utilization and vitamin B12 synthesis. This finding could be used to improve the production yield of economically important products such as vitamin B12, 1,3-propanediol, and reuterin. The reuterin system is pH-dependent where under the acidic conditions monomer, the hydrated monomer, the cyclic dimer, and acetal forms are found while under the basic condition, mainly monomer and aldol dimers and trimers are formed. (Stevens et al. 2011). In organic solutions where water is absent, the dimeric and polymeric forms are predominant (Talarico and Dobrogosz 1989; Vollenweider et al. 2003).

20.3

Production

The synthesis of reuterin can be carried out by chemical and biological methods, whereas biological methods have several advantages such as mild reaction conditions, high yield, cost-effectiveness, room temperature, normal pressure, and no formation of by-products. In contrast, its chemical synthesis is much more complicated involving high reaction temperatures, high pressures, expensive processes, and the formation of by-products (Vollenweider and Lacroix 2004) which may include toxic intermediates and may require additional purification steps. The chemical methods developed by Shell and Degussa (now owned by DuPont) are known for reuterin production where petrochemical products mainly propylene and ethylene are used. In most of the chemical methods of synthesis of fine chemicals, harsh conditions are generally used and so are not preferred. Production of reuterin by microbial fermentation involving renewable resources like glycerol, glucose, as substrates are preferable provided that the desired productivity is achieved. Glycerol is one of the by-products of biodiesel production process and is a promising raw material for industrial fermentations (Clomburg and Gonzalez 2013). The biosynthesis of reuterin system is achieved by the in situ two-step process. Under anaerobic conditions, the L. reuteri metabolizes the glycerol in the MRS medium and converts it into reuterin, but bringing the low yields due to the immediate reduction to 1,3-PDO by a NADH-linked dehydrogenase. Hence, a two-step process is proposed where in the first step biomass from L. reuteri in MRS medium is obtained which in the second step is cells incubated in glycerolwater aqueous solution at room temperature at 37 °C under normal pressure (Talarico et al. 1988). Various factors such as temperature, substrate glycerol and biomass concentration, pH, cell age, and incubation time could affect the reuterin

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production seriously. Doleyres et al. (2005), studied the various factors for the reuterin using resting cells of Lactobacillus reuteri and found that a very high reuterin concentration of 235 ± 3 mM was obtained after 45 min of incubation at 30 °C in 200–400 mM glycerol at an initial biomass concentration of 1.6 ± 0.3 × 1010 cfu/mL. Lüthi-Peng et al. (2002) reported the two-step fermentation process to produce reuterin by using the resting cells of L. reuteri (ATCC 53608). Using this method, 170 mM reuterin was produced from 200 mM of glycerol aqueous medium within 2 h of incubation. The toxic effects of reuterin on the cells of Lactobacillus reuteri reduce the production of reuterin in the medium. To overcome this inhibitory effect of the product Vancauwenberge et al. (1990) developed a new strategy for the production of reuterin in the presence of semicarbazide. This method could reduce the inhibitory effects on cells and increase the yield of reuterin. In another study, Stevens et al. (2013), reported the addition of bisulfite during reuterin production by resting cells L. reuteri specifically at pH 5.8, and increased glycerol conversion to reuterin. Furthermore, the cells were active after three consecutive production cycles. To enhance the production of reuterin, the producing bacterial strain shall tolerate its high amount so as to avoid feedback inhibition while the use of modern techniques such as genetic engineering could be effectively used to avoid the undesirable by-product formation. Table 20.1 represents the biotechnological production of 3-hydroxypropionaldehyde using the different bacterial strains.

20.4

Mode of Action

The aldehyde and hydroxyl groups are present in the structure of reuterin. In presence of water, the aldehyde group of reuterin being highly reactive forms supplementary compounds such as 3-HPA dimer, hydrate, and the toxic compound acrolein (Fig. 20.1). Because of the complexity of reuterin system, the mode of action by which reuterin exerts its antimicrobial effects is difficult to determine and yet not clear. Schaefer et al. (2010), reported the aldehyde form of reuterin is the bioactive agent that induces the oxidative stress response to bacteria by modifying thiol groups and reducing glutathione. The activity of reuterin is rendered by the presence of aldehyde group and not the toxic acrolein produced in the water. The dimeric form of reuterin may also exert antimicrobial activity by blocking the enzyme ribonucleotide reductase, which is required for DNA synthesis (Vollenweider et al. 2003). Talarico and Dobrogosz (1989) explained the broadspectrum effects of reuterin possibly due to the inhibition of ribonucleotide reductase. To address the question of how the reuterin system exerts its antimicrobial effect on the bacteria and biological molecules Vollenweider et al. (2003), studied the dynamics of the reuterin system (mainly composed of a dimer, and hydrate) using electrospray ionization mass spectrometry and 13C NMR. The analysis showed that reuterin system consists of a concentration-dependent distribution of the three compounds. The 13C NMR spectrum analysis suggested that the high concentration HPA system, HPA dimer was predominant than HPA hydrate and 3-HPA (reuterin).

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Table 20.1 Biotechnological production of reuterin (Vollenweider and Lacroix 2004) Sr no. 1

Name of bacteria Lactobacillus sp.

Source –

Glycerol 9 g/L

Reuterin 7.1 g/L

Klebsiella pneumoniae NRRL B-199 (ATCC 8724) Enterobacter agglomerans CNCM1210 Lactobacillus reuteri 12,002 Lactobacillus reuteri ATCC 53608

–

30 g/L

13.1 g/L

–

725 mM

30 mM

Pig intestine –

25% (wt/vol)

4000 AU/mL

97 mM

107.5 mM

6

L. reuteri (ATCC 53608)

–

65.95 mM

7

Lactobacillus coryniformis CECT 5711 Lactobacillus reuteri CECT 925 T Limosilactobacillus reuteri SD2112 Lactobacillus reuteri INIA P579

Goat’s milk cheese Pig faeces

20 mM glucose, 200 mM glycerol 250 mM

–

Martín et al. (2005)

250 mM

–

Human intestine –

200 mM

170 mM

100 mM

1.3 M

11

L. reuteri INIA P572 & L. reuteri INIA P579

–

50 mM

12

Lactobacillus reuteri BPL-36

360 mM

13

Lactobacillus reuteri PRO 137 L. reuteri BR201

Human infant fecal sample –

5.5 mM in cheese 1.5 mM in yogurt 89.63 mM

Rodriguez et al. (2003) Cleusix et al. (2007) Montiel et al. (2014b) Langa et al. (2013)

45.7 mM

300 mM

Infant feces –

500 mM

378 mM

235.9 mmol

250 mmol

–

6.8 g/L

13.4 g/L

Caucasian male’s faeces

350 mmol/L

212.74 mmol/ L

2

3

4 5

8 9 10

14 15 16 17

L. reuteri CGMCC 1.3264 L. diolivorans LMG 19668 Limosilactobacillus reuteri DPC16

Reference Slininger et al. (1983) Slininger and Bothast (1985) Barbirato et al. (1996) El-Ziney et al. (1999) OrtizRivera et al. (2017) Luthi-Peng et al. (2002a, b)

Mishra et al. (2012) Tobajas et al. (2007) Ju et al. (2021) Soderling et al. (2011) Castellani et al. (2021) Sun et al. (2022a)

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However, at a lower concentration of HPA system HPA hydrate was predominant. Later, Vollenweider et al. (2010), proposed that the antimicrobial activity of 3-HPA leads to the depletion of free SH- groups in GSH and proteins, resulting in the disparity of the cellular redox status leading to cell death. In addition, the reuterin system containing acrolein is the active compound responsible for the detoxification of HCAs conjugation, a possible human carcinogen, and possesses strong antimicrobial activity attributed to reuterin (Engels et al. 2016). However, a detailed study is needed to elucidate the mode of action of reuterin in vivo as well as in vitro. The reuterin system is made up of different compounds, their exact concentration in an aqueous solution and the mode of action of individual compounds and their cumulative action needs to be accurately determined.

20.5

Stability

The stability of reuterin depends on several parameters, such as pH, salt concentration, and temperature. Reuterin undergoes spontaneous dehydration in aqueous solutions and forms acrolein, which is a toxic substance that brings bitterness in wine (Bauer et al. 2010a, b). The reuterin system is highly soluble in water and is resistant to proteases, nucleases, and lipases (Stevens et al. 2011). Ju et al. (2021), measured the effect of temperature and time on the amount of reuterin degradation into acrolein where they observed that degradation was temperature dependent which increased with increase in temperature. Similar results were obtained by Engels et al. (2016), who observed no acrolein production from reuterin formed at 4 °C. Castellani et al. (2021) determined the stability of reuterin in the sterile-filtrated reuterin supernatant even after several freeze-thaw cycles of -20 °C and 4 °C remained stable until day 35 followed by a slow decrease. Soltani et al. (2021), evaluated the stability of reuterin under the stressed gastrointestinal conditions where reuterin could sustain (>90%) without either degradation or bioconversion. The high stability of reuterin is beneficial and a desired characteristic for the host in suppressing the pathogens in the gastrointestinal tract where reuterin is present. Lüthi-Peng et al. (2002), reported the stability of reuterin system in water, milk, and MRS medium. They observed that reuterin remained unaltered upon storage at refrigeration temperature as well as at 15 °C when stored in water for 48 h. While in the case of milk and MRS medium, rapid loss of reuterin was found at higher temperature of incubation. Hence, the reuterin system is having higher stability in an aqueous solution than in milk and MRS medium. Yogurt is a widely consumed dairy product across the world. Reuterin can be added externally to food products as a purified ingredient or reuterin-producing bacteria could be supplemented in yogurt that could produce reuterin in situ. Langa et al. (2013) reported in situ production of reuterin by Lactobacillus reuteri strains in cheese and yogurt. In view of higher production of reuterin, addition of glycerol in yogurt or in any fermented milk product could be an option, but cannot be an alternative for higher reuterin production. Pilote-Fortin et al. (2021), investigated the stability of reuterin when added to milk before yogurt processing where reuterin concentration decreased by 90% at

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Table 20.2 Stability of the reuterin system under different conditions (Sun et al. 2022b) Media Water, milk, and MRS

Reuterin concentration 2.6–6.0 mM

Water at 37 °C

Not stated

Cell culture

1.3 mM

In vitro model

270 mM

Yogurt

10 mM

Sorghum silage and glycerol for L. reuteri SO23 Glycerol aqueous solution Coconut milk and glycerol

Not stated

4 mM 0.14 mM

Stability No loss of reuterin at 4 °C and 15 °C till 48 h Reuterin was much more unstable in milk and MRS+ Stable at pH 2 and pH 6.5 not stable Stable for 24 h

Method of detection Colorimetric assay, Derivative spectroscopy

HPLC and FT-IR HPLC

Stable at stressful GI conditions Stable 4° and 21 °C at 15 days Stable at 21 days

RP-HPLC

Stable at 4 °C for 4 week Stable at 4 °C for 30 days

HPLC

HPLC Not stated

Photometric method

References Luthi-Peng et al. (2002a, b)

Talarico et al. (1988) FernándezCruz et al. (2016) Soltani et al. (2021) Pilote-Fortin et al. (2021) González et al. (2019) Ju et al. (2021) Mauro and Garcia (2019)

21 °C, but this degradation was slower at 4 °C. Some examples on reuterin stability studies are depicted in Table 20.2. Until now to our knowledge, natural resistance mechanism to reuterin has not been reported by any bacteria while no genes have been identified that conferred resistance to other organisms. This validates the role of reuterin as an antimicrobial agent as well as its stability.

20.6

Toxicity

The reuterin system contains acrolein which is a main toxicant causing its adverse effects on DNA, proteins, endoplasmic reticulum, immune system, membrane damage, and also its oxidative stress (Zhang et al. 2018). When compared with acrolein, reuterin is less toxic. Reuterin exhibits antimicrobial activity against pathogens present in foods and so can be applied for food preservation (Sun et al. 2022a, b). Furthermore, the concentration of reuterin 40 mg/mL has no negative effect on skin (Soltani et al. 2022). The effect of reuterin activity on human health is perilous and so usages in the food materials and pharma industries remain questionable. In vitro studies of reuterin show no hemolytic activity and human epithelial cell line Caco-2 remains viable up to 10.8 mM concentration of reuterin (Soltani et al.

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2021). The knowledge about the toxicity of reuterin is inadequate that intensifies the demand for more research. However, reuterin producer Lactobacillus reuteri is having GRAS status and hence is the organism of choice to be used in food products in the capacity of a probiotic microorganism. Additionally, the jeopardy posed by the adverse interactions between dietary compounds and drugs that are metabolized by cytochromes P450 enzymes could be a matter of concern (Fernández-Cruz et al. 2016). This demands the judicious experiments to be conducted to understand the reuterin toxicity and its metabolic fate or questionable interactions to validate reuterin as a food biopreservative. Although reuterin is a good candidate in food and pharma sector as a preservative, it is not yet commercially available possibly due to its incompetency in achieving the regulatory requirements. Reuterin although observed to be produced by GRAS Lactobacilli; it needs to attain safety regulations for food, pharma, and veterinary applications.

20.7

Applications

Owing to good antimicrobial activity and high stability, reuterin is applied as a food preservative in numerous types of foods under food processing conditions. It has a high potential to increase the shelf life of food without affecting quality and any kind of harm. Pathogenic microorganisms are the main cause of different foodborne diseases after the intake of contaminated foods. The food preservatives prevent the growth of pathogenic microorganisms and help in preserving the food over a period without altering its properties. Recent techniques have been developed to ensure safety, nutritional importance, and sensory characteristics of foods (Quinto et al. 2019). The antimicrobial activity of purified reuterin was evaluated against L. monocytogenes, a Gram-positive organism found in cold-smoked salmon where the pathogens were inactivated under moderate temperature and extended the shelf life of cold-smoked salmon (Montiel et al. 2014b). The reuterin system was also tested against the contaminant Escherichia coli in ground beef under refrigerated storage conditions for 20 days where the contaminant was eliminated in ground beef (Muthukumarasamy et al. 2003). A variety of methods have been reported for the decontamination of meat surfaces. Hernández-Carrillo et al. (2021), developed an edible coating system using pectin, lemon essential oil, and reuterin. The author employed this system in strawberry preservation for 31 days of storage. He observed that reuterin-based coating could inhibit Penicillium conidia for more than two logarithmic cycles without quality reduction. Montiel et al. (2014a), employed a new method using reuterin in combination with high hydrostatic pressure of 450 MPa for 5 min which extended the shelf life of cold-smoked salmon for about 35 days when stored at 4 °C. The Lactobacillus reuteri strain 12,002 was isolated from a small pig intestine that could produce reuterin in the two-step fermentation process which was checked as a preservative at a concentration of 100 AU/g for meat storage especially raw ground pork that resulted in an inhibition of the growth of E. coli after 1 day of storage at 7 °C while reuterin at a concentration of 250 AU/g reduced the number

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Table 20.3 Preservation of animal products using the reuterin system (Sun et al. 2022a, b) Reuterin amount 16 mM

Meat & fish product Cooked ham

16 mM

Cooked ham

5 mM

Raw chicken legs Chicken carcasses Sea bass fillets

0.5 mM 0.49, 0.55 g/L Spray dried reuterin 4.9 mM

Antimicrobial activity against L. Monocytogenes, S. Enteritidis, and E. coli O157:H7 TVC

Mix with HHP at 450 MPa for 5 min HHP at 450 MPa for 5 min

TVC

Lactic acid (RL), microcin J25

Salmonella enterica

Lactic acid or microcin J25 None

Pseudomonas spp.

Tuna burger

Pseudomonas spp.

Chicken meat

Campylobacter jejuni

Modified atmosphere packaging (MAP) None

Reference Montiel et al. (2015) Montiel et al. (2016) Zhang et al. (2022) Zhang et al. (2021) Angiolillo et al. (2018) Angiolillo et al. (2017) Asare et al. (2020)

growth of L. monocytogenes after 1 week of storage at 7 °C (El-Ziney et al. 1999). Kuleaşan and Çakmakçı (2002), observed that reuterin along with nitrate inhibits the growth of L. monocytogenes on the surface of beef sausages. Table 20.3 shows recent examples of reuterin system as a preservative in meat and fish products. The reuterin-producing probiotic strain Lactobacillus reuteri is commonly used as a sustained source of reuterin in food products. The production of reuterin in situ by supplementation of food plus food-grade glycerol provides a continuous supply of reuterin to act as antimicrobial compound. The antimicrobial potential of reuterin has been successfully employed in milk, yogurt, and cheese, however its application is not yet widely accepted. Arqués et al. (2008b) studied the inhibitory activity of reuterin combined with lactoperoxidase system on E. coli and S. enterica. It exerted a strong bactericidal activity at 4° as well as complete elimination of pathogenic bacteria in milk at 8 °C. Langa et al. (2013) explored in situ reuterin production in a yogurt model system, in which L. reuteri survived to produce reuterin. The displayed reuterin concentrations up to 5.5 mM in cheese and up to 1.5 mM in yogurt were reported. Ortiz-Rivera et al. (2017) extracted the reuterin from Lactobacillus reuteri ATCC 53608 in fermented milk product which could inhibit pathogenic and spoilage bacteria and hence used as a biopreservative without altering quality parameters. The reuterin 107.5 mM in glycerol aqueous solution and in fermented milk product of 33.97 mM obtained during this study. The optimized concentration of reuterin in cheese could control unwanted microorganisms and maintain the quality of dairy products but at the cost of developing of red color in cheese, owing to the supply of

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glycerol in the medium (Martín-Cabrejas et al. 2017), which could be unacceptable. Alternatively in situ production of reuterin together with glycerol prevented the late blowing defect of cheese caused by Clostridium tyrobutyricum (Gómez-Torres et al. 2014). In another study, the symbiotic effects of reuterin blended with nisin, lacticin 481, or enterocin AS-48 could inactivate the growth of Listeria monocytogenes and Staphylococcus aureus completely in milk (Arqués et al. 2011). The reuterin system containing acrolein with higher concentrations was tested and observed to reduce the microbial population of Enterobacteriaceae, yeasts, and molds in minimally processed fresh-cut lettuce during 13 days of refrigerated storage (4 °C) (Asare et al. 2018). Some of the examples of reuterin used as healthpromoting activities are listed in Table 20.4. In vitro studies conducted by Kim et al. (2022) examined the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus strains on human colorectal carcinoma RKO cells and in vivo xenograft models. Both strains show strong anti-tumor effects in human colorectal carcinoma RKO cells. Furthermore, in vitro studies show that L. reuteri FLRE5K1 isolated from healthy mouse kidneys significantly reduce the incidence of melanoma cancer and prolonged the survival of tumor-carrying mice (Luo et al. 2020). In another study conducted by Bell et al. (2022) examined that reuterin could decrease colon tumor growth in healthy intestinal microbiome through selective protein oxidation and inhibits ribosomal biogenesis and protein translation in vivo and in vitro. The consumption of probiotic lozenges L. reutericontaining lozenges has also improved pregnancy gingivitis in women (Schlagenhauf et al. 2016).

20.8

Conclusion

The aqueous system of reuterin consists of a different derivative that shows remarkable activities toward the various microorganisms. Various methods have been reported to produce reuterin. Many times, biotechnologically produced biomolecules have been advantageous compared to chemical methods and so true in the case of reuterin also. The reuterin system synthesis gets affected by various factors such as temperature, cell age, pH and medium composition, etc. Reuterin system may also find more applications in health care, the food sector as well as the chemical industry as feedstuff. Due to the complex nature of the reuterin, an effective method to analyze all components of the system is necessary. The mode of action of reuterin also needs to be elaborated to know the exact mechanism. The above problems need to be solved for further research on the production of reuterin. However, the antibiotic nature of the reuterin molecule is the most lucrative characteristic since it can inhibit the growth of fungi, pathogenic bacteria, and viruses, which makes it attractive for use in the food preservation and healthcare sector. Most of the reuterin producers are probiotic bacteria such as Lactobacillus spp. which have GRAS status and are safe for consumption by humans.

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Table 20.4 Various health-promoting activities of reuterin (Sun et al. 2022a, b) Health-related activities Oral health

Oral health

Reuterin system Reuterinrelated compounds (RRC-01, 02, and - 03) In situ

Studies done by In vitro

Activities against Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277

Reference Fujiwara et al. (2017)

In vitro

Van Holm et al. (2022)

Bertin et al. (2017) Xu et al. (2022)

In situ

In vitro

P. gingivalis P. intermedia F. Nucleatum A. Naeslundii A. viscosus (anti-biofilm) Campylobacter spp.

In situ

In vitro

E. coli O157:H7

Purified reuterin isolated from Lactobacillus reuteri (LR 21) In situ production of reuterin from Lactobacillus reuteri DPC16

In vitro

Clostridium perfringens

In vitro (simulated gastrointestinal tract)

Trypanocidal agent

In situ

In vitro

Escherichia coli O157: H7, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes Trypanosoma brucei

Trypanocidal agent

In situ

In vivo

Trypanosoma brucei

Chicken intestinal microbiota Bovine gastrointestinal tract Human and chicken gastrointestinal tract Gastrointestinal tract

20.9

Asare (2019)

Zhao et al. (2012)

Yunmbam and Roberts (1992) Yunmbam and Roberts (1993)

Future Perspectives

The reuterin is a multi-component system produced by Lactobacillus reuteri strain in the majority that exerts its broad-spectrum antimicrobial action. But, most of the reported papers show only antibacterial and antifungal activity against diverse microbes and fail to investigate the interaction or reaction of various food components with reuterin. Further research is necessary to elucidate the behavior of reuterin components with the food ingredients. The productivity of reuterin needs

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to be increased by optimizing the culture condition, purification, stability, etc., through the use of genetic engineering techniques and overall fermentation methods. Reuterin in combination with other antimicrobial molecules may increase its antimicrobial effects on different pathogenic bacteria. The practical applications of reuterin system in food and medical fields need more data on their toxicity for safe use. In the agriculture sector food safety could be effectively addressed through reuterin system. Although the reuterin system has many health-promoting activities, it still lacks the data on clinical studies. The reuterin system has potential to commercialize in food products. Further research on exploring the efficacy of reuterin system as a therapeutic agent against various diseases is essential. Acknowledgments The authors would like to thank Rajiv Gandhi Science & Technology Commission, Mumbai, for financial support and the FIST grant of DST, New Delhi, for infrastructural facilities provided to the School of Life Sciences and School of Chemical Sciences of KBCNMU.

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Van Holm W, Verspecht T, Carvalho R, Bernaerts K, Boon N, Zayed N, Teughels W (2022) Glycerol strengthens probiotic effect of Limosilactobacillus reuteri in oral biofilms: a synergistic synbiotic approach. Mol Oral Microbiol Vancauwenberge JE, Slininger PJ, Bothast RJ (1990) Bacterial conversion of glycerol to betahydroxypropionaldehyde. Appl Environ Microbiol 56(2):329–332 Vollenweider S, Lacroix C (2004) 3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production. Appl Microbiol Biotechnol 64(1):16–27 Vollenweider S, Grassi G, König I, Puhan Z (2003) Purification and structural characterization of 3-hydroxypropionaldehyde and its derivatives. J Agric Food Chem 51(11):3287–3293 Vollenweider S, Evers S, Zurbriggen K, Lacroix C (2010) Unraveling the hydroxypropionaldehyde (HPA) system: an active antimicrobial agent against human pathogens. J Agric Food Chem 58(19):10315–10322 Walter (2008a) L. reuteri is an obligate hetero-fermentative lactobacilli and a natural inhabitant of the gastrointestinal (GI) tract of animals and humans Oh et al., 2010 Walter J (2008b) Ecological role of lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol 74(16):4985–4996 Xu Y, Wang Y, Ding X, Wang J, Zhan X (2022) Inhibitory effects of reuterin on biofilm formation, quorum sensing and virulence genes of Clostridium perfringens. LWT 162:113421 Yang KM, Kim JS, Kim HS, Kim YY, Oh JK, Jung HW et al (2021) Lactobacillus reuteri AN417 cell-free culture supernatant as a novel antibacterial agent targeting oral pathogenic bacteria. Sci Rep 11(1):1–16 Yunmbam MK, Roberts JF (1992) The in vitro efficacy of reuterin on the culture and bloodstream forms of Trypanosoma brucei brucei. Comp Biochem Physiol C Comp Pharmacol Toxicol 101(2):235–238 Yunmbam MK, Roberts JF (1993) In vivo evaluation of reuterin and its combinations with suramin, melarsoprol, DL-alpha-difluoromethylornithine and bleomycin in mice infected with Trypanosoma brucei brucei. Comp Biochem Physiol C Comp Pharmacol Toxicol 105(3): 521–524 Zeng AP, Biebl H, Schlieker H, Deckwer WD (1993) Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzym Microb Technol 15(9):770–779 Zhang YJ, Li S, Gan RY, Zhou T, Xu DP, Li HB (2015) Impacts of gut bacteria on human health and diseases. Int J Mol Sci 16(4):7493–7519 Zhang J, Sturla S, Lacroix C, Schwab C (2018) Gut microbial glycerol metabolism as an endogenous acrolein source. MBio 9(1):e01947–e01917 Zhang L, Ben Said L, Diarra MS, Fliss I (2021) Inhibitory activity of natural synergetic antimicrobial consortia against Salmonella enterica on broiler chicken carcasses. Front Microbiol 12: 656956 Zhang L, Said LB, Diarra MS, Fliss I (2022) Effects of bacterial-derived antimicrobial solutions on shelf-life, microbiota and sensory attributes of raw chicken legs under refrigerated storage condition. Int J Food Microbiol 383:109958 Zhao Q, Maddox IS, Mutukumira A, Lee SJ, Shu Q (2012) The effect of cell immobilization on the antibacterial activity of Lactobacillus reuteri DPC16 cells during passage through a simulated gastrointestinal tract system. World J Microbiol Biotechnol 28(10):3025–3037

Seaweed Farming: An Environmental and Societal Perspective

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Meenakshi Singh, Sahil Kapoor, Trisha Bagchi, Sanchita Paul, and Surojit Kar

Abstract

Asia holds the top position in seaweed production creating a pathway for India to utilize its available coastal shorelines and excel in seaweed farming in the coming years. Given the small number of commercial species cultivated on large scale to meet the rising global demand, India can fill the emerging gap by extending the commercialization of unexplored or wild seaweeds to generate revenue under the umbrella of blue biotechnology. Keeping this in mind, the present review discusses the upstream processing of seaweeds, involving different farming techniques, harvesting strategies, extraction methods and purification of bioactive compounds. Further attention is drawn to various applications for pharmaceutical, food and health sector that covers environmental aspects like pollution management and mitigation measures as well as existing societal benefits. The popularity of seaweed products is rising across geographic regions that can be leveraged by promoting training programs on cultivation. Many state agencies, government institutes and private bodies support local farmers with funding opportunities and technical expertise. The blue resolution programme facilitated in India will pave the way forward to unlock the full potential of natural resources in coastal regions.

M. Singh (✉) Department of Ecology and Biodiversity, Sustaina Greens LLP, Vadodara, Gujarat, India S. Kapoor Department of Botany, Goswami Ganesh Dutta Sanatan Dharma College, Chandigarh, Punjab, India T. Bagchi · S. Paul · S. Kar Department of Botany, West Bengal State University, Barasat, West Bengal, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_21

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Keywords

Seaweed farming · Industrial applications · Societal benefits · Environmental aspects · Training programs

21.1

Introduction

Seaweeds are marine, benthic algal macrophytes with diverse phylogenetic lineages but are superficially classified into three classes namely Chlorophyta, Phaeophyta and Rhodophyta (Ganesan et al. 2019). Seaweeds are vital source of global aquaculture and fall under the most traded food commodity after crabs, barbels and other cyprinids (Gao et al. 2022). They play multiple roles in ecological, provisional and regulatory services, such as eutrophication mitigation, CO2 sequestration, greenhouse mitigation, shoreline protection and habitat provision (Pörtner et al. 2019). The last few decades have witnessed few constraints in scaling up the sustainable production of seaweed to feed the ever-growing human population (Hamid et al. 2018; Liu et al. 2019). Moreover, the exponential surge in global demand for seaweed has further increased after the pandemic because of its high nutritional potential. According to a recent study conducted by Research and Market in 2021, the seaweed market is forecasted to reach a CAGR of $23.2 billion by 2028 (https:// www.researchandmarkets.com/r/58s3hh). At present 49 countries are involved in seaweed cultivation, however, Asian countries account for 97% of global manufacturing, majorly from China, Japan, Indonesia, and Korea. As per Persistence Market Research’s study on the global seaweed cultivation market, between 2022 and 2032, the market is expected to rise at a CAGR of 7.8% (https://www. persistencemarketresearch.com/market-research/seaweed-cultivation-market.asp). India is ranked 13th with 5.3 thousand wet tonnes of seaweed harvested, and about 0.2% of the world’s seaweed production (TIFAC 2018). With a coastline coverage of approx. 8000 km, India has invested in the blue revolution to achieve a target of at least one million tonnes a year by 2025 in the seaweed farming sector (Jayaprakash et al. 2017). The Indian coastal areas with suitable conditions for seaweed farming exist in Tamil Nadu, Lakshadweep, Andaman Nicobar Islands, Gujarat, Kerala, Telangana, Orissa, Karnataka, Andhra Pradesh and Goa (Ganesan et al. 2019). The investment in the seaweed manifesto will benefit to overcome climate-induced mortality by developing heat-resistant strains and establishing a functional seed bank, and also by creating awareness about lost folk remedies and coastal cuisines using seaweeds. This practice will remarkably influence the local fisherman community and revive age-old indigenous food habits. Also, it would enable sustainable green jobs in rural coastal areas and generate revenue by trading seaweed-based products (https://foodtank.com/ news/2021/01/indias-blue-revolution-targets-investments-in-seaweed/). Hence, government initiatives and joint efforts of policymakers, stakeholders and experts will create lucrative opportunities for seaweeds market segments, specifically vegan

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food and beverage, extraction of hydrocolloids, animal feed, agriculture, and other applications for human consumption (Marcus 2013). The remarkable journey of seaweed as a “Medical food” for its pharmacologically and biologically vital compounds had created a progressive impact on food quality. The presence of a variety of minerals, vitamins, protein, bromine, iodine, and bioactive ingredients, namely polysaccharides, minerals, fatty acids, vitamins, pigments, etc. makes seaweed a popular food substitute and herbal medicine (Mostafavi and Zaeim 2020). Seaweeds are a good source of polysaccharides such as fucoidan, agar, carrageenan, ulvan, alginate, and laminarin, which are used in different industries like food, textile and dairy pharmaceuticals as stabilizing, thickening and gelling agents (Zhao 2016). Based on the diverse applications about 844 species are cultivated on Indian shorelines, however, only 60 species are commercially exploited for the phycocolloids (carrageenan, alginate, agar) production for different applications (Wassie et al. 2021). Around 43 Indian industries are involved in the seaweed business and various government bodies like State Fisheries Department, CSIR-CSMCRI, Department of Fisheries of Coastal States and NFDB provide guidance and financial support for site selection based on the availability of wide but shallow coastal areas with market demand, salinity concentration, temperature, depth of the water during low-tide, level of transparency, and mild water currents (http://dof.gov.in/sites/default/files/2020-07/Seaweed_ Cultivation.pdf). This review represents an overview of different uses of seaweeds ranging from industrial, environmental, and societal perspectives. The current status of seaweed farming in India and all over the globe based on different technologies in its farming practices is represented. Later, it briefly mentions the ongoing programmes on seaweed cultivation in India and other countries to unlock the untapped potential by overcoming some technical challenges. This review will facilitate the designing of draft policies in the blue revolution to match the seaweed market trends and forecasts.

21.2

Upstream Processing of Seaweed

21.2.1 Seaweed Farming Principle and Cultivation Techniques Seaweeds are typically present in the benthic area as they must receive sunlight passing through the water to carry out photosynthesis and grow on the substrate with their root-like holdfasts. The seaweed takes all its nutrients and dissolved gases from the seawater through its entire body (Liu et al. 2020). But seaweed is not at all an obligate benthic organism. It has three exceptions to growing without benthic substrates. Firstly, they can be free-floating, with the help of an air bladder, i.e., they are halopelagic. As an example, we can name the two species Sargassum natans and Sargassum fluitans (Choi et al. 2020). The second type of seaweed propagules and become attached to a floating substrate like a drifting log, buoy and its rope or nets. The last one is grown in a provided adequate circulation in the tank or in

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confined places without attachments. They all can grow if they get the required sunlight, water, and nutrient. Before starting the seaweed, the culture there should be pre-cultivation choices like biohazard controls, herbivory, fouling, strong currents, storm, and other vulnerabilities which are difficult to control but can be minimized. Seaweed has bio-extractive nature and uses to feed fish with unused feed. No pesticides or other chemicals are used in this culture which proves the eco-friendliness of seaweed farming (Kim et al. 2018). Some critical steps for initiating seaweed farming (Liu et al. 2020) are as follows: (a) Characteristics of coastal environments are evaluated including farming site, water, climate, biodiversity, quality, access and facilities, socio-economic, legal, and cultural characteristics, and manpower availabilities. (b) Select the right place and specimens for farming for the intended purposes. (c) Selection and implementation of reproduction, propagation, and traditional methods to safeguard sufficient number and quality, optimal spatial arrangements, and density of propagules in the cropping season for biosynthesis while assisting the growth quality under biotic and abiotic conditions. (d) Harvesting and bringing it to land. (e) Post-harvesting handling and making the product market ready. Seaweeds are generally cultivated using several cultivation techniques including line cultivation, floating raft cultivation, net cultivation, submerged hanging lines, tank cultivation etc. (Admassu et al. 2018). Some of the major techniques and methods have been well elaborated (Table 21.1). Kaliaperumal and Chennubhotla (2017) have reported 870 species of seaweeds from Indian coastal areas including Lakshadweep and Andaman-Nicobar Islands. Over the last 20 years, several cultivation technologies have been developed in India for the collection of diverse industrially important seaweeds, such as along the south coast of India, floating rafts are used for cultivation of commercially important seaweed species (Ganesan et al. 2019). Recently, CSIR-CSMCRI Bhavnagar, India, has devised the tube net method for successful cultivation of Gracilaria dura and Kappaphycus alvarezii on the Gujarat coast (Ganesan et al. 2019).

21.2.2 Harvesting Strategy Harvesting of cultivated seaweeds depends on the farmed crop quality, specimen, methods used, intended purpose, operating scale, technological availability, water, and climatic conditions. There are two types of harvesting: total harvest and partial harvest. In total harvest technique, ropes, nets, frame/cage material, seaweed specimens etc., are used to grow seaweeds like Laminaria, Eucheuma and Kappaphycus. The partial harvest includes only newly growing seedlings from initial planting or previous harvest leaving sufficient plant parts allowing them to re-grow, and known as multiple harvests, e.g., Porphyra, Gracilaria and Sargassum (Mantri et al. 2020). In total harvest technique, maximum gains are attained at the

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Table 21.1 List of different techniques and methods used for the cultivation of seaweeds Techniques Line cultivation Off-bottom

Submerged hanging line Floating cage Net cultivation Floating raft cultivation

Tank or pond culture

Kelp forest or seaweed prairies Freefloating rafts Rock-based farming

Methods Seaweeds are attached on a rope of 10 m–50 m with the spacing of 0.5 m–1 m arranged parallelly Seaweed seedlings were planted closely 15–20 cm apart at the shallow bottom of waterline and tied with nylon ropes while maintaining 1 m distance between them Planting on nearshore mid water line and submerged to few meters at high tides and exposed in low tides Planting near the surface, slightly submerged and does not need any anchoring A net tube is attached to a bamboo raft and submerged to a certain depth Planting on a rope or net that is floating and framed with bamboo or other material in a shape. Arranged vertically or angled to intercept solar radiation Cultured in a controlled condition and adequate space for intended production purposes. Mainly for wastewater biofiltration tanks or canals of pisciculture to remove and reuse excess nutrients of the water Direct planting to the seafloor or the artificial substrate to the floor for neutral and natural growth Like line or net cultivation with or without shaped frames and left free-floating It is an onshore farming where tanks/ponds/lagoons are prepared to submerge pieces of seedlings that are attached to the rock with an elastic band and let exposed in natural water and climate for a few weeks. Once the seeding fixes its point, it is shifted to controlled conditions of light, pH, seaweed density, and CO2 in greenhouse

Examples G. edulis

Eucheuma and Kappaphycus

Laminaria saccharina

K. alvarezii, E. denticulatum K. alvarezii, G. dura Kappaphycus sp.

Caulerpa and Ulva

Ecklonia sp.

Laminaria, Undaria, Porphyra Spinosum sp., Chondrus crispus

References Racine et al. (2021), Bhuyan et al. (2021), Duarte et al. (2017), Xiao et al. (2021)

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end of growing season because seedlings crop can suffer from seasonal changes. For example, Kappaphycus harvesting is generally done at 40–60 days intervals but the carrageenan is normally found in the older tissue of the seaweed. The harvested line that holds the older seaweeds used to pass through the line stripper. This removes all the tissues and accumulates them in one place. Getting new tissues with the older ones makes sense rather than obtaining only new tissues (Murugan et al. 2013; Rajaram et al. 2020). A partial harvest is friendlier to the farmers as it allows multiple harvests; counting several crops without gaps in a year decreases complete losses, farming costs and biotic stresses, and promotes a year-round supply of seaweedbased products. Machine-based harvesting of seaweeds is chiefly regarded as a costeffective and time-efficient technique. However, manual harvesting is generally preferred over machine-based harvesting as it promotes careful off-site separation of undesired sea debris and gives high quality to the material. In mechanical harvesting, boats are used to replace the whole setup to the land and remove the ropes, nets, and other materials through mechanical cutters. The total operation is either motorized or hand-operated. Then, the harvested materials are shifted to the shore and unloaded with cranes (in case of large seaweeds) and then placed on a cart or conveyor belt for cleaning and further processing (Rajauria et al. 2016). Hand harvesting is a greater income source. To maintain a healthy and sustainable resource, appropriate and well-maintained tools are important, e.g., the drag-rake is used to harvest Chondrus crispus which is a carrageenan resource. If the rake is not maintained well, it can damage the standing stock. A sickle is used to cut in Scotland, Neijri in Japan, and Corran in Ireland is used to cut the delicate mosses. In India, a sickle is used as a tool of hand harvesting of the seaweeds. To harvest natural seaweed bed, a certain amount of time and material is needed to extract the resources successfully. Gathering all the harvested material by foot needs extra labor whereas importing it with a boat or seaweed trawler is easier mainly by tide times. The high current of water contributes to huge productivity. Transportation to processing, all the procedure is maintained precisely. In 1986, the harvesters use Norwegian suction with the 40–60% exploitation rate. Using this method Saccharina latissima is harvested. The crochet hook rotates around fronds and uproots it. The long handled serrated steel rakes are attached to a boat which moves slowly through the seaweed canopy. The rakes cut the floated seaweeds. This method removes bulk of upper distally end canopy with a huge biomass. The leftover of the weeds allows robust regrowth twice in a year. The rockweeds are harvested with rockweed rakes (Seghetta et al. 2016; Park et al. 2017). In India, majority of the seaweeds are harvested manually as it is an important source of income for coastal fisherwomen and fisherman (Ganesan et al. 2019). The Palk Bay coast and Gulf of Mannar are extremely productive regions for collection of commercially important alginophytes and agarophytes (Ganesan et al. 2019). The Bamboo raft system is the best method for harvesting of commercially important Indian seaweed species (Ganesan et al. 2019).

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21.2.3 Extraction Techniques Seaweeds are having crucial metabolites such as proteins, lipids, minerals, cellulose, and natural pigments. Moreover, seaweeds are rich source of polyunsaturated fatty acid, linoleic and α-linoleic acid. It helps in preventing diabetes, cardiovascular diseases, and osteoarthritis. The phycocolloid ulvan fiber extracted from seaweed species has high water retention capacity and widely used in intestinal transit dysfunction. It can also help in reducing the cholesterol, LDL, and triglycerides level while it increases the serum level of HDL cholesterol. Seaweeds extracted proteins have high concentration of essential amino acids compared to traditional plant protein resources. The cellulose and the polysaccharide extracted from seaweeds are converted into ethanol, levulinic acid derivatives such as ethyl levulinate and are used as diesel and biodiesel additives (Mantri et al. 2022; Ganesan et al. 2011). These above-mentioned bioactive compounds are extracted from diverse seaweed species using several advanced techniques as elaborated in Table 21.2. Seaweeds are mainly collected for the extraction of industrially valuable phycocolloids including carrageenan, alginate, and agar. Numerous research groups in India are actively engaged in the fabrication of photobioreactors for increased seaweed biomass production. Moreover, they are developing novel biorefinery processes for the extraction of extremely valuable seaweed-based products (Ganesan et al. 2019).

21.2.4 Purification Strategy Several techniques have been optimized for the purification of economically relevant bioactive compounds from seaweeds. However, chromatography is considered a gold standard method for the purification of the seaweed supernatants and crude material components. This technique indicates the separation of components from a solution depending on their inter-relationship by mobile and stationary phases. Mobile phase passes the components through the system whereas stationary phases held the remains at one place firmly by a support. The chromatographic methods are categorized into different types comprehended in the table below (Table 21.3).

21.3

Application of Seaweed

Seaweeds have immense applications in diverse sectors as illustrated in Fig. 21.1, and well elaborated in sections below.

21.3.1 Industrial Application of Seaweeds (a) Agar: Agar is a long-chain water-soluble polysaccharide that is mainly found in red algae. Agar consists of agaropectin and agarose and is used in the production

Enzyme-assisted extraction

Acid extraction

Alkali extraction

R-phycoerythrin extraction

Techniques Hot water extraction

Methods This is used for extraction of compounds by degrading the cell wall. The specimen is washed with distilled water and dried (60 °C), homogenized, filtered, and soaked in hot water for 3 h (100 °C/ 33.3 g/L) Red algae taken, cut into pieces, grinded by using mortar pestle, transferred to a 250 ml beaker, buffer added and thawed in -20 °C, 37 °C solution is centrifuged at 8000 rpm for 15 min. Precipitated pellets are discarded and supernatants are assayed by using spectrophotometer frequencies 562, 615 and 652 nm This is used to separate –OH ions of the base that interfere with the linkage of hydrogen in the polysaccharide release in the solvent. Dried algae is treated with ethanol (95%) at 60 °C for 2 h and then at 90 °C in distilled water for 2 h HCl breaks cell components and releases polysaccharides into the solvent. 0.1 M HCl in room temperature 4 h in 1:30 (w/v) algae: Water Enzyme degrades seaweed cell wall and releases the contents in the solvent. Xylanase, viscozyme, cellulose, ultrafine, flavoursome, termamyl, protamex, and neutrase are used for extraction purposes. 2 g sample is added in 50 ml distilled water and incubated for 10 min, dried (60 °C), milled to 0.4) that are highly beneficial for human health (Kumar et al. 2011). Matanjun et al. 2009 showed that three seaweeds (C. lentillifera, S. polycystum and E. cottonii) collected from the coast of North Borneo have high content of dietary fibers, macrominerals (Na, Mg, K and Ca), microminerals (Se, Fe and I), ascorbic acid, alpha-tocopherol and crude protein content. Ajayan et al. (2003) found that twenty species of seaweed collected from the Kerala coast contained high concentration of fatty acid (linolenic acid, oleic acid) and essential dietary elements (C, Mg, N, Mn, Cu and Zn). K. alvarezii collected from southeast coast of India showed significant amount of amino acid (lysine), fatty acid (linoleic acid), anthocyanin, total carotenoids and total phenolic content (Rajaram et al. 2021). Recently, Arisekar et al. (2021) showed that seaweeds dwelling in the Gulf of Munnar region are safe for human consumption as well as production of pharmaceutical products and food supplements. The potential health benefits of different seaweed species have been further elaborated in Table 21.6. Therefore, the seaweed-derived products have tremendous potential in mitigating several lifestyle-associated disorders.

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Table 21.6 The health benefits and biochemical constituents of different seaweed species Seaweed Green seaweed

Brown seaweed

Species E. compressa

C. lentillifera C. racemosa var. laetevirens C. racemosa var. clavifera E. compressa E. linza E. tubulosa S. polycystum

E. cava

S. fusiforme S. fluitans S. horneri

Red seaweed

Biochemical constituents Iron, calcium, dietary fibers, vitamins Dietary omega-3 PUFA

Health benefits High nutritional value

References Mamatha et al. (2007)

Ameliorate cardiovascular diseases and reduce LDL cholesterol

Nagappan and Vairappan (2014)

Phenolic compounds

Antioxidants

Ganesan et al. (2011)

Microminerals (se, Fe and I), dietary fibers Microminerals (Na, mg, K and ca) Phlorotannins and sterols (ergosterol and fucosterol) Sulfated polysaccharides

High nutritional value

Matanjun et al. (2009)

Antioxidant properties

Li et al. (2009)

Anti-coagulant, anticancer, antiinflammatory, antiviral and hepatoprotective activity Angiogenesis Immunomodulation Gastric mucosal protection Antioxidant and cardioprotective

Sun et al. (2018), ChaleDzul et al. (2017), Sanjeewa et al. (2017) Jintang et al. (2010), Matou et al. (2002), Shibata et al. (2000) Rajaram et al. (2021)

Prevention/control of hypertension and type II diabetes α-Glucosidase inhibitory activity

Harnedy and Fitz Gerald (2013) Kim et al. (2008)

A. nodosum F. vesiculosus Cladosiphon okamuranus

Fucoidans

K. alvarezii

Fatty acid (linoleic acid), anthocyanin, Total carotenoids, Total phenolics Protein hydrolysates

Palmaria palmata Grateloupia elliptica

2,4,6tribromophenol and 2,4-dibromophenol

21.3.3.2 Potential Health Risk The extensive release of heavy metals in water resources from agricultural runoff, industrial waste, domestic sewage, and other anthropogenic and natural activities have led to wide scale contamination of aquatic ecosystems (Foday et al. 2021; Afzal

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2018). These heavy metals can easily bioaccumulate in the food chain on virtue of its non-biodegradable nature. The heavy metals namely Cd, Cr, As and Pb causes severe marine pollution as well as several human health issues, including cancer, lung and brain damage, nausea, and toxicity through generation of reactive oxygen species and oxidative stress (Ajjabi and Chouba 2009; Abu Al-Rub et al. 2004; Argun et al. 2007; Yang et al. 2015; Tchounwou et al. 2012). Recently, it has reported that edible seaweeds dwelling in untreated wastewater from industries, agricultural field, and mining area bioaccumulate alarming levels of toxic heavy metals (Pb, Cd and As), pesticides, dioxins, radioactive isotopes, and ammonium, which are considered to have several adverse effects on human health, namely cardiovascular, kidney and CNS disorders (Martín-León et al. 2021, Murai et al. 2021, Kumar et al. 2020, Tchounwou et al. 2012, Van der Spiegel et al. 2013). The cell wall of seaweed is composed of cellulose, monomeric alcohol, alginate, laminarin, protein and deprotonated sulphate that serve as a perfect binding site for metal ions (Gupta and Rastogi 2008; Pradhan et al. 2017). Recently, Sundhar et al. (2020) revealed that seaweeds (G. acerosa and S. wightii) collected from the Gulf of Mannar contain organochlorine pesticides above the maximum residual level that is detrimental to marine environment and human health. Some species of seaweeds including U pertusa, Caulerpa and S. confusum are reported to contain several toxic compounds such as galactolipids, sulfolipids, caulerpicin and sargalin (Naidu et al. 1993). Kamala-Kannan et al. (2008) found that the U. lactuca collected from Pulicat Lake contains high amount of toxic heavy metal (Pb: 8.32 μg/g). The ten species of seaweeds collected from the Palk Bay coast (South-eastern India) contain Cd above the maximum permissible limit as suggested by CEQG and WHO (Rajaram et al. 2020). Paz et al. (2019) reported that the edible Asian seaweeds contained maximum amount of Pb (0.40 mg/ kg dw), Al (38.9 mg/kg dw) and Cd (0.59 mg/kg dw). Martín-León et al. (2021) demonstrated that Asian seaweed Hijiki and Nori contains high levels of toxic heavy metal (Cd), whereas Asian Wakame seaweed contains maximum amount of Pb. A recent study demonstrated that the Indian consumers were most concerned about the heavy metal contaminants in seaweeds (Gajaria and Mantri 2021). The bioaccumulation of heavy metals and other toxic compounds in seaweeds is a matter of great concern (Kumar et al. 2020). Hence, it is imperative to perform the toxicological and interventional studies to assess the potential health risk and put forward safe consumption of seaweeds.

21.3.3.3 Seaweed-Associated Bioeconomy Seaweeds have cosmopolitan distribution from Antarctic, Artic to tropical sea (Gajaria and Mantri 2021). Seaweed-based products are considered as precious commercial commodity in several south-east Asian countries (Neish et al. 2017). The phycocolloids (Agar, alginates, and carrageenan) extracted from seaweeds has driven major economic benefits in various industries including medicinal, personal healthcare, nutraceutical, and food-additive industries (Hafting et al. 2015). Recent years have witnessed a dramatic increase in the trade of various seaweeds in some maritime nations (Mantri et al. 2019). Globally, there is a huge upsurge in

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the demand of seaweed-based products (Gajaria and Mantri 2021). For instance, recent years have seen an immense increase in the consumption of seaweed as processed ingredient in sushi and snack food items across Australia, Norway and India (Mamatha et al. 2007; Altintzoglou et al. 2016; Birch and Bonwick 2019). It has been estimated that nearly 500 million tonnes of seaweed biomass may be produced by 2050 and the global market is expected to reach $ 22.13 billion by 2024 (Cottier-Cook et al. 2016; FAO 2021). However, in the most recent years, the global production of seaweeds has relatively declined due to slow growth output in tropical seaweeds species and decreased production in southeast Asian countries (FAO 2021). Currently, Seaweed mariculture is considered to have a huge potential in uplifting the socio-economic status of low-income fisherman. Young entrepreneurs are hugely benefited from commercial seaweed farming. For instance, the cultivation of Kappaphycus species in India has generated 7,65,000 man-days of employment and annual turnover of approximately `2 billion (Mantri et al. 2019). The department of Science & Technology (DST), India under the women scientist program has offered a tremendous opportunity for empowerment of coastal inhabitants of Kerala through large-scale cultivation of high-value K. alvarezii for various industrial purposes (Bindu and Levine 2011). Sundhar et al. (2020) reported that the cultivation of G. verrucosa (Huds) Papenfuss in Chilika Lake offers a sustainable source for livelihood generation in coastal areas of Orissa, India. Subba Rao et al. (2006) reported that mariculture of H. musciformis has promising potential for income generation of coastal inhabitants of southeast India. Indian government has implemented several rural employment schemes in seaweed farming for uplifting the coastal economy (Mantri et al. 2020). Mantri et al. (2019) reported that nearly 5000 women in India earn their livelihood from seaweed industries. Moreover, seaweed harvesting and post harvesting activities have an immense potential for generating employment for another 20,000 coastal inhabitants. Several students have reported that the commercial cultivation of Gelidium sp., Kappaphycus sp. and Gracilaria sp. has a huge potential in livelihood generation for diverse Indian coastal communities (Rameshkumar and Rajaram 2019). The blue revolution flagship programme of Government of India also supports seaweed cultivation and seaweed-resource management for diversification of livelihood in coastal communities (Bhuyan et al. 2021). As the marine ecosystem of Indian coastline promotes abundant growth of seaweeds, the seaweeds collection has been a major source of income for the Indian coastal community. Several studies have demonstrated that seaweeds have remarkable potential in global food industry and Indian market as a commodity product (Gajaria and Mantri 2021; Jha et al. 2021). Numerous field and experimental cultivation trials have uncovered the huge commercial potential of Kappaphycus cultivation in Indian coastal regions and global industries (Bindu and Levine 2011). Recently, G. acerosa and Gracilaria have fetched highest price of about $628–1884 per dry tonnes in the local market. India has witnessed a commendable increase in import–export of agar on global scale (Mantri et al. 2019).

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Seaweed experts have demonstrated an immense scope in the farming of industrially valuable Gracilaria species in Indian coastal areas (Mantri et al. 2019). Recently, the coastal community of the Gulf of Mannar region has exported a large quantity of Saragassum species to seaweed-based industries on commercial scale (Rameshkumar and Rajaram 2019). The recent advances in down-stream processing of seaweeds have accentuated the progress in commercialization of seaweed-based products (Ganesan et al. 2019). Seaweeds have profound scope in modern small enterprises (Gajaria and Mantri 2021). Recently, Government of India has included seaweed culture in its “blue revolution” flagship programme to promote new ventures in seaweed-based industries (Jha et al. 2021). These studies validate the fact that the seaweed-based farming and seaweed-derived products have prodigious prospects in socio-economic development and accomplishing global future goals.

21.4

Past and Ongoing Programs to Promote Seaweed Cultivation

Seaweed cultivation as food and food ingredients in Asia-Pacific, particularly in China, Indonesia, the Philippines, Korea, and Japan, has huge potential. A variety of seaweed is explored as a fuel because of their high photosynthetic efficiency and their ability to produce lipids, a biodiesel feedstock that is expected to open new avenues for seaweed biomass manufacturers. To achieve the target of doubling up seaweed production of one million tonnes by 2025 by investing $86.8 million under the Blue Revolution scheme is proposed in India. This policy framework will improve the livelihoods of coastal communities (https://foodtank.com/ news/2021/01/indias-blue-revolution-targets-investments-in-seaweed/). Moreover, a few startups have gained international investments in their prototypes, to accelerate seaweed harvesting journey. For instance, Sea6 Energy indigenous seaweed harvester named SeaCombine can reduce the manpower work and make the process more efficient and timesaving. Another startup, Zaara Biotech based in Thrissur is involved in producing nutritional seaweed cookies to tackle food crisis and malnutrition. Further, government is providing subsidies to the fisherman communities or farmers involved in seaweed business for setting up seed bank, nurseries, processing, and marketing units, besides giving training on various cultivation techniques. This will empower unemployed youth, women and local communities by lending approvals on 100 per cent FDI through automated paths in aquaculture. The valuable chemicals like antioxidants, and pigments as natural additives have high applicability in the nutraceutical, cosmetics, pharmaceutical industries that further instigates seaweed farming potential and opportunities (https://cir.nii.ac.jp/crid/15739503 99231627264). Also, the development of offshore marine macroalgae farms could play a major role in sustainable energy production from organic matter. The use of special textile mats can facilitate the cultivation of seaweed and increase production significantly (https://agrinews.in/india-plans-to-increase-seaweed-output-at-11-5lakh-tonnes-in-five-years/). The list of companies involved in production of seaweed-based products has been further elaborated in Table 21.7.

Cargill Inc.

DuPont de Nemours, Inc.

Irish seaweeds

Beijing Leili marine bioindustry Inc. (Leili group)

8

9

10

11

6

7

SNAP natural products and alginate (P) ltd Sarda biopolymers private limited

Name of companies Aquagri processing private limited Marine hydrocolloids

Kerala nutraceuticals Pvt. ltd. Srinivasa marine chemicals Atlantic Mariculture

5

4

3

2

Sr. No. 1

Sargassum sp.

Chondrus crispus

Seaweed

Chondrus sp., Gigartina sp., Iridaea sp., and Eucheuma sp.

S. wightii A. nodosum Chondracanthus chamissoi

S. wightii

E. cottony

S. wightii, Turbinaria sp.

G. acerosa

Name of Seaweeds K. alvarezii

Table 21.7 List of companies using Seaweeds in their product formulation

Crop bio-stimulant

CIFTEQ® seaweed Nutridrink Seaweed fertilizer, seaweed extract Acadian SeaPlus™ nutraceuticals & cosmetics Satiagel, Aubygel, Satiagum, and seabird brands Confectionery, beverages, frozen desserts, and seafood segments Kelp seaweed capsules

Carrageenan

Agar, agarose, Gellan gum, chitin and chitosan Alginate

Product Carrageenan

United Kingdom China

United States

United States

Canada

India

India

India

India

India

Country India

https://irishseaweeds. com/ http://www.leili.com/

https://www.dupont. com/

https://www. acadianseaplants.com/ https://www.cargill.co. in/

https://www. sardabiopolymers.com/ carrageenan-powder/ Gopalakrishnan et al. (2020) www.seaweedindia.com

https://snapalginate.com/

References http://aquagri.in/ seaweed-cultivation/ https://meron.com/

630 M. Singh et al.

Ascophyllum nodosum, Chondrus crispus

Palmaria palmata

The Cornish seaweed company The seaweed company

VitaminSea seaweed

Z company

Mara seaweed

16

18

19

20

17

15

Qingdao gather Great Ocean algae industry group co., ltd Seaweed energy solutions AS

14

Alaria esculenta

Alaria sp., Fucus sp., Laminaria sp., Undaria sp., Codium sp., Gracilaria sp.

Saccharina latissimi, Alaria esculenta, Laminaria hyperborea, Laminaria digitata, Palmaria palmata, Ulva lactuca Mixed kelps and sea greens

Eucheuma cottonii

Pacific harvest

13

Saccharina latissima, Alaria esculenta, Palmaria palmata Undaria sp.

Maine Fresh Sea farm

12

Wild North Atlantic whole leaf Kelp powder Irish Moss powder Dulse seaweed flakes

Organic edible Cornish seaweed Seaweed-based biostimulant solution

Seaweed solutions

Seagreen broth, bread, and smoothie Food items like cakes, smoothies, soups & stocks Seaweed salad, seaweed feed and seaweed fertilizer

Scotland

Netherlands

United States

United Kingdom Netherlands

Norway

China

New Zealand

Portland

https://www. cornishseaweed.co.uk/ https://www. theseaweedcompany. com/ https:// vitaminseaseaweed.com/ https://www.z-company. nl/?s=seaweed https://maraseaweed. com/

https://seaweedsolutions. com/

https://maineseafarms. com/ https://pacificharvest.co. nz/ https://en.judayang.com/

21 Seaweed Farming: An Environmental and Societal Perspective 631

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21.5

M. Singh et al.

Strategies to Overcome Technical Challenges

The coastline supports the exuberant growth of different seaweed populations and has a huge economic magnitude. However, there are many challenges that need to be addressed to achieve the set targets. The first biggest challenge is Climatic disasters like Tycoons/Cyclones/Tsunami/Hurricanes which negatively impacts the onshore facilities and led to decreased crop yield and low quality (Arisekar et al. 2021). The second major challenge is conflict with water-use in coastal areas, as mostly fishing activities are carried out in sea leaving less space for designing floating beds for growing seaweeds. Third challenge that impacts seaweed yield is eutrophication caused by discharge of sewage or industrial waste rich in Nitrates and Phosphates into the sea by municipalities, industries and factories located near to seashore. Fourth challenge comprised of development of Wind-power generating stations near the coast affecting algal farm infrastructure (Henriques et al. 2019). The fifth challenge is monsoon dependence for seaweed cultivation to withstand rough sea conditions in high tide fluctuations and seawater turbulence while producing good biomass yields (Ganesan et al. 2019). The sixth challenge is maintaining the quality of thallus of seed stock that can withstand high temperatures, tolerant to infestations and grow faster in limited space (He and Yarish 2006). The seventh challenge is overcoming epiphytic attack and removal of fouling algae from the water (Correa et al. 2001). In this case, the symptoms are white spots that occur on the branches of seaweed which soon spread and cover the entire plant. The best way to get rid of the disease is to cross-cultivate seaweed growing in one place with other, also replacement of culture strains with wild plants from same region might prove effective (Capo et al. 1999; Strain et al. 2007). The last but not the least challenge is to compile the seaweed strain diversity and their mapping on coasts using GIS technology that will aid in formulation of efficient policies and can be easily accessible to research community.

21.6

Conclusion

The untapped diversity of seaweeds still needs to be explored for filling up the gap areas in seaweed commercialization and bioproduct development. The persistent efforts of scientific institutes should be incentivized for strain improvement, early detection of disease and tolerance to epiphytes. Moreover, the joint collaborations between academia and private enterprises must be promoted and financially supported by boosting the biosecurity and strengthening of local value chains and product diversification. The ecological benefit provided by seaweeds can combat major issues like ocean acidification, eutrophication, hypoxia, and reduce sea debris by oxygenation. This will further help in establishment of biobanks and improvise the existing farming practices, both for upstream and downstream operations.

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Development of New Molecules Through Molecular Docking

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Charu Jaiswal, Kushal Kant Pant, Ravi Kiran Sriniwas Behera, Renu Bhatt, and Vikas Chandra

Abstract

Molecular docking is an in silico method that involves positioning 3D structure of ligand and target (derived from the databases) in different orientations. Thousands of molecules that can be natural products, synthetic compounds, or often the approved drugs available in the databases can be used for virtual screening, a process where the ligand molecules are docked against the target to look for optimal conformation. It is a crucial step in the computer-aided drug designing process, as following this step, further screening and development of the desired molecule can be performed. The prerequisite for the development of new drug molecule is a known structure of target against which the drug is to be developed. After the screening process for optimal conformation, a potent lead compound can be identified through the scoring function. A significant concept termed as structure-ctivity relationship (SAR) provides a clue of properties of a molecule based on its structure. It enables the identification of the desired positions on a molecule for modification in order to improve the pharmacological properties of the molecule such as efficacy, potency, solubility, and pharmacokinetics etc. Lead discovery ensues a process that involves the development of a drug candidate that once tested through bioinformatics tools can be further taken for preclinical studies, followed by clinical trials. The results of clinical trials are crucial in making decision on whether to release the drug into the market. Moreover, once hit compounds are identified, lead molecules can be chosen to perform lead optimization, which involves maximizing the interactions between

C. Jaiswal Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India K. K. Pant · R. K. S. Behera · R. Bhatt (✉) · V. Chandra (✉) Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 P. Verma (ed.), Industrial Microbiology and Biotechnology, https://doi.org/10.1007/978-981-99-2816-3_22

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the ligand and selected target molecule to enhance its pharmacological activity and reduce the side effects. Keywords

Ligand · Target · Receptor · Lead · Drug · In silico

Abbreviations 3D ADMET CADD CDC CoMFA CoMSIA CSP CVS ESBL GOLD GUI GWOVina HADDOCK HTS LBDD MCS MD MLR NMR PCA PDB PDBQT PLS QSAR RCSB RMSD RMSF RNA SAR

Three-dimensional Absorption, distribution, metabolism, excretion, and toxicity Computer-aided drug design Centers for Disease Control and Prevention Comparative molecular field analysis Comparative molecular similarity indices Conformationally sampled pharmacophore Consensus virtual screening Extended-spectrum β-lactamases Genetic optimisation for ligand docking Graphical user interface Grey wolf optimizer Vina High ambiguity driven protein-protein docking High-throughput screening Ligand-based drug design Maximum common substructure Molecular dynamics Multiple linear regression analysis Nuclear magnetic resonance Principal component analysis Protein Data Bank Protein Data Bank, Partial Charge (Q), & Atom Type (T) format Partial least squares analysis Quantitative structure-activity relationship Research Collaboratory for Structural Bioinformatics Root-mean-square deviation Root-mean-square fluctuation Ribonucleic acid Structure-activity relationship

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Introduction

Molecular docking is a mechanism wherein various natural or chemical compounds are docked against a target molecule; hence, it provides a way that can be used for the generation of novel compounds called lead compounds that can significantly serve for the treatment of various diseases, of which one of its use is the generation of lead compound against multidrug-resistant bacteria. With time, many bacteria have become resistant due to the overuse of antibiotics. For this reason, discovering and testing the drugs directly in the laboratory is not feasible. In silico method emerged as an alternative to ease such circumstances, where prior to actual lab testing of the drug, the lead compound (that can act as a potential drug candidate) is first identified using computer-aided drug designing and analyzed for its effect on the biomolecular system using molecular simulation studies. This chapter focuses particularly on the identification of lead compounds using structure-based drug design (SBDD). The bacterial species of Proteus, Klebsiella and Escherichia coli are responsible for nosocomial infection (Jacobsen and Shirtliff 2011; Schaffer and Pearson 2015; Majumder et al. 2018), which is one of the major concerns as per CDC. These gramnegative bacteria produce enzymes called extended-spectrum β-lactamases (ESBL) (Al-Zarouni et al. 2008; Pasta et al. 2008; Slama 2008; Aladag and Durak 2009). ESBL synthesizing bacteria are majorly accountable for nosocomial infections as it enables the hydrolytic cleavage of cephalosporins, monobactams, and penicillins (Aladag and Durak 2009; Al-Zarouni et al. 2008). These bacteria utilize various virulence factors to establish and flourish in host resulting in its virulent behavior (Schaffer and Pearson 2015). Most of the nosocomial infections caused by the ESBL-producing bacteria are resistant to antibiotics hence presenting a major concern to human well-being. Molecular docking is a method where ligand conformations taken within binding sites of target macromolecules are explored. Therefore, the primary factor determining SBDD’s success is its scoring functions. There are numerous software applications that are used to perform structure-based virtual screening (SBVS), they all employ different algorithms. Consensus virtual screening (CVS), a novel SBDD technique, has been applied in certain research during the past few years to improve SBDD accuracy and lower the number of false positive results obtained. The access of a 3D structure of target molecule is a need for using SBDD. 3D structures of molecules are kept in a few virtual databases, like the Protein Data Bank (PDB). The 3D structure cannot always be obtained experimentally; in this case, a protein’s 3D structure may be predicted from its amino acid sequence according to the homology modelling methodology. This chapter provides an overview of SBDD using CADD tools, the areas where CADD tools help SBDD, and the current approaches that are taken into account to shorten the timing and expense of drug development. Even though it was ineffective initially, this random screening method has helped to identify a number of significant substances (Maia et al. 2020). To speed up the development of antibiotics and other drugs, computational techniques are helpful tools for analyzing and directing studies. The two main categories of computer-aided drug design (CADD) methods currently in use are

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structure-based drug design (SBDD) and ligand-based drug design (LBDD). To pinpoint essential locations and interactions crucial to each macromolecular target’s specific biological functions, SBDD techniques examine the three-dimensional structural data of target macromolecules (e.g. proteins, RNA). This information may subsequently be utilized to develop antibiotics or other medicines. The goal of LBDD is to identify known ligands against target and establish a structure-activity relationship (SAR) between physiochemical characteristics and activity of ligands. This knowledge may be used to improve existing medications or to help create new medications with increased activity (Yu and MacKerell 2017).

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Computer-Aided Drug Design

The process of generating new medicines requires a significant investment of time and money, which is intriguing for the pharmaceutical sector. Computer-aided drug design (CADD) is a widely utilized technique to reduce expense as well as time of the medication development procedure. In rational approach, CADD is an extremely helpful technique to decrease the amount of time needed for novel drug candidate identification, characterization, and structure optimization (Ekins et al. 2000, 2002; Van De Waterbeemd and Gifford 2003; Ekins 2006). CADD makes it possible to focus studies more effectively, which helps speed up developing new medications and lower the cost of development. It is an emerging technique for discovering or designing candidate drugs and can be used to identify potential drug before validation in wet lab settings. CADD can provide atomic level SAR that eases the process of drug designing, hence can reduce the cost of production (Schneider and Fechner 2005; Yu et al. 2013). Studying the molecule at the atomic level significantly contributes to understand an appropriate drug design to overcome the limitation of antibiotic resistance, by finding out a new target based on the SAR analysis and ultimately helps in the development of an effective drug (Yu and MacKerell 2017). As mentioned earlier, drug designing or development can be achieved via two approaches that are SBDD and LBDD. Both the approaches are integrated with quantitative structure-activity relationship (QSAR). The details of LBDD and SBDD are given in the following sections. An outline of the CADD process is depicted in Fig. 22.1.

22.3

Ligand-Based Drug Design (LBDD)

In LBDD approach, the ligand of a drug is known, i.e. the relationship between the physicochemical properties and the activity of the drug is established known as the QSAR (Macalino et al. 2015; Yu and MacKerell 2017; Surabhi and Singh 2018). Pharmacophore model is generated using the structure of a ligand to bind an active site based upon the assumption that the compounds that have similar structures will exhibit similar biological action and interaction with target molecule as well. This is a common method in ligand-based drug design, which is used when the

22

Development of New Molecules Through Molecular Docking

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Fig. 22.1 An outline of the computer-aided drug design (CADD) process

three-dimensional (3D) structures of prospective therapeutic targets are not known. A significant and frequently used tool in LBDD is three-dimensional QSAR. Pharmacophore modelling is another such tool. Using these tools characteristics of association between drugs and ligands can be understood with predictive models appropriate for finding optimum lead molecule (Acharya et al. 2011).

22.4

Structure-Based Drug Design (SBDD)

SBDD is a promising, reliable and practical in silico method. In order to determine the optimum conformation in which the two molecules interact and create stable complex, SBDD applies scoring functions to calculate strength of non-covalent associations. SBDD aims to find out the information on the 3D structure of the target which can be a protein, enzyme, or RNA. This helps to identify the sites significant for the particular biological function (Yu and MacKerell 2017). Initially, the cycle comprises the selection of the structure that has been isolated from X-ray crystallography, homology modelling, and NMR spectroscopy. Once appropriate structure is determined, compounds are ranked on the basis of steric hindrance and the intermolecular interactions of molecules with active site of target protein, after which the biochemistry assays are applied on top-ranked compounds (Surabhi and Singh 2018).

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22.5

C. Jaiswal et al.

Steps of SBDD and Lead Compound Identification

SBDD entails a number of stages, including the selection of target, the identification of binding sites, preparation of ligand library, docking, and scoring functions (Wang et al. 2018) followed by molecular dynamics (MD) simulation study to validate the effect.

22.6

Preparation of the Ligand Library

It is the initial stage in SBDD where the structure is derived from X-ray, and NMR submitted data at RCSB PDB (Wang et al. 2018). Homology can be performed if the structure of protein is unavailable on databases. Other methodologies such as threading approaches (Jo and Cheng 2014; Wang et al. 2014; Jo et al. 2015), and de novo folding (Adhikari and Cheng 2017; Fischer et al. 2017; Karakaş et al. 2012) can often be used in case target structure is not available. While generating lead compound against drug- resistant bacteria, usually the protein responsible for resistance of that particular bacteria is targeted.

22.7

Binding Site Identification

Approaches such as site-directed mutagenesis or X-ray crystallography can be used for determining the binding sites in the target molecule (Pan et al. 2017). The binding sites are generally present within the target protein. This in turn will lead to identification of optimal pharmacophores for the novel ligand by determining properties like post-docking dynamics, protein ligand association, hydrogen bonding, etc. (Zhang et al. 2016). The binding site can be determined via literature survey (Gomez-Gutierrez et al. 2017). Few of the tools such as DoGSite Scorer server (Volkamer et al. 2012), CASTp (Sahu et al. 2017), NSiteMatch (Sun and Chen 2017), metapocket (Huang 2019), LISE (Xie et al. 2013), and MSpocket (Zhu and Pisabarro 2011) can often be used for deriving the information about the binding sites. The bulky ligands that are to approach the binding site may be filtered out during the process of lead identification (Wang et al. 2018). To build the library, ligand molecules may be chosen from preexisting ligands, or databases (Irwin et al. 2012; Tonghui et al. 2017; Kontoyianni 2017). Additionally, ligands should be examined for “Lipinski’s rule of five” restrictions that says the value of molecular mass should be