Grand Challenges in Fungal Biotechnology (Grand Challenges in Biology and Biotechnology) 3030295400, 9783030295400

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Grand Challenges in Biology and Biotechnology

Helena Nevalainen   Editor

Grand Challenges in Fungal Biotechnology

Grand Challenges in Biology and Biotechnology

Series editor Pabulo H. Rampelotto

More information about this series at http://www.springer.com/series/13485

Helena Nevalainen Editor

Grand Challenges in Fungal Biotechnology

Editor Helena Nevalainen Department of Molecular Sciences Macquarie University Sydney, New South Wales, Australia

ISSN 2367-1017 ISSN 2367-1025 (electronic) Grand Challenges in Biology and Biotechnology ISBN 978-3-030-29540-0 ISBN 978-3-030-29541-7 (eBook) https://doi.org/10.1007/978-3-030-29541-7 © Springer Nature Switzerland AG 2020 The chapter “Fungal Biotechnology in Space: Why and How?” is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter. This work is subject to copyright. All rights are reserved 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 Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

The twenty-first century is facing a number of challenges in the areas of health, developing sustainable bioeconomy, facilitating agricultural production and securing practices that support a cleaner environment. While there are chemical solutions to some of the challenges, finding biobased solutions is becoming increasingly important. Nature has produced organisms that can perform some truly amazing chemistry at a level of sophistication that human chemists simply cannot match. Filamentous fungi that have earned monikers like ‘the forgotten kingdom’ and ‘the highly productive black box’ are one such group of organisms of which the full potential is yet to be exploited. Some of their properties, such as the extraordinary capacity to secrete enzymes into the external environment, have been harnessed for the production of industrial enzymes and for purposes such as hydrolysis of cellulosic biomass for biofuel production. Efficient improvement of current strains is pending on the success of key enabling molecular technologies including highthroughput screening and molecular technologies enabling targeted editing of the fungal genome, discussed in this book. The 18 chapters in the book are organized into four parts. The first part opens the discussion by providing an overview of global challenges and describes some of the approaches taken towards utilizing fungi as food and their capabilities for degradation of environmental pollutants. Hydrolysis of plant and algal biomass is discussed with a view of obtaining valuable products thereof and current challenges in the process. The second part is dedicated to key enabling technologies for fungal strain improvement providing discussion on the importance of understanding the genetic constitution and gene regulation in the organism of interest and advances of highthroughput screening of fungal strains. The third part touches upon the economic potential of fungal biotechnology taking the reader through the laboratory to the market place and explores fungi-based solutions in the area of agriculture. The fourth and final part looks into emerging opportunities in the fungal biotechnology. This part introduces small molecules produced by fungi and highlights the broad scope for new applications of fungal biotechnology on both Earth and the Space.

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This book is thought-provoking reading for research scientists and biotechnologists in academia and industry, readers particularly interested in fungal biotechnology and also those working within the broader area of microbial biotechnology. With stand-alone chapters written in an accessible language, the book is also recommended as a reference text for decision-makers in government and non-governmental organizations in their efforts to foster the development of cleaner technologies and global bioeconomy. The solutions that nature has developed to cope with challenging biochemical problems provide inspiration for further innovations in industrial processing technologies, food security and environmental protection amongst other challenges. Sydney, Australia

Helena Nevalainen

Contents

Part I 1

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Fungal Biotechnology: Unlocking the Full Potential of Fungi for a More Sustainable World . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lene Lange, Jane W. Agger, and Anne S. Meyer

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Fungal Attack on Environmental Pollutants Representing Poor Microbial Growth Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dietmar Schlosser

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The Biotechnology of Quorn Mycoprotein: Past, Present and Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jack A. Whittaker, Robert I. Johnson, Tim J. A. Finnigan, Simon V. Avery, and Paul S. Dyer The Current Biotechnological Status and Potential of Plant and Algal Biomass Degrading/Modifying Enzymes from Ascomycete Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ronald P. de Vries, Aleksandrina Patyshakuliyeva, Sandra Garrigues, and Sheba Agarwal-Jans

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Fungal Biotechnology and the Global Challenges

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Developments in Key Enabling Technologies

Genetic Transformation of Filamentous Fungi: Achievements and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexander Lichius, Dubraska Moreno Ruiz, and Susanne Zeilinger Bottlenecks and Future Outlooks for High-Throughput Technologies for Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . Kyle Rothschild-Mancinelli, Susanne M. Germann, and Mikael R. Andersen

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Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories . . . . . . . . . . . . . . . . . . . . . . . José Arnau, Debbie Yaver, and Carsten M. Hjort Meeting a Challenge: A View on Studying Transcriptional Control of Genes Involved in Plant Biomass Degradation in Aspergillus niger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jing Niu, Arthur F. J. Ram, and Peter J. Punt

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Molecular and Genetic Strategies for Enhanced Production of Heterologous Lignocellulosic Enzymes . . . . . . . . . . . . . . . . . . . . Sophie A. Comyn and Jon K. Magnuson

Horizontal Gene Transfer in Fungi . . . . . . . . . . . . . . . . . . . . . . . . Erin L. Bredeweg and Scott E. Baker

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Spotlight on Class I Hydrophobins: Their Intriguing Biochemical Properties and Industrial Prospects . . . . . . . . . . . . . . . . . . . . . . . . Paola Cicatiello, Ilaria Sorrentino, Alessandra Piscitelli, and Paola Giardina

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Branching Out: Emerging Opportunities

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Towards Bioeconomy: Potential of Fungal Biotechnology

The Economic Potential of Arbuscular Mycorrhizal Fungi in Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maya Benami, Yochai Isack, Dan Grotsky, Danny Levy, and Yossi Kofman

Part IV

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An Aroma Odyssey: The Promise of Volatile Fungal Metabolites in Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Victoria L. Korn, Sally Padhi, and Joan W. Bennett Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Martin Hofrichter, Harald Kellner, Robert Herzog, Alexander Karich, Christiane Liers, Katrin Scheibner, Virginia Wambui Kimani, and René Ullrich Progress and Research Needs of Plant Biomass Degradation by Basidiomycete Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miia R. Mäkelä, Kristiina Hildén, Joanna E. Kowalczyk, and Annele Hatakka TCA Cycle Organic Acids Produced by Filamentous Fungi: The Building Blocks of the Future . . . . . . . . . . . . . . . . . . . . . . . . . J. Stefan Rokem

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Opportunities for New-Generation Ganoderma boninense Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisha Govender, Wong Mui-Yun, and Robert Russell Monteith Paterson Fungal Biotechnology in Space: Why and How? . . . . . . . . . . . . . . Marta Cortesão, Tabea Schütze, Robert Marx, Ralf Moeller, and Vera Meyer

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Part I

Fungal Biotechnology and the Global Challenges

Chapter 1

Fungal Biotechnology: Unlocking the Full Potential of Fungi for a More Sustainable World Lene Lange, Jane W. Agger, and Anne S. Meyer

1 Introduction Fungi have a paramount presence in the common global food culture heritage and are essential for making bread, beer and wine, as well as products such as tempeh (by Rhizopus oryzae or R. oligosporus), miso/tamari (species of Aspergillus) and red yeast (Monascus spp.), widely consumed in Asia. They also provide unique characteristics and taste to a series of cheese products, e.g. blue vein cheese (Penicillium roquefortii and other Penicillium spp.) and brie and camembert cheeses (P. candidum, P. camemberti). The control of infectious diseases through use of penicillin is harnessing the rich fungal metabolome, and so are the many other fungal-derived drugs (e.g. cephalosporin, cyclosporine, statins and strobilurins). As an example, control of hyper-cholesterol in humans may be highlighted, where use of the fungal metabolite statin (and its derivatives) is among the globally most commonly used type of drug. The so-called white biotechnology is an established and growing business. It includes novel production (by filamentous fungi) of technical compounds, such as itaconic, gluconic and citric acid, and is hereby along with production of industrial fungal enzymes dominating the market of fungal biotechnology. The next steps in white biotechnology are already on their way. The producers of these components (often in collaboration with academia) are developing the business by expanding it into a production of platform chemicals and fungalderived building blocks for materials, many of which could potentially replace petroleum-based chemistry (by drop in products) but also lead to developing materials with new value-adding functionalities. Notably, the residual fungal biomass

L. Lange (*) LLa-BioEconomy, Research & Advisory, Valby, Copenhagen, Denmark J. W. Agger · A. S. Meyer Department of Biotechnology and Biomedicine, DTU Bioengineering, Technical University of Denmark, Lyngby, Denmark © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_1

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from such commercial-scale fungal productions is in general not upgraded to highervalue products; most common use is as feedstock for biogas production. Fungal enzymes are considered essential for converting lignocellulose and other types of plant biomass to valuable products (Gupta et al. 2016). Fungal genomes appear an almost inexhaustible source for discovery of new enzymes, when considering how many enzymes are already found, described and brought in use, from tapping into only a minute part of the fungal kingdom. However, use of fungi today goes far beyond these examples and includes employing fungi as cell factories or as production hosts for manufacture of different products and metabolites (Amor et al. 2016; Martins-Santana et al. 2018). The recent literature is abundant with new genomic analyses revealing the huge potential of using fungal biosynthetic gene clusters for production of different natural compounds (e.g. Li et al. 2016; Nielsen et al. 2017). A noteworthy classical case is production of human insulin (in baker’s yeast), griseofulvin (an antifungal antibiotic) and an array of enzymes (and blends of enzymes) produced recombinantly in primarily Aspergillus spp. and Trichoderma spp. (see Fig. 1.1). In the twentieth century, use of fungal products has moved substantially into a new industrial era. From approximately 1985 onwards, the industrial biotechnology sector developed methods for large-scale production of a wide range of enzymes, making it possible for agroindustrial sectors in particular, such as textile and leather industries, to switch from chemical processing to the milder, more environmentally benign enzyme processing. In parallel, processes for commercially viable fungal production of chemicals were developed. Now the world is at the doorstep of the new bio-based society, where fossil-based products are to be replaced by bio-based products. We have entered the postgenomic era offering new methods for genomic investigations and genetic engineering, hence paving the way for developing sustainable biological solutions to important problems to take over from the environmentally burdensome use of synthetic chemicals. In this chapter, we provide an overview of the many areas where fungi and fungal products (metabolites and enzyme proteins) can contribute to solving a wide spectrum of the current global societal challenges. However, we go beyond such an overview in offering updated information and visions of the future that new breakthroughs in fungal biotechnology can open up for solving important global challenges. The areas of climate change mitigation and adaptation, where fungi and fungal products could contribute significantly, are very broad. They include many areas of more value adding than production of biofuels for substituting for fossil-based products. Energy is too low in price to be commercially viable to produce as the only value-added product from biorefinery conversion of a given biomass. Notably, we will have less focus on the field of emerging threats from fungi, so excellently reviewed recently by Matthew et al. (2013).

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Fig. 1.1 “Fungal Hall of Fame” illustrating the five types of fungi, which are the most important players in industrial lignocellulose biorefinery processing: (a) Aspergillus oryzae, which along with (b) A. niger and A. nidulans, are the most widely used monocomponent enzyme production organisms. (c) Trichoderma reesei is, due to its exceptional secretion capacity, the preferred production host for enzyme blends specifically designed for efficient biomass conversion. (d) Saccharomyces cerevisiae, the organism of choice for production of ethanol from the biomass conversion-derived sugar platform. Pichia pastoris, the expression host most often used for producing laboratory-scale volumes of newly discovered enzymes, to facilitate characterization and evaluation of the new enzymes for industrial potentials. (e) Myceliophthora thermophila (along with another thermophilic fungus, Thermoascus aurantiacus), are representing alternatives to production by species of Aspergillus. Credits: (a) Courtesy of Reinhard Wilting, Novozymes A/S; (b) from Read ND, in Electron Microscopy of Plant Pathogens (Mendgen K, Lesemann D-E, eds.), Springer-Verlag, Berlin, Germany, 1991, with permission; (c) U.S. Department of Energy Office of Science (http://www.jgi.doe.gov/sequencing/why/Treesei.html); (d) Sciencephoto.com; (e) courtesy of de Vries R, CBS-KNAW, Fungal Biodiversity Centre, The Netherlands (Source: Lange L: Fungal enzymes and yeasts for conversion of plant biomass to bioenergy and high-value products. Microbiol Spectrum 5(1): doi: https://doi.org/10.1128/ microbiolspec.FUNK 2016)

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2 New Tools and Technologies: Making It Possible to Do More The new bio-based bioeconomy era is built on a strong foundation of technologies for large-scale, biological (fungal or bacterial) recombinant production of stable proteins. Developing the industrial biotechnology sector to be a competitive business segment has required process conditions and yields to be refined beyond what had been seen as possible just 25–30 years ago. These efforts were made out of necessity in order to match the cost structure of industrial chemical processing of bulk products. However, the core technologies for biological production and bioprocessing are now complemented by a broad set of newer technologies. The development of the field of genomics has been of paramount importance and includes affordable and fast genome sequencing, affordable and efficient production of codon-optimized synthetic genes, provision of shortcuts and robust systems for successful recombinant production of enzyme proteins and, more specifically, optimized strains of, e.g. the yeast expression system (Pichia pastoris), for use in research laboratories. Similarly, the development within the field of metabolic engineering has led to advanced production of higher-value products in, e.g. baker’s yeast. New gene editing technologies such as CRISP-Cas 9 will add momentum to this field. However, synthetic biology goes beyond this in using more disruptional, molecular technologies for creation, e.g. of new types of cells and new types of functions, not found in nature. Genome sequence analysis for direct sequence-based annotation of carbohydrate active enzymes and discovery and characterization of new enzyme activities are continuously being refined through a range of different approaches [blast searches, sigP, pFam, HMM, dbCAN, PPR (Busk and Lange 2013), Hotpep (Busk et al. 2017) and, the newest, SACCHARIS (Jones et al. 2018), dbCAN2 (Zhang et al. 2018) and lastly CUPP (conserved unique peptide patterns), an automated, peptide-based functional annotation method (Barrett and Lange 2019)]. The most recent version of the dbCAN2 server (Zhang et al. 2018) integrates other tools for annotation of carbohydrate active enzymes (CAZymes): (1) HMMER for annotated CAZyme domain boundaries determination according to the dbCAN CAZyme domain HMM database, (2) DIAMOND for fast Blast hits in the CAZy database and (3) Hotpep for short conserved motifs in the Peptide Pattern Recognition (PPR) library (Meaning of this list of acronyms appears from the quoted papers). All these approaches to sequence analysis are tailor-made to be able to derive information and knowledge from genome data. Emphasis, attention and focus over the last decades have been on a rapidly growing number of genome-sequencing projects; notably, the 1000 fungal genome project was made possible by a broad concerted effort from European and American mycologists https://genome.jgi.doe. gov/programs/fungi/1000fungalgenomes.jsf; also sequencing efforts by the Beijing Genomics Institute (https://www.bgi.com/global/) have contributed significantly. The focus on DNA sequencing has pushed transcriptomics a little out of focus and has almost obliterated the use of cDNA technologies. However, there is likely to

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be renewed interest and refocus on transcriptome studies (by RNA sequencing) and on proteome studies, especially for eukaryotes (including fungi). Understanding of the transcriptome and proteome opens for insight into functionality and action mechanisms of the proteins. It hereby provides significant input to (1) discovery of new mechanisms and strategies used by the fungi in nature to, e.g. degrade plant materials (e.g. Kuuskeri et al. 2016; Miyauchi et al. 2018); (2) understanding and combatting plant diseases (e.g. Collins et al. 2017); and not least for (3) achieving significant advances in the next-level biotechnological processes, i.e. improved production and use of enzymes and metabolites in bioprocessing. Research support functions have been established and widely used globally [CAZy (Carbohydrate-Active Enzymes database), JGI (Joint Genome Institute), EBI/EMBL (European Bioinformatics Institute), dbCAN (database for automated carbohydrate-active enzyme annotation), BGI (Beijing Genome Institute)]. Furthermore, as regards research-based culture collections, valuable collections have in recent years been given up due to change in priorities combined with high maintenance costs. However, many have succeeded to modernize their technologies and collections (taking onboard also molecular approaches), thus upholding their position as invaluable assets for both basic and applied research. Another strong trend in methodological approaches, which function as enabler for the new bio-based era, is in a holistic approach aiming for conceptually increased biological understanding. This can be done by integrated use of biological methods along with structural biology methods and with refined chemical/physical methods like NMR, MS and bioimaging. Additional trends are the move from studying a genome in isolation to studying comparative genomics and, not least, studying interactions between organisms, as well as gene regulation and prediction of gene function. By all such measures, it is possible to convert the overwhelming amount of data into information and even to new biological understanding. Rapid growth of computer power and speed and ease of bioinformatics analysis enable us simultaneously to analyse all published fungal genomes (approx. 2500) on a normal laptop computer. Seen from an applied perspective, development of the biomass conversion processes as a basis for the new bioeconomy has moved ahead quickly and efficiently due to the pre-eminent CAZy database. Furthermore, enzyme performance has been improved through sophisticated and integrated uses of 3D structures, protein engineering, artificial evolution, machine learning and robotics. This rapid increase in versatility and accessibility of methodological tools and approaches has enabled the overall field of mycology to reach a higher level, not only generating new data but also providing relevant information to enable us to answer conceptually interesting mycological biological questions. In the last decade, we have moved from developing tools and methods to using them in an integrated manner: bringing us “back to biology”. However, unlocking the full potential of the fungal contribution to the new bio-economy will require many more biotechnology breakthroughs. Five general trends are noteworthy (see Table 1.1).

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Table 1.1 Five key fungal biotechnology trends in relation to exploitation of fungi and fungal enzymes and metabolites for solving societal challenges No. I

II

III

IV

V

Principle Combine datasets, e.g. genomic, transcriptomic, proteomic and phenotypic data Combine genomics and explorative experimental methodological approaches

Expand recombinant production (and use) to complex proteins (e.g. membrane proteins, molecular pumps) Broaden enzyme classification and functional annotation to more enzyme classes, going beyond carbohydrate active enzymes

Elucidate not only the organismal composition but also the molecular function of microbiomes, including not only prokaryotes but also eukaryotes

Goal Identify genetic basis for organismal and molecular diversity; and gain improved insight into protein function Discover new enzyme functions, interactions and mechanisms. Find and understand synergies between exoproteome and exometabolome Learn from and harness nature’s microbial strategies to solve problems by new transformative uses of biology-based principles Expand options for exploiting enzyme catalysis in new processes enabled by function-targeted (in silico and experimental) discoveries making more types of proteases and lipases available for biorefinery upgrade beyond lignocellulose Expand the knowledge base of biological microbiome strategies and functions, leveraged to, e.g. facilitating development of food and feed ingredients with prebiotic, gut-health-promoting effect

I. Making it possible to combine many types of data sets, not just genomes but also transcriptomic, phenotypic, physiology (anaerobic or aerobic), lifestyle (saprotrophic, biotrophic, etc.), substrate specificity and affinity, and making it possible to predict the composition of the fungal interaction secretome through using state-of-the-art sequence analysis, e.g. the CUPP method (Barrett and Lange 2019). II. Combining several types of methodological approaches, e.g. experimental enzymology, NMR, mass spectrometry, bioinformatics, chemistry and separation and recovery technologies. III. Expanding recombinant production capabilities to express enzymes and handle metabolites far beyond the beaten path of core sets of ascomycetous products; even industrial use of the membrane-bound proteins is within reach! IV. Expanding the types of enzymes, we are able to handle; here, we need to make huge explorative steps, taking in new virgin land within proteases, lipases, esterases and transferases. V. And not least, enabling us to master understanding the composition, function and use of the complexity of microbiomes often composed of a broad organismal and funcional biodiversity of fungi, protozoa and bacteria.

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3 Learning from Nature The methodological approaches to understanding the role of fungi in nature are legio, and the data generated risk overwhelming us, blocking the view of patterns and new conceptual understanding of essential but complex biological features and integrated interactions. For moving quickly ahead, it is clever and smart to collaborate across different disciplines to learn what nature has developed to perfection in the fungal kingdom over evolutionary time. Such interdisciplinary research may target new high-throughput methods, e.g. in the form of developing new expression platforms, as recently done using Saccharomyces cerevisiae for fast assessment of fungal natural products (Harvey et al. 2018). Learning from nature may also involve understanding species diversity by comparing genome sequences (predicted genes), protein function and enzyme profiles against phenotype of fungi (see, e.g. Vesth et al. 2018). Knowledge about fungal physiology in nature combined with use of novel functional classification systems for enzymes can identify hot spots for discovery of specific functions of enzymes and metabolites (see, e.g. Busk and Lange 2013; Agger et al. 2017). Of crucial importance is also detailed reaction analysis of relevant enzymes to provide the decision base for possible practical exploitation in various biorefinery processes (see, e.g. Mosbech et al. 2018; von Freiesleben et al. 2018). We can provide shortcuts to understanding molecular functions by studying signature species, which have distinct physiological specialization (as done, e.g. in the recent studies by Collins et al. 2017; Kuuskeri et al. 2016; Miyauchi et al. 2018). We can recognize differences and similarities between anaerobic and aerobic life forms and compare ancient zoosporic fungi with the dikaria asco- and basidiomycetes to unravel the basal and extremely diverse part of the fungal tree of life (Fig. 1.2). We can observe patterns of optimized use of CBMs, linkers and LPMOs, in specialized fungi, and we can elucidate the wonders of the sophisticated co-evolution of the fungus-gardening termites and leafcutter ants (see Fig. 1.3); and what is more, we can harness the wealth of enzyme discoveries from such systems, which represent nature’s prototype of the yellow and the green biorefinery, termites and ants, respectively. In all of such ways, we can learn and make use of genomics data much better by learning from nature. We can further identify new ways to harness the mechanisms and products used by fungi in nature to help develop a more sustainable future.

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Fig. 1.2 Phylogeny of the cellulose active enzyme, glycohydrolase 5 (GH5) of early lineage fungi. The GH5 (EC 3.2.1.4) 1,4 β-endoglucanases of the chytrid R. rosea are embedded in an “all-fungal” clade, containing also ascomycetous, basidiomycetous and zygomycetous GH5 endoglucanases. GH5 endoglucanases of anaerobic rumen fungi (Neocallimastigomycota) are found in a clade dominated by rumen bacterial GH5 proteins. Besides these two major occurrences of early lineage fungal GH5, one copy of R. rosea GH5 is found in a predominantly bacterial GH5 clade (to the left) together with one GH5 protein from a zygomycetous species (Umbelopsis ramanniana) and an Archaea (Pyrococcus abyssi) GH5 protein. Two possible horizontal gene transfer events are indicated (between rumen bacteria and rumen fungi and between rumen bacteria and rumen protozoa). Colour code: purple, early lineage fungi; red, Zygomycota; green, Ascomycota; orange, Basidiomycota; blue, bacteria; turquoise, Archaea; dark green, plants; black, animals (Source: Lange, L., Pilgaard, B., Herbst, F.-A., Busk, P.K., Gleason, F., Pedersen, A.G., 2018. Origin of fungal biomass-degrading enzymes: Evolution, diversity and function of enzymes of early lineage fungi. Fungal Biology Reviews. https://doi.org/10.1016/j.fbr.2018.09.001

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Fig. 1.3 Leafcutter ants are growing and feeding a fungal mycelial colony in their subterraneous nest. Enzyme analysis of the layers of the leafcutter ant’s fungal garden has documented that the symbiosis between the leafcutter ant the fungus enables full decomposition of the lignocellulosic biomass (Source: Lange, L and Grell M: The prominent role of fungi and fungal enzymes in the ant–fungus biomass conversion symbiosis. Appl. Microbiol. Biotechnol. 98, 4839–4851, 2014. DOI https://doi. org/10.1007/s00253-014-5708-5)

4 Global Challenges Where Fungi Can Contribute to Solutions 4.1 4.1.1

Climate Change: The Role of Fungi in Mitigation and Adaptation Fungal Enzymes

Microbial enzymes are essential for developing and producing bio-based substitutes for fossil-based materials, chemicals and fuel. In nature, evolution of efficient biomass-converting enzymes is an integrated part of the evolution of the entire fungal kingdom. The heterotrophic fungal lifestyle relies on plant materials being broken down into smaller building blocks that provide the basis for the fungus to grow and reproduce. Further, through evolution, filamentous fungi have developed the most efficient secretion mechanism to make enzymes available for breaking down the substrate into which fungal hyphae grow. Large-scale production of

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industrial enzymes is competitive (also when sold as bulk products) due only to efficient secretion of the enzymes, paving the way for low-cost protein recovery. The biomass conversion needed to make new bio-based products as substitutes for fossil-based products builds on the success of the industrial biotechnology, which is substituting chemical processing in forestry and agroindustries (e.g. textiles, juice and paper and pulp). Development of industrial biotechnology required optimized recombinant production of fungal enzymes at an industrial scale. Species of Aspergillus and Trichoderma became the production hosts of choice. Globally only rather few production hosts have been developed for large-scale use. The canonical fungal species in the biological production “Hall of Fame” are few (see Fig. 1.1). Furthermore, sophisticated protein engineering and the integrated use of artificial evolution enabled the production of even more efficient and stable enzymes, which were suitable for use in industrial processes and for global commercialization as bulk products. The new bioeconomy, which will produce bio-based products as substitutes for fossil-based products, builds on such biotechnological methods. The enzyme part of the classic industrial (white) biotechnology is, basically, the era of one gene, one protein, one product, one solution. However, enzymatic conversion of complex and often recalcitrant biomass (e.g. lignocellulose or keratin) for the new bioeconomy requires not just one enzyme at a time but an entire, carefully designed blend of enzymes (Merino and Cherry 2007). The marketed enzyme blends for biomass conversion are usually produced in one multigene recombinant production host (Trichoderma spp.; the Accellerase type of blends (Dupont) and the Cellic-CTec type of blends (Novozymes)). Notably, producing recombinantly one enzyme at a time would be too costly (e.g. in fermentation tank year investments) and would therefore not comply with the cost structure of bulk enzymes. However, optimizing both the blend composition and production technology remains a biotechnology challenge: the enzymes needed for biomass decomposition must be identified, and their combined action must be optimized to constitute “minimal cocktails”, i.e. the minimal number, the minimal levels and the optimal combination of the best performing enzyme activities for the purpose (Bunterngsook et al. 2018; Meyer et al. 2009). Pretreatment procedures need to be adjusted to ensure enzyme access to substrate. The blend needs to be designed; the production host needs to be constructed. Fermentation conditions need to be optimized so that a bottleneck is not introduced, by a single enzyme being present in too low amounts, which will limit commercial use of the blend. Furthermore, in the years to come, enzyme blends must be developed for decomposing the entire spectrum of new types of biomasses, residues and sidestreams with valorization potentials, hereby going far beyond the wheat straw, corn stover and wood chips currently exploited. There are many biotechnology challenges and opportunities for optimizing even further the production of bio-based substitutes for fossil-based products, and new industrially relevant kinetic phenomena, hypotheses and mechanisms underlying concerted enzyme functions for efficient conversion of complex biomass continue to emerge (Kari et al. 2018; Lange et al. 2016a, b; Pierce et al. 2017). Three examples are chosen to illustrate the new biotech opportunities for further improvement of enzymatic conversion of biomass.

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1. The recent discoveries that the lytic polysaccharide monooxygenases (LPMOs, belonging to the auxiliary activity enzymes) play a key role in natural (primarily fungal) degradation of recalcitrant lignocellulosic. This has opened the way for a whole, new approach to improved efficiency in biomass conversion (Bissaro et al. 2018). However, to unlock the full potential of the LPMO technologies is not a trivial matter. Most LPMOs are difficult to express recombinantly, and process efficiency will most likely benefit from including not just one but a number of synergistic LPMOs in the blend. Nor is it straightforward to make an informed choice of which LPMOs to select because the different functions of the many LPMOs often present in efficient fungal biomass degraders are not yet fully elucidated. As an example of LPMO complexity, it has recently been established that in 98% of publicly available fungal genomes, the number of AA9 (LPMO) genes is larger than the sum of the genes encoding for GH6 and GH7 cellulases (Lenfant et al. 2017), and, e.g. a white-rot basidiomycete fungus, Schizophyllum commune has 22 AA9 encoding genes (Ohm et al. 2010; Knabe et al. 2010). 2. The importance of the CBM (carbohydrate-binding module) for enzyme function has been extensively studied (see, e.g. review by Shoseyov et al. 2006). New progress within CBM technology has revealed unexpected new functions that do not relate to the original biological roles of the enzymes (Armenta et al. 2017). CBM engineering could lead to improved blends of cellulose-degrading enzymes. This could, for example, be achieved by introducing different types of CBMs in different positions (N- or C terminal, or both) bound to linkers of varying length, but this remains an underexploited field. 3. Construction of integrated and flexible biorefinery conversions that allow recovery, for example, of protein content, first and finally convert only the fibres into a sugar platform for production of chemicals, materials and fuel. Further, through a cascading conversion of the biomass, other components of the plant biomass could be converted into higher-value products such as feed additives from the hemicellulose and functional materials (e.g. binders) from the lignin (Silva et al. 2018).

4.1.2

Significant Reduction of CO2 Emissions per Ton Food and Feed Produced

Significant reduction of CO2 emissions of each ton of food or feed is possible by efficiently using all parts of the plant products harvested. This can be achieved by cutting down the huge losses that occur all over the world in food and feed value chains. Currently as much as 35–50% of the total harvest is going to waste globally: protein rots in the field. Industrial sidestreams burden waste water streams instead of being used as raw materials for new food, feed, other materials and energy; upgraded use of the organic part of municipal waste and sludge is not pursued even though technologies have been developed, such as production of organic acids, building blocks for bioplastics, oils, etc. Fungal enzymes are essential for the majority of such

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upgrading processes and can improve the overall efficiency of our use of biological resources.

4.1.3

Significant Reduction of Methane Emissions

Significant reduction of methane emissions from meat and dairy production must be attempted. So far, efforts directed towards reduction of methane from ruminants have primarily been conducted with the aim of improving feed conversion efficiency. However, at time of writing, the focus is changing and is now also directed towards reducing methane from cows in order to deliver the required and demanded reduction of greenhouse gas emissions from this type of agricultural production. Basically, two different approaches can be pursued (independently or combined) for reducing methane from cattle production: one is through targeted genetic cattle breeding; the other is the feeding strategy approach. For the latter, fungi are key in two different ways: the rumen fungi are part of the healthy and non-methaneproducing rumen microbiome; and fungal enzymes could possibly also be of importance in developing new (prebiotic), methane-reducing feed additives for ruminants. The enzymes of anaerobic, zoosporic rumen fungi have been studied in detail in the past and provided interesting results and insights; this field has recently been revitalized in relation to possible utilization for biogas production (see, e.g. Cheng et al. 2018). However, the understanding of the role of rumen fungi in the rumen microbiome has not yet benefitted significantly from the development of genomics. The majority of the microbiome studies of livestock gut and rumen microflora have included only the bacteria; the fungi (and the protozoans) have been excluded by the sequencing protocol followed, which has presented genomic DNA of prokaryotes, bacteria and archaea only for rumen microbiome sequencing. From earlier studies of rumen fungi, we know that rumen fungi inhabit the small rumen particles (primarily composed of plant cell wall materials, at μm or mm scales); the fungal rhizoids open up the plant cells and thus expose much more of the plant biomass polymers to bacterial decomposition (Nagpal et al. 2009). The cellulose-degrading enzymes of the rumen fungi, recently reviewed (Lange et al. 2018), form an intrinsic and complex molecular structure, named the fungal cellulosome. In the cellulosome, all the enzymes needed for breaking down plant cellulose polymers are integrated. The catalytic part of the rumen biomass-degrading enzymes has been shown to be of bacterial origin, acquired by the rumen fungi from the rumen bacteria through horizontal gene transfer (Haitjema et al. 2017; Lange et al. 2018). However, surprisingly, the dockerin and the structuring components of the fungal cellulosome were by Haitjema and coworkers found to be of de novo fungal origin and do not resemble any bacterial structure. Further studies and feeding experiments are needed to reveal if methane reduction can be achieved by adding components with prebiotic effect to animal feed, thus stimulating the non-methaneproducing part of the microbiome (including the rumen fungi). In addition, the prebiotic feed additive could be further enforced by adding a probiotic element,

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for example, by enriching the rumen fungal microflora by adding inocula of rumen fungi (Nagpal et al. 2009).

4.1.4

Improved Resource Efficiency Can Free Land for Forestry and Biodiversity

Using fungal enzymes to convert hitherto underutilized biological resources to feed could lead to indirect land use changes and free land for other purposes such as for CO2 sequestering (by planting more trees) or for providing space for biodiversity to flourish. Currently, around 75% of all arable land in the world is used for production of animal feed. If we succeed in converting especially industrial sidestreams into nutritious animal feed (e.g. upgrading the protein fraction of rapeseed press pulp or sunflower press pulp to protein-rich animal feed), we will be able to free more land for growing forests and lessen the pressure on rainforest to be converted into agricultural land. This will in itself be one of the most efficient routes to follow in order to mitigate climate change. The biotechnology challenges to achieve this are covered above. However, new and improved technologies are not sufficient. We must also change the policies and the incentive structure to ensure climate change mitigation to happen.

4.1.5

Using Misplaced Carbon Resources to Produce Single-Cell Protein

There could also be a possible role for fungi in the new negative emission technologies (NET). The world is facing serious difficulties in keeping global warming under 1.5  C. This calls not only for a reduction in the greenhouse gas emissions but also that we introduce new technologies which give a net deficit of emissions, i.e. by drawing more emissions out of the atmosphere through carbon-capturing technologies. A highly interesting use of captured methane is achieved by using the methane as a substrate for growing bacteria and thus producing single-cell bacterial protein (see https://www.unibio.dk/). This type of sustainable NET approach, carbon capture and use (CCU), has already been upscaled. The bacterial protein-rich, methanebased animal feed product has been approved in the EU since 1995 according to Commission Directive 95/33/EC. However, new opportunities can be developed that use the resources even more efficiently. It would most likely be feasible and efficient to grow biomass, for example, of fungal baker’s yeast, on the residues of the bacterial biomass after the protein has been extracted from the bacterial biomass. Fungi can produce new protein content provided minerals, and C and N sources (plus some accessible sugars to get started) are available. Further, and most importantly, baker’s yeast (called yeast cream when used for animal feed) has already been documented to be a highly nutritious feed, for example, for pigs. Large-scale application of yeast biomass from biological production has been used widely in Denmark for more than a decade as feed in Danish pork production.

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New Fungal Products Are Instrumental for Climate Change Adaptation

Climate change-induced impact on patterns of rainfall is challenging agriculture in many parts of the world. The latest reporting from IPCC (Intergovernmental Panel on Climate Change) reporting specifically underlines that there is a high risk of reduced agricultural yields, for example, in sub-Saharan Africa, and an IPCC expert group has identified West Africa as a particular “climate change hot spot” to experience significant negative impacts from climate change on crop yields and production (Hoegh-Guldberg et al. 2018; Masson-Delmotte et al. 2018). The need for adaptation to global warming and unpredictable rainfall is pressing, and additional compensating food and feed resources may be needed. However, developing and using new fungus-derived products can play a significant role for both adaptation and compensation. More specifically, new products, similar in effect to the JumpStart product (Novozymes A/S), could be instrumental in also adaption to the occurrence of drought conditions in the critical period after crop seeds have germinated. JumpStart is a fungal inoculant sown together with the seed. The causal agent, a filamentous fungus (a species of Penicillium), establishes its mycelium around the roots of the crop seedlings where the mycelium functions as a set of add-on filamentous “roots” and increases water efficiency. Furthermore, the selected types of Penicillium species have one more significant specialized characteristic. The fungi impact on the soil around the roots, solubilize the hitherto non-accessible soil resources of phosphorous and thus make phosphorus more accessible for the plant. This de facto leads to increased nutrient efficiency of the plants. Together, these two effects—increased water efficiency and improved nutrient efficiency—act as a plant strengthener and enable the plant to have greater tolerance towards periods of especially “spring” drought. Based on this very promising example, one additional climate positive effect of fungal inoculants can be hypothesized. Wheat plants have been shown to be highly sensitive to rise in temperature. So far, breeding efforts have not been successful in overcoming this detrimental risk imposed by global warming. It is apparently an inherent characteristic of the wheat plant to be sensitive to higher temperatures especially during flowering, an inherent wheat trait, which is found difficult to change through breeding. Based on the positive effect of Penicillium inoculum (described above) experiments could be done to elucidate if plant strengthening fungal inocula (or a combination of fungal and bacterial inocula) could have a positive effect on wheat plants through conferring a higher tolerance of elevated temperatures. For more details of how to get more out of the harvest, waste significantly less, and thus compensate for decrease in crop yield due to climate change-challenged agriculture, see below (Sect. 4.2).

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Understanding the Threat of Loss of Biodiversity Due to Global Warming

In recent years, new epidemic diseases caused by fungi have been spreading within wild populations of bats, toads and snakes; the diseases move and spread to such an extent and speed that they threaten habitat biodiversity, survival of populations and species and may be even be a threat to signature life forms. It is still not clear if such epidemics are due to changes in the fungal pathogens becoming more aggressive through mechanisms such as enzyme mutations or HGT, horizontal gene transfer (Rosewich and Kistler 2000), or are due to climate change possibly weakening animal host defence responses (or a combination of both). Increased molecular understanding of fungal pathogenesis and epidemiological spread of such fungal infections is a necessity for creating a basis for forming a strategy for how these new diseases may be combatted. In recent years, the most severe fungal epidemic has been the attack by the chytrid Batrachochytrium dendrobatidis on toads, which is endangering populations of toads in California, for example. Similarly, the white nose fungal-based syndrome has spread in population of bats, such as those on the east coast of the USA, and most recently, the fungal snake disease has raised alarm and concern in the USA, Australia and Europe (Franklinos et al. 2017). The white nose syndrome has been carefully studied to reveal that the causal agent is the ascomycete fungus Geomyces destructans (Lorch et al. 2011). Very recently, another new disease is spreading, invading and weakening populations of wild snakes (named snake fungal disease, SFD). The causal agent of SFD is believed to be the fungus Ophidiomyces ophiodiicola (formerly known as Chrysosporium ophiodiicola) (Lorch et al. 2015). Also, for this potentially devastating fungal attack on wild snakes, we still do not know, whether it has developed due to changes taking place in the fungal pathogen (e.g. through developing or acquiring more potent proteases, which enables it to also invade intact and healthy snake skin) or whether SFD attacks are due to climate change, making the snakes more susceptible to skin-invading fungi. Concerted efforts should be invested into understanding these globally most devastating, life-threatening attacks on vertebrates in general. Such increased understanding could help us be prepared for the situation where fungal dermatophyte attacks on humans also become more serious due to global warming (maybe in synergy with chemical pollutants).

4.2 4.2.1

Feeding a Rapidly Growing Global Population Using the Biological Resources More Efficiently

The most straightforward efforts to enable us to feed the rapidly growing global population is to use a greater part of the bio-resources, which now go to waste. This can be done by applying the set of (fungal-derived) enzymatic biomass conversion

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technologies developed within the scope of the new bioeconomy for upgrading residues, sidestreams and wastes. The result would be significantly improved resource efficiency, with far more responsible use of global bio-resources where crop residues and industrial sidestreams are seen as new raw materials for producing more animal feed and food. By doing this, one could also focus in an integrated manner on developing a new type of biorefinery products—the much-needed soil improvers. The three most well-known soil improvers already in use are (1) the product “biochar”, (2) the residual after biogas production and (3) the organic part of the sludge after waste water treatment (holding a significant amount of phosphorous, upconcentrated in the microbial biomass). Furthermore, fractions of partially degraded lignocellulose, after the free sugars have been used for production of bioethanol, materials or chemicals, may be optimized for use as soil improvers as well. Through employing soil improvers, the fertility of an extensive portion of the world’s agricultural land, which currently does not receive sufficient fertilizer, could increase, even in a cost-efficient manner, upgrading available local bioresources. The best way of communicating the immense potential within this area is by giving concrete examples. An industrial example of improved use of biological resources is found in the dairy sector. Here, use of the leftover products from cheese manufacturing, the whey, has provided basis for a wide range of new higher-value products such as food ingredients for premature babies, pregnant women, infants or athletes. Whey-based products are also used in ice cream, in “light” products as well as in low-fat cheese (see, e.g. Arla Foods, https://www.arla.dk) (see also Nordic Council of Ministers 2017). Another very successful industrial development is the transformation of potato starch production to a modern biorefinery process (see, e.g. http://www.kmc.dk/). Fungal (and bacterial) enzymes are also cornerstones for such upgraded use of industrial sidestreams from, for example, forestry, textiles, brewery, milling, etc. However, it can also be the fungal organism itself, which is used directly as the new biomass. A striking example of value-added use of an underexploited bio-resource is from Ghana (Adu-Amankwa 2006) where cassava peel is used as substrate on which to grow baker’s yeast and in that way produce nutritious, protein-rich animal feed based on the fungal biomass. A similar concept has been developed in Norway to produce protein-rich yeast cream for salmon fed by growing yeast (Candida utilis) on wood paste mixed with brown algae; the algae are added to provide an N-supply for the yeast growth (Sharma et al. 2018). The potentials are numerous within the broad field of upgrading sidestreams from processing products from agriculture, fisheries and forestry. In a nutshell, this is the most promising field of all efforts to transform the world towards a more responsible consumption of global bio-resources, lifting the biomass resource from wood paste to protein-rich, fungal-based healthy animal feed. And, basically, the technologies are ready now for local development and upscaling.

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Develop, Recover and Refine Local Protein Resources

A significant part of the European meat production is based on imported protein-rich feed products such as soy. This import takes place at the same time as significant local European protein resources are either underexploited or go to waste. Such new local sources of proteins can come not only from plants but also from fungi, algae, insects, invasive animals, etc. The current European focus is on growing more local protein-rich plant crops; however, fungal enzymes also have a role to play in such attempts. A current trend is to consider changing agricultural practices to grow green grass and clover (as feedstock for a green biorefinery) instead of cereals in the northern part of Europe. The green biorefinery provides a shortcut to improved use of the land through longer growing seasons and to reduced pollution from agricultural land due to more active root systems over a larger part of the year. The processing of the green grass and clover is simple—screw press processing—and results in a juice and a pulp fraction. Here again, fungal enzymes can play a role. Besides precipitation of the soluble enzymes present in this juice, an additional 40% protein can be extracted from the green pulp (Dotsenko and Lange 2017) by a simple and low-cost protease enzyme treatment using a broadly acting protease (Savinase from Novozymes A/S; this enzyme is a Bacillus-derived subtilisin protease). The search for increased use of alternative local protein resources is moving also beyond protein-rich crops. Sources of new and alternative proteins can also come from macro- or microalgae [extracted by fungal enzymes (Dotsenko and Lange 2017)], from insect larvae (Belghi et al. 2018), from starfish (Mazorra-Manzano et al. 2018; Sørensen and Nørgaard 2016) or from fungal single-cell proteins (Sharma et al. 2018). Examples of production of single-cell fungal proteins are given under Sect. 4.1.5.

4.2.3

Gut-Health-Promoting Food Ingredients and Animal Feed Additives

Genome sequencing of gut microbiomes coupled with the need for improved health and welfare in both man and animal has led to concerted efforts to develop new types of food ingredients and animal feed additives. Gut-health active compounds were, however, known before sequencing of the microbiomes was performed. The most well-known concepts of such first-generation gut-health products were nutritional fibre products from corn, which were especially commonly used in the last century in India, or more specifically products such as XOS and FOS, acronyms for xylanbased oligosaccharides and fructose-based oligosaccharides, respectively. A new generation of such products has recently emerged, which again is partially based on use of fungal enzymes. The evidence-based development of such new gut-health ingredients has been made possible by and coincided and in synergy with sequencing of the gut microbiome.

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Feed ingredients with prebiotic action are coming within reach at the same time as the world is experiencing a serious rise in antibiotic resistance believed to be caused by rising use of antibiotics in both industrial livestock production and as drugs for combating infectious diseases in man. Concerted efforts are now being invested in trying to develop feed products, which could strengthen the animal gut flora and reduce the need for (also prophylactic) antibiotic treatments. The results are very encouraging. Short oligosaccharides prepared by enzyme treatment of polysaccharides (using fungal endo-xylanases, Dotsenko et al. 2018) have been shown to have a positive effect on the composition of pig gut flora. Such results have been obtained by in vitro fermentation of pig gut microbiome. Similarly, co-fermented and enzyme-treated seaweed and rapeseed press pulp have been shown in large-scale, on-farm experiments to lead to significant improvement of the pig gut flora (fermentation expert https://fermentationexperts.com/). Such initiatives are put in the ambitious perspective, enabling reduced use of antibiotics in industrial meat production, and as a means towards substituting also for zinc treatment. Overall, the field of prebiotic and probiotic feed ingredients as mode of actions for improved gut health is moving forward (de Lange et al. 2010). Such results may usher into a new era within animal feed, which paves the way for reducing and replacing the use of antibiotics in livestock production, including all types of non-ruminant animals, pigs, chicken and fish, and possibly also suitable for cattle with a prebiotic perspective also for reducing methane emission (see Sect. 4.1.2). In human health, numerous but still preliminary results from medical studies suggest that a healthy gut flora lowers the risk of lifestyle diseases as well as of a number of other types of serious diseases, not least inflammatory bowel disease. As a consequence, of such highly promising and inspiring results achieved from gut microbiome studies, a series of new gut-health-promoting food ingredients are being developed. The most promising effects seem to come from combining the effect of both prebiotic compounds (e.g. produced by fungal enzymes and favouring the healthy part of the gut microbiome) and a probiotic component that adds live bacteria, such as lactic acid bacteria, to the feed additive. Further improvement may be achieved by administering the probiotic inoculum so that it is accompanied by its favourite substrate and by ensuring presence of enzymes to convert feed substrates into more compounds of prebiotic effect, while even better results may be achieved by including microbial products, which also have additional anti-inflammatory effect. So far, the fungal role has primarily been the use of fungal-derived enzymes to prepare optimized food and feed additives. However, the next steps (for both feed and food ingredients) are to test whether (a) fungal cell wall-derived products could have an even stronger effect than the products derived from plant cell walls (i.e. notably whether the fungal cell wall has relevant chitin, mixed linked-glucans and beta-glucans, that may be instrumental for giving the prebiotic effect) or (b) whether fungal probiotic inoculum, for ruminants, for example, could have beneficial effects on the level of methane emission (and feed conversion).

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Strengthening Human Health Through Fermented Food

Scientific investigations in the last century already supported the conclusion that intake of fermented food as part of your daily staple food could have significant positive effect especially for the health of children. More specifically, such intake could prophylactically increase tolerance (foster robustness) to diarrhoea and save children from dying from diarrhoea if infected (e.g. by using traditional yeastfermented food in West Africa). Similarly, positive results have been achieved with regard to documented low risk of microbes (e.g. mycotoxin-producing species) contaminating the food fermentations when locally developed cultural traditions for preparing fermented food are followed. A modern trend in western cuisine is also to use more fermented food, including partially adopting the Asian tradition of using fungi for preparing fermented food. Food fermentations often include many different types of fungal yeasts as well as mucoromycotous fungi (such as Rhizopus oryzae), ascomycetous fungi (e.g. Aspergillus) and/or bacteria such as LAB (lactic acid bacteria). It would be highly interesting to be able to measure possible effects of intake of such modern fungal food fermentations on the human gut flora and to see whether even further improvements could be achieved by adding prebiotic-acting polysaccharide oligomers prepared from fungal cell wall materials to the fermentation. The simplest and cheapest shortcut to improved public health would seem to be a daily intake of even minor amounts of gut-health-stimulating compounds (a good choice could be fungal and/or bacterial fermented seaweeds with your morning yoghurt). Diet amendments could also be a shortcut towards making affordable health improvements in lower income and rural areas by reintroducing locally prepared gut-stimulating food and feed ingredients, made even safer by the use of modern quality control measures to avoid contamination by mycotoxin producing microbes.

4.2.5

Mushrooms and Tasty Climate-Friendly Food from Coffee Grounds

Spent coffee beans are one of the most underutilized biological resources. More than 99% of the biomass is left-behind, when the coffee has been made and the coffee ground is discarded. SME initiatives in several countries in both Europe, Asia and Africa have started to use spent coffee grounds as a new growth substrate, for example, for growing oyster mushroom (Pleurotus ostreatus) (see, e.g. Pleissner and Venus 2017; Pauli 2017; and Beyond Coffee, https://www.beyondcoffee.dk). However, even after the mushrooms have been harvested, the greater part of the coffee ground substrate is still left behind. A new project and business trend has been initiated that aims at converting the entire coffee ground biomass into a tasty food ingredient. The basic principle is that the fungal mycelium, which has efficiently invaded the coffee grounds even before the fruiting bodies are harvested, can be regrown and the entire biomass converted into a food ingredient, for example, of

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bread, soups or sauces through enzymatic decomposition (based on enzymes from P. ostreatus itself). With good regrowth of the mushroom mycelium, the entire integrated plant/mushroom biomass can by consecutive steps of biological processing (e.g. fermentation) be converted into an umami-tasting protein-rich ingredients (Lange, Lau and Wejendorp, unpublished). The next step in this endeavour could be to develop more types of fungal biomass, grown on clean and healthy organic sidestreams. Mushroom-based, umami-tasting products can make vegetarian and vegan-based food a very different experience, with appeal to and entice more consumers to switch to a more climate-friendly food source. Gourmet taste and climate-friendliness are combined.

4.2.6

Upgrading Keratinaceous Biomass Using Fungal Enzymes

Feather from chickens and bristles from pigs are globally very abundant protein-rich waste streams. When attempts are made to use these wastes for animal feed, either the processing is incomplete (e.g. by alone grinding) and leaves the material almost non-accessible to the animal metabolism or the wastes are pretreated so harshly by chemical and physical means that most of the essential, branched amino acids are destroyed and the nutritious value lowered significantly. We have identified fungal species (such as the ascomycete Onygena corvina) which in nature grow specifically on keratinaceous materials such as horn and feather (see Fig.1.4a), and we have unravelled which specific enzymes (keratinolytic proteases) the fungus is using to degrade the recalcitrant keratin product (Fig. 1.4b; Huang et al. 2015; Lange et al. 2016a, b). Enzymatic degradation of the recalcitrant keratinaceous materials is a very complicated process requiring a number of different types of proteases (at least one endo-acting and one exo-acting protease, and one protease/peptidase, acting on shorter peptide oligomers). Furthermore, we have documented that both keratindegrading fungi and the skin-invading dermatophytic fungi have an overrepresentation of the special AA11 type of the LPMO enzymes (Busk and Lange 2015). The studies of enzyme degradation of keratinaceous biomass were carried forward to testing the feed value of decomposed pig bristles. Encouraging results were achieved (Kang et al. 2018; Falco et al. 2019) and when seen in a comparative perspective, the fungal enzymes seem to be more slow acting than the bacterial enzymes. Several bacteria have been identified that have matching sets (as compared to fungal enzymes) of efficient proteases with keratinolytic effects. It thus seems feasible to carry out biological conversion of the recalcitrant protein resources locked up in hitherto underutilized protein resources such as feathers, hair, hooves and bristles. However, several biotechnology challenges remain, and we have still not found a suitable and efficient way for also using the LPMO enzymes for á possibly even more efficient conversion of keratin into nutritious animal feed.

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Fig. 1.4 (a) The keratin-invading, ascomycetous fungus, Onygena corvina here shown growing on horn; O. corvina are also described to grow on other keratinaceous materials in nature, such as feathers. (b) The proteinaceous structure of keratin can be decomposed by a synergistic effect of three proteases: excellular endoproteases (S8), exoproteases (M28), oligopeptidases/ metalloproteases (M3) and a sulphite/disulphide reductases (Source: Lange L, Huang Y and Busk PK: Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Appl. Microbiol. Biotechnol. 100: 2083–2096, 2016)

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The Important Role of Fungi for Improved Health Mycotoxins and Discovery of New Fungal-Based Drugs

Certain genera of fungi have a high diversity of potent, biologically active secondary metabolites. This includes the highly toxic mycotoxins (some of which are more toxic to man than any man-made synthetic toxin); however, it also includes a very wide and hitherto not fully exploited field of fungal products with potentials for being developed into new fungal drugs, of relevance for curing not just infectious diseases but also cancer, high blood pressure and CNS imbalances. The source of many such biologically active metabolites is mostly filamentous fungi. It can be seen as an inherent part of the filamentous fungus lifestyle; it secretes metabolically active enzymes, breaking down the substrate in a wide circle around the growing hyphal tip. This means that it is a competitive advantage for the fungus that it can protect all such affected part of the now microbially accessible substrate for its own consumption. It protects its “lunch package” for any other fungus or bacteria or maybe even against insects trying to invade and exploit what the fungus has broken down. In the coming years, it is of paramount importance that the research in studying biologically active secondary metabolites of fungi is strengthened as research field. We can globally expect the threat of epidemic infectious diseases is growing due to climate change, and due to the current rapidly growing threat of resistance developing against the antibiotics now in use. Already now >30,000 people die in Europe every year due to infections which cannot be cured due to acquired antibiotic resistance. Resistance also to fungicides is developing to be a very serious problem; immunocompromised patients may be dying from fungal attack after they were successfully cured from, e.g. leukaemia. The research challenges in the field of discovering new drugs are plenty; however, so are the new methods available. Especially the possibility of combining a wide range of both biological and non-biological research methods in an integrated manner in drug discovery seems very promising. Fungal biotech combined with bioimaging, NMR, MS, immunology, single-cell DNA and RNA sequences and proteomics methods is exciting. All are supported by a wide and sophisticated development of bioinformatics and big biodata crunching IT. It is a field, which calls for urgency, but also for collaboration. The silo structure, separating mycologists, medical doctors, bioinformatics and chemists, must be broken down in order to be able to move fast forward here.

4.3.2

Biological Plant Protection as a Substitute for Chemical Pesticides?

Recently, cases have been identified of drinking water reservoirs and wells contaminated by breakdown products from agricultural pesticides. Among such pesticides,

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several have been found to have possibly adverse effects on human health, immunomodulation and brain development and to reduce fertility, increase risk of cancer, etc. (Bjørling-Poulsen et al. 2008). Several research-funding calls have been addressing this societal challenge of how to regain and ensure future clean drinking water resources all over the world. One such avenue is to attempt to develop biological plant disease control measures. Specific strategies aim at strengthening the plant’s own defence instead of killing the pathogens or discovering new bio-based, non-hazardous compounds from which to make new and safer substitutes for chemical pest and disease control in plants. Here also, fungi and fungal products will be one of the strong cards to play. This may be done either by finding highly specific bioactive molecules that affect only pathogen- or pest-related targets or by plant-strengthening fungi (maybe in mixture with, e.g. yield improving bacteria) that can be distributed along with the seed or used as foliar sprays for increasing tolerance to infections.

4.3.3

Purifying Water by a Fungal-Derived Membrane-Bound Protein

Clean fresh water resources are in strong demand globally, yet are locally scarce and threatened with depletion at many places in the near future. Climate change-derived global warming and scarcity of rainfall, deforestation and threatening desertification are adding to the severity of this problem. A new bio-based approach to purifying water has been developed. The method ensures that all infectious propagules as well as contaminating (possibly toxic) chemicals and minerals (including salts) are removed from the water. The basic principle of this method comes from nature. All living cells have transporters into and out of the cells. One type of transporter allows only the transport of water sensu stricto. This means that the only item or molecule that can pass through such proteinaceous transporters is the molecule H2O. The specific type of transporter molecule upscaled for industrial use has been derived from a fungal yeast (Bomholt et al. 2013). Development of this product has overcome hitherto insurmountable obstacles. The fungal molecule, named aquaporin, is a membrane-bound protein complex, which therefore requires very different methods of handling as compared to normal secreted or cytoplasm-soluble proteins; until recently membrane-bound proteins could not be recovered, purified and characterized by standard protein recovery methods. However, the Danish company Aquaporin has in collaboration with University of Copenhagen, Institute of Biology, developed technologies, which enable industrial-scale use of the recombinantly produced aquaporin molecule inserted in specific membranes or filters and thus facilitate aquaporin-based water purification technologies (Bomholt et al. 2013). This approach is being developed, for example, for the Chinese market, where inside out aquaporin lined straws are assembled in a filtering unit fitted underneath the kitchen sink also in private homes. The development of this first industrial-scale use of a fungal membrane-bound protein could pave the way for new uses of other fungal membrane-bound proteins (such as enzymes) already shown to have interesting activities but not yet ready for upscaled use, due to their membranebound nature.

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New Ways of Controlling Fungal Diseases in Man and Animals

Fungal diseases are notoriously difficult to control, because of all human microbial pathogens (bacteria, mycoplasma and viruses), they are by far the ones most closely related to their hosts. Over the decades during which chemo-drugs and x-ray treatments have been administrated for combatting cancer and curing AIDS patients, fungal infections have remained life-threatening, especially for immunocompromised patients. However, recently a new serious and unfortunately often also lifethreatening obstacle has arisen—antibiotic resistance among the fungal infectious agents—which is emerging steadily and more frequently. More and more observations, albeit mostly based on circumstantial evidence, suggest that the abundant use of the rather few efficient antifungal pesticides in agriculture is posing a risk of fungal infections in humans becoming non-curable due to acquired drug resistance. The need for new types of control measures for combatting fungal diseases is pressing, and reserving specific types of fungicidally active drugs for humans only is urgent.

5 Future Perspectives 5.1

Perspectives for Fungal Biotechnology Developments Needed for Unlocking the Full Potentials of Fungi

Methodological progress of applied relevance is both needed and within reach for many areas of fungal biotechnology. Examples feature transforming genomic (and meta-genomic) data to information and biological insight relevant for science and for applied use, and developing cross-methodological approaches (spanning bioinformatics, genomics, transcriptomics, proteomics, metabolomics, mass spectrometry, NMR and bioimaging) to provide basis for increased understanding of fungal pathogenesis of man, animal (especially the epidemics among vertebrates) and plants (e.g. new epidemics of cereal rusts deserves attention). An integrated methodological approach to increased understanding of development of fungal resistance towards fungicides, in parallel with discovery efforts for finding new approaches to combating fungal diseases, is expected to become more important already during the next decades under climate change conditions. Proactive mycological research efforts are in demand to ensure that the fungi (and other eukaryotes) are included in the upcoming broad spectrum of metagenomics microbiome studies of the phyllosphere, rhizosphere, gut channels, rumen microbiota, skin, the mucous surfaces of animals, etc. Mycological focus needs to be broadened to include more prominent studies of what fungi do, hereby complementing the molecular tools for identifying who the fungi are (species identification and phylogeny). A prominent step here is to include an integrated methodological approach in studying the fungal exoproteome and exometabolome

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(here including use of new digitalization, bioinformatics, machine learning and big data options). By doing this, increased understanding of fungal phenotype can be achieved, including elucidation of how fungi interact with their host and substrate, with other fungi and with competitors, as well as symbionts. A well-known but underexploited aspect of fungal biotechnology is to see the residual fungal biomass (from fungal production or from biorefinery fermentations) as a raw material for new high-value products. Several components of the fungal biomass can be made use of: fungal proteins can be used as both food and feed ingredients; fungal cell wall materials can be used for their health-promoting effect (e.g. the β-glucans); and the small oligomers made from the fungal polymers can have prebiotic effect for improved gut health of both animals and man. Fungal hyphae may also be used as basis for new design types of materials. It would be timely and value adding for society as well as for climate change mitigation (and also stimulating the new biobased business segment) to give priority to the next steps in developing the white biotechnology (fungal production of bio-based chemicals and materials and use of enzymes for processing of biobased non-food products). Fungal production, albeit already 2–3 decades under development, can be (by use of more advanced methods) even further improved. In the future, industrial-scale microbial fermentation could span from pure culture fermentations (e.g. for enzyme production), over spontaneous-mixed culture (as used in anaerobic digestion), all the way to complex microbiome fermentations. More over, microbiome fermentation could benefit from the flexibility in adapting to the day-today variations in composition of the household waste. Future may show if synthetic biology methodological approach will be required to discover, describe and understand the various microbiome (interactions and regulatory) networks to a level sufficiently deep to be able to control microbiome fermentations in industrial scale. This can contribute to using the biomass materials even more efficiently and pave the way for faster introduction of a wider range of bio-based products and biological processing, being the most efficient vehicle for phasing out our dependency and use of non-renewable resources.

5.2

Perspectives for What Fungi Can Do for a Better World

For feeding the world, fungal enzymes and fungal proteins can contribute significantly to improved use of biological resources or, put more specifically, can enable upgraded use of crop residues and industrial sidestreams from processing of agricultural, fishery and forestry products. However, fungal contributions to feeding the world go beyond nutrition. Fungal enzymes are essential for assisting production of new types of gut-health-promoting food ingredients and food additives. When employed in this way, fungal enzymes will likely be essential for serious reduction in the use of antibiotics in the livestock for meat production and similarly, of significant importance for combatting lifestyle diseases through improved gut health

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in humans. New drugs and new ways of production of biological drug candidates can also be developed from and by fungi. Strategies used by fungi in nature can inspire the use of molecular mechanisms for production of clean water from salty or dirty water. Development of a new generation of fungal plant strengtheners, which may hopefully pave the way for reducing use of pesticides and for adapting to and compensating for the expected climate change-challenged food production in many parts of the world is also a genuine option. In the bio-based society, we need to acknowledge and know all the good things we actually can obtain from fungi and mycology. However, we also need to be able to recognize and communicate where fungi are our foes. The new, spreading fungal diseases in specific groups of vertebrates (toads, snakes and bats) may be a warning signal of possible future growth in the incidence of fungal infections also in humans, pets and livestock. The threat of new infectious diseases becoming more serious due to climate change is made even more grave by the increasing occurrence of antibiotic resistance. Discovery of new types of fungal-derived drugs as well as new antifungal drugs is becoming all the more urgent.

5.3

Perspectives for Communication and Cross-Disciplinary Collaboration

We want to emphasize the importance of and need for communicating broadly the wonders of the role of fungi in nature. Good communication includes visualization of how improved understanding of the fungal life form can be more innovative, and work more efficiently in a cross-disciplinary manner, drawing on many other fields of expertise, to unlock the potential of fungi to develop new sustainable solutions to problems of global and local importance.

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Belghi I, Liland S, Waagbø R, Biancarosa I, Pelusioc N, Yanxian Lid S, Krogdahl I, Locka EJ (2018) Potential of insect-based diets for Atlantic salmon (Salmo salar). Aquaculture 491:72–81 Bissaro B, Várnai A, Røhr AK, Eijsink VGH (2018) Oxidoreductases and reactive oxygen species in conversion of lignocellulosic biomass. Microbiol Mol Biol Rev 82. https://doi.org/10.1128/ MMBR.00029-18 Bjørling-Poulsen M, Raun-Andersen H, Grandjean P (2008) Potential developmental neurotoxicity of pesticides used in Europe. Environ Health 7:50 Bomholt J, Hélix-Nielsen C, Scharff-Poulsen P, Pedersen PA (2013) Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. PLoS One 8:e56431 Bunterngsook B, Laothanachareon T, Chotirotsukon C, Inoue H, Fujii T, Hoshino T, Roongsawang N, Kuboon S, Kraithong W, Techanan W, Kraikul N, Champreda V (2018) Development of tailor-made synergistic cellulolytic enzyme system for saccharification of steam exploded sugarcane bagasse. J Biosci Bioeng 125:390–396 Busk PK, Lange L (2013) Function-based classification of carbohydrate active enzymes by recognition of short, conserved peptide motifs. Appl Environ Microbiol 79:3380–3391 Busk PK, Lange L (2015) Classification of fungal and bacterial lytic polysaccharide monooxygenases. BMC Genomics 16:368 Busk PK, Pilgaard B, Meyer AS, Lange L (2017) Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function. BMC Bioinformatics 18:214 Cheng YM, Shi Q, Sun R, Liang D, Li Y, Jin W, Zhu W (2018) The biotechnological potential of anaerobic fungi on fiber degradation and methane production. World J Microbiol Biotechnol 34:155–163 Collins C, Hurley R, Almutlaqah N, O’Keeffe G, Keane TM, Fitzpatrick DA, Owens RA (2017) Proteomic characterization of Armillaria mellea reveals oxidative stress response mechanisms and altered secondary metabolism profiles. Microorganisms 5:60–76 de Lange CFM, Pluske J, Gongac J, Nyachotid CM (2010) Strategic use of feed ingredients and feed additives to stimulate gut health and development in young pigs. Livest Sci 134:124–134 Dotsenko G, Lange L (2017) Enzyme enhanced protein recovery from green biomass pulp. Waste Biomass Valor 8:1257–1264 Dotsenko G, Meyer AS, Canibe N, Thygesen A, Nielsen MK, Lange L (2018) Enzymatic production of wheat and ryegrass derived xylooligosaccharides and evaluation of their in vitro effect on pig gut microbiota. Biomass Convers Biorefin 8:497–507 Falco FC, Espersen R, Svensson B, Gernaey KV, Lantz AE (2019) An integrated strategy for the cost-effective production of bristle protein hydrolysate by the keratinolytic filamentous bacterium Amycolatopsis keratiniphila D2. Waste Manag 89:94–102 Franklinos LHV, Lorch JM, Bohuski E, Fernandez JR-R, Wright ON, Fitzpatrick L, Petrovan S, Durrant C, Linton C, Baláž V, Cunningham AA, Lawson B (2017) Emerging fungal pathogen Ophidiomyces ophiodiicola in wild European snakes. Sci Rep 7:3844 Gupta VK, Kubicek CP, Berrin JG, Wilson DW, Couturier M, Berlin A, Filho EXF, Ezeji T (2016) Fungal enzymes for bio-products from sustainable and waste biomass. Trends Biochem Sci 41:633–645 Haitjema CH, Gilmore SP, Henske JK, Solomon KV, de Groot R, Kuo A, Mondo SJ, Salamov AA, LaButti K, Zhao Z, Chiniquy J, Barry K, Brewer HM, Purvine SO, Wright AT, Hainaut M, Boxma B, van Alen T, Hackstein JHP, Henrissat B, Baker SE, Grigoriev IV, O’Malley MA (2017) A parts list for fungal cellulosomes revealed by comparative genomics. Nat Microbiol 30:17087 Harvey CJB, Tang M, Schlecht U, Horecka J, Fischer CR, Lin H, Li J, Naughton B, Cherry J, Miranda M, Li YF, Chu AM, Hennessy JR, Vandova GA, Inglis D, Aiyar RS, Steinmetz L, Davis RW, Medema MH, Sattely E, Khosla CST, Onge R, Tang Y, Hillenmeyer MEHEX (2018) A heterologous expression platform for the discovery of fungal natural products. Sci Adv 4:5459

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Matthew C, Fisher D, Henk A, Briggs C, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ (2013) Emerging fungal threats to animal, plant and ecosystem health. Nature 484(7393):186 Mazorra-Manzano MA, Ramírez-Suárez JC, Moreno-Hernández JM, Pacheco-Aguilar R (2018) Chapter 17: Seafood proteins. In: Proteins for food processing, Woodhead publishing series in food science, technology and nutrition, 2nd edn. Woodhead Publishing, Sawston, pp 445–475 Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Adv Biochem Eng Biotechnol 108:95–120 Meyer AS, Rosgaard L, Sørensen HR (2009) The minimal enzyme cocktail concept for biomass processing. J Cereal Sci 50:337–344 Miyauchi S, Rancon A, Drula E, Hage H, Chaduli D, Favel A, Grisel S, Henrissat B, HerpoëlGimbert I, Ruiz-Dueñas FJ, Chevret D, Hainaut M, Lin J, Wang M, Pangilinan J, Lipzen A, Lesage-Meessen L, Navarro D, Riley R, Grigoriev IV, Zhou S, Raouche S, Rosso MN (2018) Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass. Biotechnol Biofuels 11:201 Mosbech C, Holck J, Meye AS, Agger JW (2018) The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch. Biotechnol Biofuels 11:71–79 Nagpal R, Puniya AK, Griffith GW, Goel G, Puniya M, Sehgal JP, Singh K (2009) Chapter 17: Anaerobic rumen fungi: potential and applications. In: Khachatourians GG, Arora DK, Rajendran TP, Srivastava AK (eds) Agriculturally important micro-organisms, vol 1. Academic World International, Dubai, pp 375–393 Nielsen JC, Grijseels S, Prigent S, Ji B, Dainat J, Nielsen KF, Frisvad JC, Workman M, Nielsen J (2017) Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nat Microbiol 2:17044 Nordic Council of Ministers (2017) 25 cases for sustainable change. Available online. Accessed Dec 2018. https://doi.org/10.6027/ANP2016-782 Ohm RA, de Jong JF, Lugones LG, Aerts A, Kothe E, Stajich JE, de Vries RP, Record E, Levasseur A, Baker SE, Bartholomew KA, Coutinho PM, Erdmann S, Fowler TJ, Gathman AC, Lombard V, Henrissat B, Knabe N, Kües U, Lilly WW, Lindquist E, Lucas S, Magnuson JK, Piumi F, Raudaskoski M, Salamov A, Schmutz J, Schwarze FW, van Kuyk PA, Horton JS, Grigoriev IV, Wösten HA (2010) Genome sequence of the model mushroom Schizophyllum commune. Nat Biotechnol 28:957–963 Pauli G (2017) World of mushrooms. In: Milenkovic I, MIlosavljevic I (eds) Mushroom cultivation manual for the small mushroom entrepreneur. Ekofungi, Belgrade, pp 4–8 Pierce BC, Agger JW, Wichmann J, Meyer AS (2017) Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase. Enzyme Microb Technol 98:58–66 Pleisner D, Venus J (2017) Beyond coffee production –utilization of coffee residues. Atlas of Science, another view on science. http://atlasofscience.org Rosewich UL, Kistler HC (2000) Role of horizontal gene transfer in the evolution of fungi. Annu Rev Phytopathol 38:325–363 Sharma S, Hansen LD, Hansen JØ, Mydland LT, Horn SJ, Øverland M, Eijsink VGH, Vuoristo KS (2018) Microbial protein produced from brown seaweed and spruce wood as a feed ingredient. J Agric Food Chem 66:8328–8335 Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev 70:283–295 Silva COG, Vaz RP, Filho EXF (2018) Bringing plant cell wall-degrading enzymes into the lignocellulosic biorefinery concept. Biofuels Bioprod Biorefin 12:277–289 Sørensen P, Nørgaard VJ (2016) Starfish (Asterias rubens) as feed ingredient for piglets. Anim Feed Sci Technol 211:281–288

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Chapter 2

Fungal Attack on Environmental Pollutants Representing Poor Microbial Growth Substrates Dietmar Schlosser

1 Introduction In the Anthropocene, man-made environmental pollution has become a serious and global threat to live on earth. The biochemical attack on organic environmental pollutants by microorganisms can lead to the formation of organic products which are not further degraded (further on referred to as “biotransformation” or “incomplete biodegradation”) or their ultimate breakdown into CO2 and H2O (a process frequently referred to as “mineralisation”). The term “biodegradation” is not consistently used in the literature. It may exclusively address the mineralisation of an environmental pollutant or also be used in a wider sense and just refer to the disappearance of a parent pollutant. Here, we here use “biodegradation” to cover both “mineralisation” and “biotransformation” and “mineralisation” if unambiguity is needed and confusion with “biotransformation” has to be avoided (Harms et al. 2017; Solé and Schlosser 2015). Microorganisms can utilise organic environmental pollutants as their sole source of carbon and energy (or, in other words, use it as a growth substrate). This process commonly results in the mineralisation of at least a structural part of the pollutant molecule, while other parts of the pollutant’s carbon are used to build up microbial biomass (Solé and Schlosser 2015). The designation “productive degradation” is widely used for processes of the aforementioned type and refers to their common characteristic that pollutants are utilised for microbial biomass production. Prokaryotes can employ different terminal electron acceptors such as oxygen but also alternatives like nitrate, Fe(III), or sulfate for an oxidative biochemical attack on pollutants, enabling their utilisation as sole carbon and energy sources under oxic and anoxic conditions (Solé and Schlosser 2015). Despite a few reported exceptions D. Schlosser (*) Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_2

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(Russell et al. 2011), fungal hydrocarbon pollutant breakdown, however, is predominantly aerobic (Harms et al. 2017; Solé and Schlosser 2015). The efficiency of productive microbial degradation of an environmental pollutant is thought to depend on a positive feedback loop between contaminant uptake and its utilisation for growth and the thereof resulting formation of the degrading microbes. Pollutant-utilising microbes would also require a minimum energy supply to maintain their metabolism and therefore a minimum pollutant flux to persist. The availability of a pollutant to an organism (“bioavailability”) hence largely influences pollutant degradation (Harms et al. 2011, 2017; Johnsen et al. 2005). The bioavailability of pollutants would also be expected to control the microbial evolution of corresponding degradation pathways, which would only proceed if sufficient benefits for the degrading organisms could be gained from degradation (Hochstrat et al. 2015; Krueger et al. 2015a). A limited bioavailability and low to sometimes even practically negligible pollutant fluxes to cells will arise when environmental pollutant concentrations are extremely low, as for the so-called environmental micropollutants; when pollutants are only poorly water-soluble and strongly sorb to environmental matrices, as for high-molecular-mass polycyclic aromatic hydrocarbons (PAHs); and when environmental pollutants are solids, thus being practically water-insoluble, as for plastics (Harms et al. 2011, 2017) (Fig. 2.1). Moreover, some environmental pollutants are quite highly oxidised, thus representing only poor donors not supporting aerobic microbial growth. Examples for such compounds include, among others, highly chlorinated hydrocarbons (Harms et al. 2011, 2017) (Fig. 2.1; Table 2.1). All in all, organic pollutants possessing one or more of the aforementioned characteristics can be considered as only poor substrates for aerobic microbial growth (Fig. 2.1). Corresponding groups of chemicals will be addressed in more detail in Sects. 1.1, 1.2, and 1.3. Bacteria often utilise pollutants as growth substrates and typically use comparatively compound-specific biochemical pathways for this. In line with an only poor substrate quality for microbial utilisation, aerobic bacterial growth on high-molecular-mass PAHs and micropollutant representatives has seldom been reported and is not known for dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), 2,4,6-trinitrotoluene (TNT), and plastics like polystyrene (PS) and polyvinylchloride (PVC) (Harms et al. 2011, 2017; Krueger et al. 2015a) (Fig. 2.2; Table 2.1). By contrast, fungal growth on environmental pollutants is limited to a much more narrow range of compounds. Prominent examples for this include the well-documented fungal growth on compounds with rather simple structures such as n-alkanes, n-alkylbenzenes, aliphatic ketones, ethylbenzene, styrene, toluene, phenol, o-cresol, m-cresol, p-cresol, and 4-ethylphenol (Harms et al. 2011, 2017). A few reports describe fungal growth on PAHs (Cerniglia and Sutherland 2010), asphaltenes (Hernandez-Lopez et al. 2015; Uribe-Alvarez et al. 2011), and polyurethanes (PUR; Russell et al. 2011). Fungal utilisation of PE as a carbon source was also reported (Sangeetha Devi et al. 2015). Nevertheless, fungi primarily cometabolise the majority of organic environmental pollutants using a comparatively nonspecific enzymatic machinery, thereby decoupling pollutant breakdown from biomass production at the expense of carbon- and energydelivering cosubstrates (Harms et al. 2011, 2017). Fungal cometabolism of,

2 Fungal Attack on Environmental Pollutants Representing Poor Microbial. . . Pollutants occuring at only very low environmental concentrations

Highly oxidised pollutants (poor electron donors)

Micropollutants BPA, CBZ, DF, SMX

EE2, DEHP, NP, TCS

PFOA, PFOS

PCDDs, PCDFs, PBDEs

Poorly water-soluble pollutants

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Pollutants being practically insoluble in water

“Classical“ pollutants

Plastics

Asphaltenes, Higher alkanes, High-molecularmass PAHs

PA, PBAT, PBT, PBS, PE, PET, PHA, PLA, PP, PS, PTT, PUR

PCBs, TNT

PVC

Fig. 2.1 Examples for organic environmental pollutants representing poor microbial growth substrates. Such pollutants comprise compounds occurring at only very low environmental concentrations (micropollutants), compounds with very low water solubility (which can be found among both micropollutants and “classical” environmental pollutants), compounds being practically insoluble in water (solid synthetic polymers ¼ “plastics”), and those being already highly oxidised, thus representing only very poor electron donors (found among micropollutants, “classical” pollutants, and plastics). BPA, bisphenol A; CBZ, carbamazepine; DF, diclofenac; SMX, sulfamethoxazole; PFOA, perfluorooctanoic acid; PFOS, perfluorooctanesulfonate; EE2, 17α-ethinylestradiol; DEHP, di(2-ethylhexyl)phthalate; NP, nonylphenol; TCS, triclosan; PCDDs, polychlorinated dibenzo-pdioxins; PCDFs polychlorinated dibenzofurans; PBDEs, polybrominated diphenyl ethers; PAHs, polycylic aromatic hydrocarbons; PCBs, polychlorinated biphenyls; TNT, 2,4,6-tronitrotoluene; PA, polyamides; PBAT, polybutylene adipate terephthalate; PBT, polybutylene terephthalate; PBS, polybutylene succinate; PE, polyethylene; PET, polyethylene terephthalate; PHA, polyhydroxyalkanoate; PLA, polylactic acid; PP, polypropylene; PS, polystyrene; PTT, polytrimethylene terephthalate; PUR, polyurethane; PVC, polyvinyl chloride

e.g. PCDDs, PCDFs, PAHs, and TNT is well established, which may even result in mineralisation if particular wood-rotting fungi such as white-rot basidiomycetes are considered (Harms et al. 2011; Cerniglia and Sutherland 2010; Chang 2008; EsteveNunez et al. 2001; Takada et al. 1996; Scheibner et al. 1997; Nakamiya et al. 2005). Fungal capacities to attack environmental pollutants representing only poor growth substrates will be illustrated starting from Sect. 2.

1.1

Pollutants Occurring in Only Trace Concentrations

The term “micropollutants” (synonymous with “trace pollutants” and “emerging contaminants”) refers to a wide array of primarily synthetic organic chemicals, which are mostly nonregulated and comprise a multitude of different structures and origins. The typically very low environmental concentrations of micropollutants have become accessible for analysis with the introduction of sufficiently sensitive mass spectroscopy-based analytical techniques only during the last few decades, thereby triggering a steadily increasing scientific, public, and legal awareness of

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs)

Polychlorinated biphenyls (PCBs)

NO2

NO2

Cl

O Cl

Cl Cl O 75 PCDD and 135 PCDF congeners with one to eight chlorine atoms at

Cl

Cl Cl Congeners with two to ten chlorine atoms attached to biphenyl

Cl

Cl Cl

2,4,6-Trinitrotoluene (TNT)

O2 N

Classes of environmental pollutants Examples/representative(s) “Classical” pollutants Explosives CH3

Formed in small amounts when organic compounds are burned in the presence of Cl, e.g. in municipal waste incinerators; also produced during manufacturing of chlorophenol and chlorophenoxyacetic acid

Primarily used as heat transfer, dielectric, and hydraulic fluids in, e.g. transformers, capacitors, and electric motors

Production and assembly of explosives and ammunition, war damages of production/ assembly sites, use of explosives and ammunition on military testing grounds

Sources/uses

Accumulation in aquatic sediments, organic soil fractions, and organisms; frequently resulting from the release of PCB-contaminated media at sites related to the manufacturing or use of PCB-containing apparatus/ equipment Accumulation and high persistence in soils, sediments, animals, and humans; resulting from the accidental release at chemical or agrochemical production sites, the use of dioxincontaminated animal feed,

Soil, surface, and groundwater contamination at production/assembly sites and military testing grounds

Contaminated environmental media

Anaemia and disturbed liver functions in humans exposed to TNT over prolonged periods, adverse effects on male fertility, skin irritation from skin contact, possible human carcinogen, animals: adverse effects on the blood, liver, spleen, and immune system Wide range of toxic effects in humans, depending on the respective PCB: carcinogenic (dioxin-like) effects caused by particular congeners, endocrine disruption (oestrogenic effects, thyroid disorder), and neurotoxicity associated with others Teratogenic, mutagenic, carcinogenic (proven for PCDDs, in particular for the most toxic TCDD and suspected in case of PCDFs), immunotoxic, hepatotoxic, and endocrinedisrupting (thyroid disorder

Toxic/adverse effects

Table 2.1 Examples of “classical” hydrophobic environmental pollutants and micropollutants and related toxic or otherwise adverse effects on organisms

36 D. Schlosser

Active pharmaceutical ingredients

Micropollutants Active pharmaceutical ingredients

Polycyclic aromatic hydrocarbons (PAHs)

H H

H

17α-Ethinylestradiol (EE2)

HO

Benzo[a]pyrene

OH

varying positions of the two aromatic rings, e.g. 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD)

Frequently applied sulfonamide bacteriostatic antibiotic, most often used in synergistic combination with

Frequently applied synthetic oestrogen in oral contraceptives

Occurs in natural oil and coal deposits, processed fossil fuels, tar, and edible oils; formed during incomplete combustion of various organic compounds: byproducts of burning of fossil fuels and wood, also found in grilled or smoked meat and in cigarette smoke

herbicides and fibre bleaching; co-occurrence of PCDFs with PCDDs

Contaminant in WWTP effluents (communal and hospital wastewaters) and receiving surface waters due

Contaminant in WWTP effluents and receiving waters due to incomplete removal in WWTPs; moderate sorptive binding to WWTP sludge and aquatic sediments

and the military use (herbicidal warfare) of dioxincontaminated chlorophenoxyacetic acid herbicide mixtures (Agent Orange) during the Vietnam War Primarily found in soil and sediments and less commonly in water, also contaminate air-suspended particulate matter; among the most widespread organic pollutants

(continued)

Xenoestrogen, acts as endocrine-disrupting chemical (EDC), contributes to the oestrogenicity of WWTP effluents, can potentially impact the sustainability of aquatic wildlife populations (e.g. by fish feminisation) Concerns related to a possibly accelerated development of bacterial antibiotic resistance

Include carcinogenic, mutagenic, and teratogenic compounds; formation of the highly carcinogenic metabolite benzo[a]pyrene-7,8dihydrodiol-9,10-epoxide during human metabolisation of the procarcinogen benzo[a] pyrene

in humans) effects in humans and animals; developmental toxicity, chloracne, and nervous system pathology in humans

2 Fungal Attack on Environmental Pollutants Representing Poor Microbial. . . 37

Alkylphenols/ alkylphenol ethoxylates

Classes of environmental pollutants

Table 2.1 (continued)

OH

N O

Nonylphenol isomers with variously branched side chains, e.g. 4-(1-ethyl1,3-dimethylpentyl) phenol (NP)

H2N

ON H Sulfamethoxazole (SMX)

S

O

Examples/representative(s)

Release of a multitude of nonylphenol isomers with branched side chains due to incomplete degradation in WWTPs of nonylphenol ethoxylate surfactants used in various cleaning agents and contained in wastewaters; analogous release of octylphenol isomers resulting from octylphenol ethoxylates present as impurities in nonylphenol ethoxylates; frequent use of long-chain alkylphenols other than nonylphenols as components of phenolic resins

the bacteriostatic antibiotic trimethoprim (abbreviations SMX-TMP or SMZ-TMP)

Sources/uses

Presence of nonylphenols and other long-chain alkylphenols in WWTP effluents and receiving waters due to their persistence in WWTPs; strong sorptive binding to WWTP sludge and aquatic sediments; may contaminate soils when WWTP sludge is used as a fertiliser

to incomplete removal in WWTPs

Contaminated environmental media

Nonylphenols (and other long-chain alkylphenols) are xenoestrogens, can act as EDCs, contribute to the oestrogenicity of WWTP effluents, potentially impact the sustainability of aquatic wildlife populations (e.g. by fish feminisation), cause sperm count reduction in male offspring of mammals when exposed during pregnancy and lactation

Toxic/adverse effects

38 D. Schlosser

O

Br

Br

Br

O

O

Phthalates, e.g. di(2-ethylhexyl) phthalate (DEHP)

O

O

Pentabromodiphenyl ether congeners (pentaBDE), e.g. 2,20 ,4,40 ,5-pentabromodiphenyl ether

Br

Br

Elevated concentrations in soil, water, sediments, sludge, air-suspended particulate matter, food, and organisms; strong biomagnification in carnivores and humans; highly persistent

Easy release because not covalently bound to plastics, accelerated release by breaking and aging of plastics, subject to biodegradation and photodegradation when released to the outdoor environment

PBDEs are used as flame retardants, with pentaBDE commonly used in polyurethane foam; released into the environment, e.g. from pentaBDE-containing products and from emissions related to their production

Used to soften plastics (plasticisers), in particular polyvinyl chloride; phased out of many products in Europe, the USA, and Canada for health concerns

Anti-androgens (EDCs)

May affect the liver, thyroid (EDC effects), and neurobehavioural development of animals

Modified with permission by John Wiley and Sons from Table 13.1, Solé M, Schlosser D (2015) Xenobiotics form human impacts. In: Krauss G-J, Nies DH (eds) Ecological biochemistry: environmental and interspecies interactions. Wiley-VCH, Weinheim, pp. 259–275 (© Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany)

Plasticisers

Polybrominated diphenyl ethers (PBDEs)

2 Fungal Attack on Environmental Pollutants Representing Poor Microbial. . . 39

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D. Schlosser

Fig. 2.2 Chemical structures of important conventional types of synthetic polymers (“plastics”). The upper structures represent plastics with hydrolysable and the lower structures those with non-hydrolysable bonds in the polymer backbone. Nylon-6 is shown as a PA representative. R1, the di-isocyanate part in PUR; R2, the polyol moiety in PUR (Krueger et al. 2015a). Adapted by permission from Springer Nature: Springer, Appl Microbiol Biotechnol (2015) 99 (21):8857–8874, Prospects for microbiological solutions to environmental pollution with plastics (Krueger MC, Harms H, Schlosser D). © Springer-Verlag Berlin Heidelberg 2015

these compounds (Solé and Schlosser 2015). Micropollutants may be released from point as well as nonpoint sources due to agricultural, industrial, and urban activities. They are more or less ubiquitously present in the natural aquatic environment and also found in, e.g. municipal sewage and sewage sludge, hospital wastewater, consumer care products, and industrial effluents (Ahmed et al. 2017; Hochstrat et al. 2015; Solé and Schlosser 2015). Conventional wastewater treatment plants (WWTPs) were not originally designed for micropollutant removal. Advanced biological or chemical treatment technologies are frequently still at the experimental stage (Cecconet et al. 2017; Ahmed et al. 2017), and constructed wetlands have only limited applicability for micropollutant removal (Verlicchi and Zambello 2014). Therefore, these compounds are still often only insufficiently retained or degraded during wastewater treatment, thus finally entering the water cycle (Ahmed et al. 2017; Cecconet et al. 2017; Hochstrat et al. 2015; Kümmerer 2011; Lapworth et al. 2012; Silva et al. 2012). Micropollutants are hence commonly detected in surface water, are less frequently found also in groundwater, and were occasionally reported even for drinking water (Kümmerer 2009, 2011; Lapworth et al. 2012). Together with their transformation products, they are reasonably suspected to cause serious ecological and human health risks already at their environmental trace concentrations. Such risks relate to, e.g. disturbances of the endocrine system of vertebrates; adverse effects on growth, reproduction, and development in wildlife; the development of hazardous microbial resistance mechanisms; and accumulation in soils and organisms (Ahmed et al. 2017; Hochstrat et al. 2015).

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A recent application-based classification of micropollutants distinguishes between pharmaceutically active compounds (PhACs), which together with various toiletries and hygienic products form the group of pharmaceuticals and personal care products (PPCPs), industrial chemicals, and pesticides (Cecconet et al. 2017; Murray et al. 2010). For instance, analgesics, lipid regulators, antibiotics, diuretics, nonsteroidal anti-inflammatory drugs, antiseptics, beta blockers, and antimicrobials are PhACs, while personal care products involve, e.g. fragrances, food supplements, and sunscreen agents (Ahmed et al. 2017; Hochstrat et al. 2015). According to the aforementioned classification, frequently detected micropollutants such as bisphenol A (BPA; e.g. used for the synthesis of certain polycarbonate- and epoxy resin-type plastics), nonylphenol (NP; resulting from incomplete degradation of nonylphenol ethoxylate surfactants during wastewater treatment), and plasticisers like di (2-ethylhexyl)phthalate (DEHP) represent industrial chemicals (please refer to Fig. 2.1 and Table 2.1 for further information). The antiepileptic carbamazepine (CBZ), the anti-inflammatory analgesic diclofenac (DF), the contraceptive oestrogen 17α-ethinylestradiol (EE2), the antibiotic sulfamethoxazole (SMX), and the antimicrobial triclosan (TCS) are PPCPs (Fig. 2.1; Table 2.1). TCS is used as a hygienic PCP, whereas all other aforementioned PPCPs at the same time represent PhACs (Hochstrat et al. 2015; Hofmann and Schlosser 2016). Another widely used classification points to the biological effect mechanism of particular micropollutants, which are referred to as endocrine-disrupting chemicals (EDCs) due to their potential to interfere with the endocrine system of vertebrates. Micropollutants such as EE2, BPA, NP, and (possibly) also TCS are therefore also termed EDCs (Hofmann and Schlosser 2016; Jahangiri et al. 2017; Ahmed et al. 2017). Micropollutants usually occur in extremely low concentrations in the aquatic environment (typically in the ng/L to the lower μg/L range), which is their most prominent common characteristic (Ahmed et al. 2017; Kümmerer 2011; Murray et al. 2010; Solé and Schlosser 2015). Such low environmental concentrations are not only an obstacle for biodegradation from the kinetic viewpoint but can also be considered as rather unfavourable for the evolution of productive degradation pathways, despite numerous micropollutants are good electron donors and potentially contain sufficient energy that could be utilised for microbial growth (Harms et al. 2011, 2017; Hochstrat et al. 2015; Krueger et al. 2015a). Micropollutants cover a wide spectrum ranging from quite hydrophilic to very hydrophobic and only poorly water-soluble compounds (Hofmann and Schlosser 2016). Related examples for compounds with a comparatively high (SMX), moderate (BPA), and very low water solubility (DEHP, EE2, NP, TCS) are depicted in Fig. 2.1. The poor water solubility and often strong sorption to, e.g. soil constituents and aquatic sediments of hydrophobic micropollutants (see also Table 2.1) result in lowered fluxes of such contaminants to degrading cells, thereby complicating biodegradation and the evolution of related pathways in addition to corresponding difficulties arising from their usually only minute environmental concentrations (Harms et al. 2011, 2017). Moreover, some emerging contaminants are highly oxidised and thus contain only very little energy that could be derived from their oxidation and be utilised for

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microbial growth. Known examples include comparatively highly water-soluble and very mobile perfluorinated alkylated substances (PFAS) such as the liver toxicants and suspected carcinogens perfluorooctanoic acid (PFOA) and its conjugate base perfluorooctanesulfonate (PFOS), which are environmentally persistent components of fire-fighting foams, certain textiles, and insecticides (Loos et al. 2013; Murray et al. 2010; Renner 2001) (Fig. 2.1). Highly oxidised compounds are also found among very hydrophobic micropollutants, examples for this being penta- to decabrominated representatives of polybrominated diphenyl ethers (PBDEs) that are used as flame retardants (Clarke and Smith 2011; Solé and Schlosser 2015) (see also Table 2.1 and Fig. 2.1). The poor electron-donating properties of such compounds clearly hamper their productive biodegradation in addition to only low contaminant fluxes to cells (Harms et al. 2011, 2017). Both highly hydrophobic compounds with a very limited water solubility and chemicals representing only poor electron donors can also be found among “classical” environmental pollutants, which are already known since a long time and will be introduced in the next sub-section.

1.2

“Classical” Environmental Pollutants with Poor Bioavailability

“Classical” environmental pollutants may be contrasted with micropollutants mainly due to the earlier onset of their scientific, public, and legal consideration, their release into the environment frequently in high quantities, and their often (but not always) high environmental concentrations (Solé and Schlosser 2015; Hochstrat et al. 2015). For instance, soil concentrations of PAHs and the explosive 2,4,6trinitrotoluene (TNT; see Table 2.1) of up to the g/kg range can be found at particularly contaminated sites (Solé and Schlosser 2015). “Classical” environmental pollutants arise from the continued production, use, and discharge of a vast variety of different chemicals. Moreover, accidents and wars (have) additionally contribute(d) to environmental contamination. For instance, heavy pollution of both aquatic and terrestrial environments has resulted from crude oil spills; spills and leaks related to the production, storage, and use of fuels; and the production and use of explosives (Hochstrat et al. 2015; Solé and Schlosser 2015). Examples for “classical” hydrophobic and therefore only poorly bioavailable environmental mass pollutants, which at the same time are energy-rich compounds and potentially represent good electron donors, include high-molecular-mass PAHs (e.g. benzo[a]pyrene; see Table 2.1), long-chain n-alkanes, and also certain asphaltene constituents (Hernandez-Lopez et al. 2015; Harms et al. 2011, 2017) (Fig. 2.1). By contrast, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), which typically occur at very low environmental concentrations, and further “classical” environmental pollutants sometimes displaying worryingly high

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43

environmental concentrations such as polychlorinated biphenyls (PCBs) and TNT combine the problem of poor bioavailability with the obstacle of being too highly oxidised to fuel microbial growth (Harms et al. 2011, 2017) (see also Table 2.1 and Fig. 2.1).

1.3

Plastics

“Plastics” is a common and generalising designation for solid synthetic polymers (please refer to Figs. 2.1 and 2.2 for further information). These are all extremely poorly bioavailable since only a tiny fraction of the polymer would come in contact with potentially degrading microbes or enzymes (Krueger et al. 2015a). Plastics hence are poor growth substrates, which also applies to those polymers containing well-biodegradable structural elements such as amide and ester bonds and despite the high energy content and favourable electron donor quality of many types of plastics (Krueger et al. 2015a). Moreover, at least plastic materials above the size of nanoplastics (da Costa et al. 2016) would be expected to necessitate the extracellular initiation of their depolymerisation, in order to yield compounds small enough for cellular uptake and further intracellular catabolism (Harms et al. 2017; Krueger et al. 2015a). Conventional plastic types such as polyethylene (PE), polypropylene (PP), polyester, polyamide (PA) and acrylic fibres (as a sum), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethanes (PUR), and polystyrene (PS; please refer to Fig. 2.2 for structural information) have contributed 116, 68, 59, 38, 33, 27, and 25 million metric tons (MMT), respectively, to a global production volume (including additives) of 407 MMT in 2015 (Geyer et al. 2017; http://advances. sciencemag.org/content/advances/suppl/2017/07/17/3.7.e1700782.DC1/1700782_ SM.pdf). The so-called bioplastics are a large family of different materials, which can be bio-based, biodegradable, or both (EUBP 2018; Andreeßen and Steinbüchel 2019). The term “bio-based” may refer to either the bio-based carbon or the bio-based mass content of the materials, and corresponding standards for measurement and product certification and labelling have now been established (EUBP 2018). The unambiguous use of the term “biodegradability” requires the specification of the conditions applied for testing. In this context European Bioplastics (EUBP), the association of the European bioplastics industry, currently recommends to test for compostability (in an industrial compost plant) according to applicable standard reference and certification procedures (EUBP 2018). The global production capacity of bioplastics is still quite small compared to the global production volume of conventional plastics. A value of about 2.05 MMT has been reported for 2017 (corresponding to only about 1% of the total plastic materials produced annually), with shares of 1.17 and 0.88 MMT for biodegradable and bio-based plastics, respectively (EUBP 2017; Andreeßen and Steinbüchel 2019).

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Fig. 2.3 Possible pathways of microbial degradation of plastics. Polymer hydrolysis is contrasted with oxidative degradation, which can transform both hydrolysable and non-hydrolysable plastics. The green lines indicate functional groups including hydrolysable bonds inside hydrolysable plastics, and the question marks denote so-far unknown enzymes. Enzymes, oxidants, and processes in red frames have been described for fungi. Cut, cutinase; Nyl, nylon hydrolase; AlkB, alkane hydroxylase; Lac, laccase; MnP, manganese peroxidase (Krueger et al. 2015a). Adapted by permission from Springer Nature: Springer, Appl Microbiol Biotechnol (2015) 99 (21):8857–8874, Prospects for microbiological solutions to environmental pollution with plastics (Krueger MC, Harms H, Schlosser D). © Springer-Verlag Berlin Heidelberg 2015

Considering their chemical structures in connection with biochemical mechanisms potentially being able of cleaving the polymer backbone, plastics could be divided into hydrolysable and non-hydrolysable polymers (Fig. 2.2). This approach seems reasonable since natural polymers like cellulose, chitin, or proteins are usually all depolymerised by various hydrolytic enzymes, which cleave the bonds linking their subunits. Therefore, synthetic polymers with hydrolysable backbone structures might also become depolymerised via initial enzymatic hydrolysis (Krueger et al. 2015a) (see also Fig. 2.3). Indeed, bioplastics reported to be biodegradable in the sense of being compostable as outlined above all belong to hydrolysable plastic types. Prominent examples include polyesters like polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), and polybutylene adipate terephthalate (PBAT; EUBP 2017, 2018) (Fig. 2.1). Other potentially hydrolysable, albeit more recalcitrant and unfortunately also more widespread plastics are the polyester PET and polymers of the PUR and PA type (see Figs. 2.1 and 2.2) (Krueger et al. 2015a). Bio-based polyesters such as polybutylene

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45

terephthalate (PBT) and polytrimethylene terephthalate (PTT; please refer also to Fig. 2.1) are classified as non-biodegradable bioplastics (EUBP 2018). Non-hydrolysable plastics possess a backbone solely built on C–C bonds and devoid of reactive groups (see Fig. 2.2), with (possibly radicalic) redox reactions remaining as the only option for polymer breakdown into smaller molecules that might be assimilated or mineralised by microbes (see also Fig. 2.3) (Krueger et al. 2015a). PE, PP, PS, and PVC are the typical and highly recalcitrant representatives of non-hydrolysable plastics (Figs. 2.1 and 2.2). These compounds share the characteristic of being inert to hydrolysis with lignin, a major constituent of plant lignocellulose and the second-most abundant natural polymer. Lignin can efficiently be depolymerised by lignin-modifying enzymes and reactive oxygen species produced by particular higher fungi (white-rot but to a lesser extend also brown-rot basidiomycetes). Non-hydrolysable plastics are however considerably harder to attack than lignin. The latter contains partly oxidised moieties, making it far more hydrophilic than plastics and lowering the redox potential required for a successful oxidative attack (Krueger et al. 2015a). A higher recalcitrance of the aforementioned plastics than of lignin could also be inferred from bond dissociation energies (BDEs) of bonds in the corresponding polymer backbones, as weaker bonds are likely more prone to cleavage: Whereas for bonds between subunits of lignin BDEs ranging from 160 to 300 kJ/mol (in case of C–O ether bonds) or 240 to 425 kJ/mol (up to exceptional 500 kJ/mol) for C–C bonds have been described, plastics like PE, PP, PS, and PVC have BDEs in the range of 330–370 kJ/mol in the C–C bonds of their backbones (350–470 kJ/mol for C–H bonds) (Krueger et al. 2015a). The high number of chlorine substituents in PVC (one on every second carbon of the backbone) in the form of C–Cl bonds (Fig. 2.2), which represent additional inert structural elements of this polymer type, determines its high degree of oxidation, its poor electron donor quality, and hence its high resistance towards oxidative degradation in addition to the obstacle of poor bioavailability (Krueger et al. 2015a; Harms et al. 2017) (Fig. 2.1).

2 Fungal Pollutant Breakdown The benefits that fungi could obtain from the utilisation of environmental pollutants as carbon, nitrogen, or energy sources (see Sect. 1; Friedrich et al. 2007; Klun et al. 2003) are obvious. Concerning the cometabolic degradation of organic contaminants, the principal function of this type of biochemical attack obviously relates to detoxification (Harms et al. 2017; Solé and Schlosser 2015). Natural toxic compounds are constituents of many fungal environments. For example, they may arise from lignocellulosic plant material utilised by saprotrophic fungi or may be synthesised by plants to defend against plant-pathogenic fungi (Morel et al. 2013; Barabote et al. 2011; Harms et al. 2017). The cometabolic mineralisation of lignin to CO2 and H2O by white-rot fungi may be considered as a peculiarity in this respect, aiming to get access to lignocellulosic polysaccharides that serve as carbon and

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D. Schlosser

energy sources (Harms et al. 2017; Solé and Schlosser 2015). Ideally, detoxifying systems should cover the broadest possible range of potential toxicants. In line with such a characteristic, cometabolism is much less compound-specific than productive degradation. Therefore, it is also operative if fungi would get into contact with xenobiotic organic environmental pollutants (Harms et al. 2017; Solé and Schlosser 2015). Like in other eukaryotes and also in bacteria, successive phases of fungal pollutant cometabolism comprise an initial biochemical attack whereby compounds are functionalised (phase I reactions), conjugate formation in order to improve water solubility, and facilitate excretion (phase II reactions) and metabolite excretion involving efflux transporters (phase III reactions) (Harms et al. 2017; Barabote et al. 2011; Solé and Schlosser 2015). The fungal pollutant-attacking biochemical machinery will be explained in more detail in the next two sub-sections.

2.1

Extracellular Attack on Pollutants

A fungal peculiarity is the nonspecific extracellular attack on environmental pollutants using radical-generating oxidoreductases, which have evolved to support fungal growth on lignocellulosic substrates (Harms et al. 2011, 2017) (see also Fig. 2.4). Laccases (EC 1.10.3.2) are prominent multicopper oxidases, which are widely distributed in fungi and frequently occur as multiple isoenzymes. These enzymes employ molecular oxygen for the direct oxidation of various phenolic compounds, aromatic amines, and anthraquinone dyes (Majeau et al. 2010). Their substrate range can be considerably expanded in the presence of certain synthetic as well as natural laccase substrates referred to as “laccase redox mediators”, which yield organic radicals upon laccase oxidation. Such radicals can subsequently attack further environmental pollutants that are not prone to direct laccase oxidation, e.g. certain PAH representatives and the antibiotic SMX (Harms et al. 2011, 2017; Margot et al. 2015). Fungal lignin-modifying class II haem peroxidases like manganese peroxidase (MnP, EC 1.11.1.13), lignin peroxidase (LiP, EC 1.11.1.14), and versatile peroxidase (VP, EC 1.11.1.16) also oxidise environmental pollutants. Extracellular members of the haem-thiolate peroxidase superfamily such as dye-decolorising peroxidases (DyP-type peroxidases, EC 1.11.1.19) and unspecific peroxygenases (UPO, EC 1.11.2.1) were shown to attack high-redox-potential pollutants more recently (Harms et al. 2011, 2017; Mäkelä et al. 2015). Other extracellular fungal peroxidases such as Coprinopsis cinerea peroxidase (CiP, EC 1.11.1.7) and Caldariomyces fumago haem-thiolate chloroperoxidase (CPO; EC 1.11.1.10) are known to act on pollutants with lower redox potentials, e.g. various phenols (Harms et al. 2011, 2017). The aforementioned fungal oxidoreductases can catalyse the initial step in pollutant metabolism and convert many micropollutants, examples for this being the PPCPs DF, EE2, and TCS and the industrial chemicals BPA and NP (Jahangiri et al. 2017; Hofmann and Schlosser 2016; Harms et al. 2011; Kabiersch et al. 2011)

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Organic pollutants

Extracellular attack

Laccases + peroxidase reactions, hydroxyl radical attack e.g. resulting in • ether cleavage • quinoid products • hydroxylation • aromatic ring fission • CO2 • oxidative coupling products

Intracellular initial attack e.g. Cytochrome P450 monooxygenases, nitroreductases

Further metabolism

Conjugate formation

Oxidations / reductions

Transferases, e.g. • O-glucoside • O-glucuronide • O-xyloside • O-sulfate • O-methyl

Mineralisation

Hydrolase reactions resulting in depolymerisation and formation of oligo- and monomers

Fungal cell

Formation of bound residues

CO2

Metabolite excretion

Fig. 2.4 Principal methods used by fungi to degrade environmental pollutants. Initial biochemical attack on pollutants may occur extra- or intracellularly. Organic metabolites arising from extracellular oxidative or hydrolytic biotransformation steps may be subject to intracellular catabolism or may form bound residues with soil constituents. Metabolites stemming from intracellular initial attack may be excreted and can then either undergo further extracellular enzymatic reactions or form bound residues through abiotic oxidative coupling. They may also be secreted in the form of conjugates (which usually persist) or may undergo further intracellular catabolism. This may result in mineralisation or, again, in metabolite excretion at various oxidation stages if subsequent oxidation is impeded (Harms et al. 2011, 2017). Modified with permission from Harms H, Schlosser D, Wick LY (2011) Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nat Rev Microbiol 9 (3):177–192

(please also refer to Figs. 2.4 and 2.5). Also, PFOA was reported to be degraded by laccase-redox mediator systems (Luo et al. 2018a, b). Extracellular fungal oxidoreductases are further known to attack “classical” hydrophobic environmental pollutants such as PAHs (Harms et al. 2011; Steffen et al. 2003; Cerniglia and Sutherland 2010) (Figs. 2.4 and 2.6). Furthermore, they can oxidise excreted pollutant metabolites arising from intracellular biocatalytic steps (Fig. 2.4). Typically, extracellular fungal oxidoreductases produce organic radicals from the respective parent compounds via one-electron abstraction. Subsequently, such radicals undergo spontaneous follow-up reactions such as quinone formation (from PAHs and also certain chloroaromatics), ether bond cleavage (e.g. in dioxins and TCS), oxidative coupling (of EDCs or PAHs), and the covalent coupling of pollutant metabolites to soil constituents (Jahangiri et al. 2017; Harms et al. 2011). Notably, organic radicals formed from pollutants representing “good” laccase substrates such as certain PhAC

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D. Schlosser Diclofenac Cl

O Cl

NH

N

Cl

OH

Oxidative oligomerisation (radical coupling)

OH

Cl

O

Hydroxylation

Cyclisation (?)

Cl

Cl

OH

N Cl

O HO NH

NH

Cl

Cl

Cl Cl

OH

HN

O

Cl

Hydroxylation

O CH3 HO

O

Cl

OH

HO

Decarboxylation (?)

Cl

OH

OH NH

Di- to tetramers

NH

Cl

Cl

O

OH O

?

Fig. 2.5 Proposed pathway of diclofenac (DF) metabolism in a freshwater-derived ascomycete. Hydroxylation reactions are indicative for the action of intracellular cytochrome P450 monooxygenase systems. The enzymes responsible for cyclisation and decarboxylation reactions remain to be defined (indicated by a question mark in parentheses, respectively). Oxidative coupling products most likely arise from extracellular laccase reactions. Reactions indicated by dashed arrows are hypothetical, and the metabolic end product(s) remain unknown (indicated by the framed question mark) (Hofmann and Schlosser 2016). Adapted by permission from Springer Nature: Springer, Appl Microbiol Biotechnol (2016) 100:2381–2399, Biochemical and physicochemical processes contributing to the removal of endocrine-disrupting chemicals and pharmaceuticals by the aquatic ascomycete Phoma sp. UHH 5-1-03 (Hofmann U, Schlosser D) © Springer-Verlag Berlin Heidelberg 2015

representatives can in turn contribute to the oxidation of contaminants that are less susceptible to laccase oxidation (e.g. TCS). The simultaneous presence of both easily laccase-oxidisable and more recalcitrant pollutants in wastewater represents an interesting option for the efficiency enhancement of laccase-based wastewater treatment approaches (Jahangiri et al. 2018). As a peculiarity, MnP can cleave the aromatic moieties of certain environmental pollutants in cell-free reactions, which may even be accompanied by the release of CO2 from such compounds (Harms et al. 2011, 2017) (Fig. 2.4). Extracellular UPOs as found among certain basidiomycetes of the Agaricales hydroxylate hydrophobic pollutants (e.g. PAHs and dibenzofuran) as well as micropollutants such as DF in an H2O2-dependent manner, thereby combining catalytic properties of peroxidases and cytochrome P450 monooxygenase systems (Karich et al. 2017; Poraj-Kobielska et al. 2013; Harms et al. 2011). Extracellular fungal oxidoreductases have also been implicated in the fungal degradation of plastics. MnP has repeatedly been reported to attack PAs such as nylon-6 and nylon-6,6 (recently reviewed by Krueger et al. 2015a). Laccase-redox mediator combinations were shown to catalyse the partial depolymerisation of PE,

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Benzo[a]pyrene

MnP MnP

Quinones

Benzo[a]pyrene 7,8,9,10-tetraol

CO2

Hydroxybenzo[a]pyrenes and trans-dihydrodiols

Glucuronic acid and sulfate conjugates

Fig. 2.6 Biochemical pathway and products reported for the cometabolic fungal degradation of benzo[a]pyrene (Cerniglia and Sutherland 2010; Steffen et al. 2003; Harms et al. 2011). Epoxide intermediates arising from initial intracellular oxidation by cytochrome P450s, which precede the formation of hydroxylated products (Cerniglia and Sutherland 2010), are not shown. Modified with permission from Harms et al. (2011) Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nat Rev Microbiol 9 (3):177–192

nylon-6,6, and the sulfonated form of PS (i.e. PS sulfonate, which was used as a proxy for PS) (Fujisawa et al. 2001; Krueger et al. 2015b). Fungal laccase activity has also been correlated with fungal PP degradation (Jeyakumar et al. 2013). Fungal extracellular hydrolases (EC 3) have also been described to act on synthetic polymers. For instance, cutinases (EC 3.1.1.74; members of the serine hydrolases superfamily) were reported to hydrolyse polyesters like PBAT, PBS, PLA, PET, and poly(ε-caprolactone), the PA nylon-6,6, and polyacrylonitrile (Krueger et al. 2015a; Baker et al. 2012; Ferrario et al. 2016; Silva et al. 2005). A serine hydrolase-like enzyme was also implicated in fungal degradation of polyester PUR (Russell et al. 2011). Fungi can attack synthetic polymers and micropollutants such as PhACs by means of hydroxyl radicals, which are extracellularly produced through Fentontype reactions. These are driven by the redox cycling of quinone/hydroquinone couples. Cell-bound enzymes are involved in such degradation mechanisms, thereby linking extracellular to cell-bound activities explained in more detail in the next sub-section.

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Cell-Bound Activities Involved in Pollutant Degradation

In fungi (and also in other eukaryotes), the first step in the intracellular biocatalytic oxidation of organic pollutants is commonly a monohydroxylation reaction based on the activation of dioxygen and the insertion of one oxygen atom into the target molecule (Harms et al. 2011, 2017) (Fig. 2.4). Cytochrome P450 monooxygenases (EC 1.14.-.-; cytochrome P450s cannot be assigned to a single EC number) typically catalyse such reactions in both ligninolytic and non-ligninolytic fungi, frequently leading to epoxidations and hydroxylations of aromatic or aliphatic structures of pollutants such as PAHs, PCDDs, alkanes, alkyl-substituted aromatics (e.g. NP), and many PhACs (e.g. DF) (Harms et al. 2011, 2017; Solé and Schlosser 2015; Sakaki et al. 2013; Hata et al. 2010; Marco-Urrea et al. 2009; 2010a, b) (see also Figs. 2.5 and 2.6). They were also implicated in the fungal hydroxylation of PCBs and of PBDEs and their partly debrominated metabolites (Xu and Wang 2014; Hundt et al. 1999; Vilaplana et al. 2013; Cvancarova et al. 2012). The fact that fungal extracellular oxidoreductases also act on many of the aforementioned environmental pollutants (please refer to the previous sub-section) raises the question of whether an intracellular or extracellular primary pollutant oxidation prevails under a particular environmental situation, which would be expected to influence the expression of degrading enzymes (Harms et al. 2011). Multiple cytochrome P450 enzymes are thought to contribute to the vast catabolic versatility of ligninolytic fungi (Syed et al. 2014) and to enable the fungal cometabolism of structurally quite diverse pollutants such as representatives of nitroaromatic and N-heterocyclic explosives, organochlorines, PAHs, a wide range of micropollutants of various uses and origins, and synthetic polymers even in compound mixture (Harms et al. 2011, 2017). Possible functions of expanded multiple cytochrome P450s found in wood-rotting fungi also have been related to the adaptation of such organisms to lignocellulosic plant biomass, where fungal substrate colonisation may have to be accompanied by the detoxification of numerous natural plant compounds (Harms et al. 2017; Syed et al. 2014; Morel et al. 2013). Cytochrome P450 monooxygenase systems also have been implicated in the promiscuity of pollutant metabolism in non-ligninolytic fungi (Harms et al. 2011, 2017). More information about fungal P450s, which not only contribute to the metabolism of xenobiotics but are also involved in, e.g. biosynthetic reactions during fungal primary and secondary metabolism, could be retrieved from D.R. Nelson’s fungal P450 page (http://drnelson.uthsc.edu/fungal. genomes.html) and the Fungal Cytochrome P450 Database (http://p450.riceblast. snu.ac.kr) (Moktali et al. 2012). Non-haem mixed function oxidases such as 2-monooxygenases (EC 1.14.13.7) are further intracellular enzymes, which hydroxylate various phenols to yield catechols. Tyrosinases (EC 1.14.18.1) are mostly intra- but sometimes also extracellular multicopper oxidases, which convert even highly chlorinated phenols into o-catechols and oxidise o-catechols to their corresponding o-quinones (Harms et al. 2011, 2017).

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The fungal breakdown of environmental pollutants can also be initiated by cellbound reductases. Nitroaromatic compounds such as TNT are reduced to hydroxylamino- and amino-dinitrotoluenes by aromatic nitroreductases, which are widespread among fungi. Reduced metabolites of nitroaromatic compounds may be excreted by fungi and are then susceptible to further enzymatic (e.g. oxidation by laccase and MnP) and spontaneous reactions (Fig. 2.4). Other fungal nitroreductases can convert more water-soluble N-heterocyclic explosives such as Royal Demolition Explosive (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) into the respective mononitroso derivatives (Harms et al. 2011, 2017). Reductive dehalogenases have been described for ligninolytic basidiomycetes and may occur also in ascomycetes. They dechlorinate chlorohydroquinones resulting from chlorophenol metabolism and possibly also chlorocatechols arising from dioxin cleavage and diphenyl ether herbicides reductively (Harms et al. 2011, 2017; Nakamiya et al. 2005). Perhaps such fungal dehalogenase systems also reductively debrominate PBDEs, as could be deduced from the identification of related biotransformation metabolites (Xu and Wang 2014). Quinone reductases are cell-bound enzymes of diverse fungal groups, with whiterot and brown-rot basidiomycetes being prominent albeit not the only examples (Krueger et al. 2016). These enzymes are involved in quinone redox cycling, which may give rise to the production of highly reactive hydroxyl radicals via extracellular Fenton chemistry. Hydroxyl radical attack on aromatic and aliphatic pollutants often causes hydroxylation and dehalogenation reactions (Krueger et al. 2016; Harms et al. 2011, 2017). The hydroxyl radical-catalysed oxidation of various PhACs and the oxidative depolymerisation of the synthetic polymer PS sulfonate could be demonstrated for white-rot and brown-rot basidiomycetes, respectively, with quinone reductase-based redox cycling driving extracellular hydroxyl radical production (Marco-Urrea et al. 2010c; Krueger et al. 2015b). Also in brown-rot fungi, attack on PS films via Fenton chemistry driven by the redox-cycling of quinones yielded superficial oxidation of the solid polymer, albeit the overall effects on the polymer were weak (Krueger et al. 2017). Quinone reductases further detoxify quinones, e.g. resulting from the extracellular enzymatic oxidation of phenolic compounds, whereby quinones are reduced back into substrates for oxidative enzymes (Harms et al. 2011, 2017). Cell-bound hydrolases such as epoxide hydrolases acting on ether bonds (EC 3.3.2.9 and 3.3.2.10 for the microsomal and the soluble enzyme, respectively) are involved in early steps of the fungal breakdown of certain pollutants addressed in the present chapter. These enzymes convert PAH epoxides arising from cytochrome P450 reactions into trans-dihydrodiols in an H2O-dependent manner (Harms et al. 2017; Solé and Schlosser 2015; Li et al. 2009) (Fig. 2.6). Nitrile hydratases belong to the group of lyases (EC 4) and catalyse the H2O-dependent conversion of nitriles into amides. Further hydrolases such as amidases and nitrilases employ H2O for the conversion of amides (which arise from nitrile hydratase reactions) and nitriles, respectively, into carboxylic acids and ammonia (Solé and Schlosser 2015). Conjugate formation, which occurs during fungal phase II reactions, is catalysed by various transferases. These often act on hydroxyl groups of pollutants and

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pollutant metabolites. The pollutant conjugates derived thereof are commonly not further metabolised but excreted, thereby removing potentially hazardous compounds from cells (Fig. 2.4). Enzymatic conjugate formation can involve, e.g. UDP-glucuronyltransferases (transferring glucuronic acid), UDP-glycosyltransferases (sugars), sulfotransferases (sulfonyl groups), acetyltransferases (acetyl-coenzyme A), methyltransferases (methyl groups), and glutathione S-transferases (reduced glutathione) (Harms et al. 2017; Solé and Schlosser 2015). For instance, the formation of methyl and sugar conjugates has been reported for the fungal metabolism of PCBs and the micropollutant TCS, respectively (Hundt et al. 2000). Sugar and also sulfate conjugates are further well-known from fungal PAH metabolism (Harms et al. 2011, 2017) (Fig. 2.6). In wood-rotting fungi, multiple glutathione S-transferases have been implicated in detoxification processes during both xenobiotics-fungus and wood-fungus interactions, just as fungal cytochrome P450s (Harms et al. 2017; Morel et al. 2009, 2013). Fungal phase III reactions aim to excrete pollutant metabolites (Fig. 2.4). Efflux transport proteins of the ATP binding cassette (ABC) and the major facilitator superfamilies (MFS), which act against various synthetic (e.g. therapeutic drugs, fungicides) and natural antifungal agents (e.g. plant-derived toxins), can contribute to such excretion reactions and thereby confer fungal multidrug resistance (Harms et al. 2017; Barabote et al. 2011).

3 Conclusions and Outlook Poor bioavailability of organic environmental pollutants is a major obstacle for their biodegradation (Johnsen et al. 2005; Harms et al. 2011). Even if organisms possessing related degradative abilities would exist in the environment and pollutants of xenobiotic origin could potentially incidentally be degraded through cometabolism, only minute pollutant amounts far below any biodegradation rate saturation would substantially decrease biological degradation rates. Non-bioplastics certainly represent a worst-case scenario in this respect. Even under optimised laboratory conditions, notable biocatalytic modifications of parent plastics could only be observed after several days to weeks (hydrolysable plastics) or within the range of several weeks (non-hydrolysable plastics) (Krueger et al. 2015a). Numerous micropollutant structures and also a considerable variety of those of “classical” pollutants is susceptible to fungal cometabolic degradation, leaving exposure of fungal degraders and their enzymes to too low pollutant concentrations as the major obstacle for biodegradation. Also some plastics, especially those polymers possessing hydrolysable bonds in their backbones, can be attacked by cometabolic degradation mechanisms of fungi, albeit the reported rates of biological modification are very low. However, in addition to the problem of extremely low bioavailability, particularly inert structural elements of certain non-hydrolysable plastics may overextend the cometabolic degradation abilities of fungi and limit or impede an incidental biocatalytic attack. For instance, only a quite weak superficial oxidation of PS

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films could be observed upon attack by hydroxyl radicals extracellularly produced by the brown-rot fungus Gloeophyllum trabeum. The related biodegradability constraints were attributed to both the very poor bioavailability of PS and its inert basic structure, which even resists the attack by hydroxyl radicals, i.e. one of the most powerful oxidants known from biological systems (Krueger et al. 2017). Poor bioavailability of organic environmental pollutants would also be expected to be unfavourable for the evolution of productive degradation pathways serving as a basis for the growth of degrader organisms, for reasons explained in Sect. 1. However, the reported growth of fungi even on extremely recalcitrant asphaltenes (Hernandez-Lopez et al. 2015) and very poorly bioavailable PUR (Russell et al. 2011) suggests that such evolutionary processes in principle, albeit perhaps rarely, exist. There might therefore be good chances to identify novel fungal degraders among so-far unexplored biodiversity, which could be of special relevance for more easily degradable polymers such as hydrolysable plastics (Krueger et al. 2015a). Such novel fungal biocatalysts and their enzymes would also be expected to boost the development of related applications in environmental biotechnology, waste treatment, and plastic waste valorisation. In favour of such possible developments, a fungal cutinase capable of degrading PET and other synthetic polyesters could be functionally expressed in the bacterium Escherichia coli (Dimarogona et al. 2015), thereby demonstrating a potential for enzyme production at industrial scale as needed for practical applications. The successful engineering of a PET-degrading bacterial enzyme led to an increase in its catalytic performance (Austin et al. 2018), suggesting enzyme engineering as a successful strategy to improve the performance also of similar fungal enzymes. Last but not least, a more profound knowledge about the options and limitations of fungal biocatalysts acting on so far only very poorly biodegradable environmental pollutants would be expected to inspire the future development of more environmentally benign and more readily degradable chemicals, including polymers. Nevertheless, avoiding the release of too many man-made persistent pollutants into the environment has to make a significant, if not the most significant, future contribution to the reduction of environmental pollutant load. Acknowledgement This work was supported by the Helmholtz Association of German Research Centres and contributes to the Chemicals in the Environment (CITE) Research Programme conducted at the Helmholtz Centre for Environmental Research—UFZ.

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Chapter 3

The Biotechnology of Quorn Mycoprotein: Past, Present and Future Challenges Jack A. Whittaker, Robert I. Johnson, Tim J. A. Finnigan, Simon V. Avery, and Paul S. Dyer

1 Introduction As the Earth’s population continues to grow at an accelerated pace, demands for many vital resources will similarly increase. The main concerns are availability of water, oil, living space and food. The latter category is a complex one as there is a need for availability of food to avoid malnutrition yet issues in developed nations of the impact of an imbalanced diet on health. In relation to food requirements, protein is an essential macronutrient for humans, as well as other animals, with the need ideally for high-quality protein in the diet. The quality of a protein source is determined by the abundance and the variety of the amino acids it contains, with up to 9 of the 20 standard amino acids being deemed essential or conditionally essential for the human diet as these cannot be readily synthesised from other precursors by mammals (Reeds 2000; Fürst and Stehle 2004). A lack of sufficient amino acid provision can impair growth and development of an organism, eventually leading to disease and death if sustained for too long. For centuries the primary source of protein in the human diet has been through the cultivation of livestock: cattle, pigs, chicken, etc. This may have been sustainable in the past, but this is no longer the case. The explosion in population growth in the twentieth century has forced the global community to consider whether traditional, meat-focussed diets continue to be achievable. For a majority of the twentieth century, the lack of adequate protein was met with the intensification and increase of livestock cultivation. This has led to a host of environmental and animal welfare issues, signposted by disease outbreaks such as bovine spongiform encephalopathy (BSE), foot-and-mouth disease, antibiotic resistance and several examples of J. A. Whittaker · S. V. Avery · P. S. Dyer (*) School of Life Sciences, University of Nottingham, Nottingham, UK e-mail: [email protected] R. I. Johnson · T. J. A. Finnigan Quorn Foods, Stokesley, North Yorkshire, UK © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_3

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zoonotic influenza (Wilesmith 1994; Gibbens et al. 2001; Reid et al. 1999; Lam et al. 2015) and by the drastic loss of wilderness and degradation of habitats leading to significant reduction of biodiversity. Coupled with this is that livestock are one of the major contributors to greenhouse gasses and thus a significant contributor to global warming. One alternative to the intensive farming of livestock is the cultivation of microorganisms with the goal of producing edible biomass. This is commonly referred to as the formation of single-cell protein (SCP), despite many production organisms being multicellular, e.g. filamentous fungi and algae. The term was coined by the Massachusetts Institute of Technology to describe products that originated from microorganisms whilst avoiding the potential negative connotations with more obvious names such as ‘microbial’ or ‘bacterial’ protein (Trinci 1992). There are two main ways in which SCP can be consumed: directly, as biomass, or as a supplement to increase the protein content of other foods. Historically, SCP has been produced mainly as feed for livestock rather than for human consumption. Important examples include Pruteen™ (Marstrand 1981) and SCP made in the Pekilo process (Koivurinta et al. 1979). More recently Calysta (http://calysta.com/) have started producing animal feed from methanotrophic bacteria. It can be argued that there is the risk that the production of SCP as a more sustainable animal feed might only serve to intensify factory farming and its attendant issues of welfare and environmental damage. However, since the advent and subsequent success of Quorn mycoprotein, more groups have recently been attempting to produce SCP for direct human consumption, including the Swedish company Mycorena (https://mycorena. com/) and the UK company 3F Bio (http://www.3fbio.com/). Several basic requirements should be considered when designing a SCP manufacturing process for human consumption. First, the manufacture of the protein by SCP must be economically viable to compete with more established meat alternatives, such as tofu and other soy derivatives, as well as meat itself. Second, the organism must be able to be put into large-scale production and grow suitably to produce sufficient quantities of the product. Third, if the product is going to be a replacement for conventional protein sources, it follows that it must be high in protein and provide several essential amino acids (Finnigan 2012). To achieve the relatively high levels of protein demanded by the human diet, SCP is preferably produced from organisms which readily produce at least 30% of their biomass as protein. Indeed, the initial screening process at Quorn™ demanded a microorganism that produced at least 45% of its biomass as protein (Moore et al. 2011). Consequently, production of SCP is restricted to a relatively small number of species. The pool of organisms is further limited due to stricter safety concerns and subsequent regulations regarding food for human consumption when compared to animal feed (Finnigan et al. 2017). Current manufacturing methods that produce SCP for human consumption use food-grade substrates (Ritala et al. 2017). In an effort to keep costs low and increase profit margins, novel processes which use alternative, inexpensive materials have been researched. An obvious and plentiful source of carbon sources includes waste materials from industries such as those involved in the production of food, cellulosic

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ethanol and paper. For example, a Finnish process termed ‘Pekilo’ was developed to produce SCP for animal feed using sugars present in sulphite liquor paper mill effluents. In this process, the filamentous fungus Paecilomyces variotii is used in a continuous fermentation process, transforming the sulphite liquor containing 32 g/L sugars into biomass with a protein yield of 55% (w/w) (Ugalde and Castrillo 2002). Pekilo SCP was initially developed and sold as animal feed, but it was also investigated as being a protein supplement in commercial meat products (Koivurinta et al. 1979). Whilst no longer operational, the Pekilo process was an important step in proving the viability of so-called waste materials being used as substrates. Indeed, much of the research into SCP production has been focussed on the use of such waste substrates: for example, lignin and lignocellulosic waste (Chrysonilia sitophila, Rodrìguez et al. 1997; Candida utilis and Rhizopus oligosporus, Yunus et al. 2015), cheese whey (Candida krusei and Kluyveromyces marxianus, Yadav et al. 2014), agricultural waste (Candida utilis, Jalasutram et al. 2013), various industrial wastewaters (purple bacteria/microalgae, Hülsen et al. 2018) as well as common food industry wastes such as apple pomace (Aspergillus niger, Bhalla and Joshi 1994), banana wastes (A. niger, Baldensperger et al. 1985) and even prawn shells (marine yeast, Rhishipal and Philip 1998). In some bacterial fermentations, the major greenhouse gas, methane, is becoming an increasingly used substrate. Methylococcus capsulatus has been demonstrated to generate bacterial protein from methane with high efficiency but requires a bacterial consortium in order for an extended production cycle to be maintained, as opposed to using a single isolate (Bothe et al. 2002). These examples show great promise for the flexibility of being able to use microorganisms to produce proteinaceous biomass from a diverse array of substrates.

2 Quorn: A Historical Perspective Despite Quorn products first appearing on supermarket shelves in the UK in 1985 [a range of savoury pies launched by J. Sainsbury (Fig. 3.1) (Trinci 1992)], development of the product had begun several decades before. Explosive population growth in the twentieth century led to predictions of global protein famine by the 1980s, if the world continued to rely on traditional protein sources and failed to develop novel protein sources (Paddock and Paddock 1967). The United Nations produced a report (Anon 1968) which, among other solutions, considered how protein produced by microorganisms might prevent such a crisis. In response, many research groups investigated the possibility of manufacturing protein sources for animal feed, primarily using bacteria. An important example was the development of Pruteen™, a pig feed manufactured by Imperial Chemical Industries (ICI), produced using the bacterium Methylophilus methylotropus fermented on methanol (Marstrand 1981).

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Fig. 3.1 The first commercial products of mycoprotein production—savoury pies by J Sainsbury (UK). The mycoprotein was described at the time as being sourced from ‘a mushroom like a tiny plant’, avoiding reference to the term ‘fungus’

2.1

The Search for an Organism

In contrast to the Pruteen™ product, Lord Arthur Rank [of Rank Hovis McDougall (RHM)] decided that a different approach was needed. In his opinion, it was more beneficial—both in an economic and societal sense—to produce protein suitable for human consumption. RHM was a major producer of cereals and derived products and therefore produced great quantities of starch that could be exploited (Finnigan 2012). Consequently, an aim laid out by Lord Rank was to discover and utilise a microorganism that would convert the plentiful starch into edible biomass, rich in protein and suitable for human consumption. It was decided that filamentous fungi would be used for several reasons. Firstly, humans have used fungi as food for centuries, with the fruiting bodies of many fungi being eaten directly or moulds being used to create several fermented foods (e.g. oncom, tempe, soy sauce, etc.). Secondly, it was proposed that the biomass produced would mimic the organoleptic properties of animal muscle and could therefore easily act as a substitute for conventional meat products in the consumer’s diet. Finally, the extraction of fungal mycelia from culture broth is relatively easy.

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The initial search for a suitable microorganism began in a field adjacent to an RHM-owned wheat starch plant in Ashford, Kent (Angold et al. 1989). The field was regularly sprayed with starch, aiming to drive selection for organisms that preferentially metabolised starch as a carbon source. This selection protocol proved to be a success, yielding a Penicillium notatum strain designated ‘C1’, an isolate which exhibited a protein profile similar to that of casein. For 3 years, advances were made in improving the fermentation process for C1. However, the project was eventually discontinued due to unsuitable growth of P. notatum in continuous culture, awarding P. notatum another runner-up prize in the annals of biotechnology following its replacement for use in penicillin production (Finnigan 2012). And thus the search continued. Between 1967 and 1972, over 3000 fungal isolates were collected by RHM from soil samples across the globe and were subsequently screened for several parameters: protein content and quality (e.g. nitrogen and α-amino nitrogen levels), growth in continuous culture, pigment production, lack of mycotoxin production and pathogenicity and more (Anderson et al. 1975; Anderson and Solomons 1984; Moore et al. 2011). Following these preliminary screens, 20 fungal isolates were chosen for feeding trials with small animals, used as preliminary toxicology screens (Moore et al. 2011). This shortlist of fungal isolates comprised a number of Aspergillus species and Neurospora sitophila, together with eight Fusarium species including F. culmorum, F. solani and F. graminearum (Anderson et al. 1975; Anderson and Solomons 1984). Finally, one strain of F. graminearum was taken forward based on the best combination of favourable protein production, suitability for fermentor growth, low odour and low toxicity (Anderson and Solomons 1984). Ironically, despite the global sampling, the ancestor to the current production strain—then termed F. graminearum A3/5—was collected and isolated from a soil sample from a compost heap in Marlow Bottom just 4 miles from the Lord Rank Research Centre in Marlow, Buckinghamshire, UK, on 1 April 1968 (G. Edwards pers. comm.). The fungus was thereafter sub-cultured twice before reference strains were deposited as F. graminearum at the International Mycological Institute, UK, in 1969 (A3/5/ 3; IMI 145425). Derivatives of A3/5/3 from lyophilised stocks have been used thereafter for Quorn™ mycoprotein production. Note that a further strain was deposited by RHM in 1971 in the American Type Culture Collection (as ATCC 20334), although this strain was later shown to differ markedly from the original A3/5/3 strain in terms of levels of mycotoxin production (Miller and MacKenzie 2000) and the precise lineage relationship of ATCC 20334 to A3/5/3 is unclear. In 1998, the Quorn™ production strain was reidentified as F. venenatum, rather than the original F. graminearum, based on molecular phylogenetic, morphological and mycotoxin analysis (O’Donnell et al. 1998). This finding was confirmed by parallel RAPD analysis of ATCC20334 compared to closely related Fusarium species (Yoder and Christianson 1998). This was an important development because F. venenatum has significantly lower potential for mycotoxin production than F. graminearum. Most recently, a genome sequence has been reported from strain ATCC 20334 (King et al. 2018). Genome analysis together with experimental work confirmed that F. venenatum possesses significantly reduced pathogenic capacity and mycotoxin

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potential compared to F. graminearum, being incapable of colonising wheat plants and only producing a superficial infection of tomato after an extended incubation time (King et al. 2018). Although the Quorn fungus was once thought to be widespread, when identified as the cosmopolitan F. graminearum, more recent field sampling suggests that F. venenatum has a rather more restricted and rare distribution (author’s unpublished results). The fungus has been reported from nature on a variety of plant species and from soils in Europe and is presumably primarily saprotrophic in nature (Nirenberg 1995; Leslie and Summerell 2006).

2.2

Development of a Commercial Product

Following the isolation and subsequent suitability trialling of F. venenatum (at that point still identified as F. graminearum), a 10-year study was performed between 1970 and 1980 to evaluate the safety of F. venenatum mycelia for human consumption. A major concern was the potential production of mycotoxins by the fungus, as many Fusarium species are well known as mycotoxin producers linked to their role as notorious phytopathogens (Nelson et al. 1993). Consequently, substantial toxicology screening was performed involving feeding trials on 11 animal species, including several larger mammalian species such as pigs and cows. The results of these studies showed that the mycelium of F. venenatum produced no adverse effects on either the animals or their offspring. The product made from the mycelium of F. venenatum was later termed ‘mycoprotein’ by the UK Food Standards Agency (Moore et al. 2011), referring to the fact that it contained relatively high amounts of protein derived from fungi, although it is important to note that the product is not exclusively protein in composition but contains additional general components of the fungal mycelium. More details on the nutritional composition of Quorn™ mycoprotein are provided by Finnigan et al. (2017). Interestingly, this appears to have been an independent reinvention of the word ‘mycoprotein’, the use of which was first recorded back in the nineteenth century referring to protein extracted from microbial biomass (Nencki and Schaffer 1880, reported by Anon 1880). Trials with 2500 human volunteers followed, which also showed that there were no immunological manifestations or other symptoms following mycoprotein consumption (Moore et al. 2011). These results were presented in a report submitted to the now-defunct Ministry of Agriculture, Fisheries and Food (MAFF, UK) in 1983, resulting in approval being granted for mycoprotein sales in 1985. Products were marketed using the brand name Quorn™, which at the time was a name (derived from a village in Leicestershire, UK) owned by RHM for a range of packet sauce mixes but which tested well in consumer research for the new range of foods made with mycoprotein. In 1994, further toxicity tests were performed and submitted to the United States Food and Drug Administration (FDA), the approval of which was required before sale of Quorn products was possible in the USA (Miller and Dwyer 2001).

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Despite RHM having discovered a satisfactory production organism, they lacked the necessary large-scale fermentation expertise required to develop a process for the creation of a commercial product. To rectify this, a 3-year collaboration was started in 1973 with DuPont, providing the necessary groundwork and knowledge to initiate the project. Quorn was initially produced using continuously stirred tank reactors. However, it was the collaboration with ICI in 1984 that led to the development of the current airlift fermentation technology (following test production in a pressure loop fermenter from the ICI Pruteen pilot plant), as well as the establishment of Marlow Foods Ltd.—the foundation of Quorn (Finnigan 2012; Seviour et al. 2013). Since then, three more airlift fermentors have been built to meet demand for the Quorn™ product, with the construction of several more planned in the future. It has been remarked that given the long history of mycoprotein development, but fairly recent commercial success, that Quorn™ might be regarded as something of both a 20- and 50-year overnight success (Trinci 1992; Finnigan et al. 2017). Whilst the project began as a solution to the impending protein crisis, by the 1980s, it became apparent that no such crisis was on the immediate horizon. Instead, Quorn mycoprotein began to supply a market that was looking for healthy new proteins with a low environmental impact. Quorn Foods now supply over 100 different products to over 17 countries worldwide including much of Europe as well as the USA and Australia and more recently into new Asian markets. Mycoprotein is at the heart of all Quorn foods with a nutritional profile that is low in fat and saturated fat, free from cholesterol as well as rich in dietary fibre and protein (Finnigan et al. 2017). In addition, the protein has been shown to be of high quality with a protein digestibility corrected amino acid (PDCAAS) value in excess of many animal proteins (Edwards and Cummins 2010) with recent research building on this to show muscle building properties in human respondents equivalent to the animal proteins that were assessed (Dunlop et al. 2017). Indeed, research carried out over the past 20 years has consistently shown that diets rich in mycoprotein can positively impact appetite, glucose homeostasis and serum lipid profiles (Denny et al. 2008). More recently, mycoprotein has again been shown to have a positive impact on appetite and insulinaemia, although no causal mechanism was established and this remains a focus of ongoing research (Bottin et al. 2016). A comparison of metabolic markers of health in respondents consuming meat- or mycoprotein-based diets showed no significant difference (Coelho et al. 2018), and the unique combination of a glucan/chitin fibre-rich protein source continues to excite the interests of nutrition researchers. In addition, lifecycle analysis of the production of mycoprotein has shown significant benefits when compared with that of animal proteins, with embedded greenhouse gas (GHG) emissions of mycoprotein production of only 1.6 kg/kg compared with beef which is at least ten times greater (Finnigan et al. 2017; Poore and Neemak 2018).

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Fig. 3.2 Picture of Quorn fermentors 2 and 3 (in foreground) and 4 (to the rear) on site at Billingham, October 2017

3 The Present 3.1

Development of an Industrial Biotechnology Process

Several fermentation systems were tested in initial trials to produce mycoprotein, namely, batch, fed-batch and continuous processes. A continuous fermentation was chosen because biomass was produced continuously over a longer period compared to batch systems, ultimately making production more efficient. Furthermore, the conditions of a continuous culture were more easily controlled than batch cultures. Until 1994, mycoprotein was primarily produced by the pilot-scale fermenter formerly used for making Pruteen by ICI. This fermenter (named Quorn 1) had a volume of 40 m3, capable of producing 1000 tonnes of Quorn™ mycoprotein per annum. In 1993, a new 155 m3 airlift fermentor was commissioned (named Quorn 2) swiftly followed by Quorn 3 (Fig. 3.2) (Moore et al. 2011). These fermentors ramped production up to 10,000–14,000 tonnes of mycoprotein per annum. Combined, these new fermentors enabled sales of mycoprotein to reach £74 million per annum in 1997. The introduction of a further fermentor ‘Quorn 4’ has enabled subsequent fulfilment of increasing demand (Fig. 3.2). However, the Quorn Foods company now finds itself in a position where there is a demand for new fermentors to be constructed in order for supply to meet demand.

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CO2 Disengagement

O2 Added

Cooling Coils

Low RNA Mycoprotein Harvest Steam In Heat Treatment Glucose and Growth Media Added

Sterile Air and NH3

Fig. 3.3 Schematic of the airlift fermenter used by Quorn Foods to produce mycoprotein. Adapted and updated from Moore et al. (2011)

The Quorn fermentors are currently the largest continuous flow culture systems in use by the biotechnology industry worldwide, using airlift technology as opposed to more traditional stirred-tank bioreactors. The airlift design offers particular benefits for mycelial production because it is a low shear design, allowing the long hyphal lengths to develop that are needed to ensure good replication of the desired meat-like textures in the final product. Airlift fermentors are also low energy input compared to stirred tank fermentors, allowing good mixing with a much lower energy footprint. In these fermentors, oxygen saturation of the media is achieved through the influx of sterile air bubbles which then mix the culture medium, as opposed to using mechanical impellers as in conventional fermentors. Most of the oxygen transfer occurs near the base of the riser, where sterile air is pumped into the vessel, taking advantage of the substantial hydrostatic pressure (Fig. 3.3). The hydrostatic pressure, together with the turbulence generated by the pumped air, provides excellent conditions for oxygen transfer from the gaseous to the liquid phase. Consequently, the riser contains a mixture of air and culture, which flow together producing an environment where air bubbles constitute up to 50% of the volume. Then further up the riser, air levels

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decrease to around 10% of the volume at the top of the riser, resulting in a lower rate of oxygen transfer. The low pressure at the top of the riser causes nitrogen, unused oxygen and CO2 to be released from the culture at the horizontal gas disengager. The culture then enters the oxygen-depleted downcomer, where oxygen is pumped in to prevent death of the culture. Once the culture passes through the downcomer, it is directed back into the riser to be charged with air once again, completing the cycle (Fig. 3.3). The relative difference in specific gravity between the oxygen-rich riser and the oxygen-depleted downcomer ensures that the hyphal filaments within the culture circulate continuously around the fermentor loop, completing one loop every 2 min for fermentors Quorn 2 and 3. It has been calculated that mycelium travels at an approximate speed of 4 m s
1, making F. venenatum one of the ‘fastest fungi’ in the world. Medium is fed into the culture at a dilution rate in the range of 0.14 to 0.20 h
1, whilst mycoprotein is harvested at the same rate, resulting in a combined output of 30 tonnes h
1 for Quorn 2 and Quorn 3 (Moore et al. 2011). The culture is incubated at 30  C by a heat exchanger that is incorporated into the riser. Ammonia is used as the nitrogen supply for growth, which is fed into the fermentor along with the sterile compressed air at the base of the riser. The supply of ammonia into the vessel is controlled by a pH monitor set to maintain the culture at pH 6.0. The fermentor is operated as a glucostat—an environment where glucose is always in excess—with the fungus growing continuously at a specific growth rate nearing its maximum specific growth rate, to drive maximum biomass production. Once the fungus has reached a steady state approximately 4 days postinoculation, hyphal biomass is continually harvested and undergoes further processing. The raw mycelium taken from the fermenter is heated to 68  C before being passed into a separate tank, for an average of 15 min, to reduce nucleic acid levels. The requirement for this will be addressed in greater detail later in this chapter. The mycelium at this stage has specific physical characteristics as a consequence of the selection process that the ancestor of A3/5 underwent. The minimum hyphal length is ideally ca. 400–700 μm, as final products made with an increasing proportion of hyphal filaments shorter than this will unfortunately present increased crumbliness (Finnigan 2012; Finnigan et al. 2017). Whilst somewhat similar to fibres in meat, the hyphae lack the cross-linking structures prominent in muscle (Thrane 2007). Therefore, egg albumin is used as a binding agent to achieve a similar texture, but other alternatives are being used for the Quorn Food company’s expanding vegan range, for example, agar and locust bean (Ceratonia siliqua) gum. Development of the final meat-like texture is achieved by freeze texturisation of the fibre-gel complex, creating hyphal laminations whose behaviour during compression-fracture during mastication is similar in manner to whole cooked muscle. For further discussion of factors such as hyphal morphology, interactions between hyphae, orientation and dispersion of hyphae and fibre alignment, which influence the creation of a meat-like texture, see the recent review by Finnigan et al. (2017).

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Biotechnology Issues of the Modern Fermentation Process

Although the fermentation conditions of Quorn™ mycoprotein have been developed over several decades, the industrial process nevertheless suffers from several problems that continue to hinder the modern industrial process. These are outlined below.

3.2.1

Carbon Source

Considering the scale of Quorn™ mycoprotein production, an obvious cost is the carbon substrate used to facilitate the growth of F. venenatum. The current Quorn™ fermentation process uses a highly refined, starch-derived glucose syrup, rather than the original concept of using starch directly in the industrial fermentation. This change can be understood by considering that growth on glucose circumvents the need for the hydrolysis of a polymer. When polymeric carbon sources, such as starch, are utilised, the rate-limiting step is no longer the rate of substrate uptake but instead the activity of the necessary hydrolytic enzymes (such as glucoamylase). As a consequence, growth on carbon sources with increasing polymerisation is likely to result in a loss of growth rate, as was indeed shown by Anderson et al. (1975) when comparing growth of the Quorn fungus on glucose, maltose or maltotriose. In the case of starch, F. venenatum possesses limited α-amylase activity, and as a result growth on starch is relatively slow. Therefore, the use of glucose was favoured in the industrial process due to faster growth rates and subsequently a more productive industrial process. The application of the glucose syrup has two significant problems: its price and its supply. Every day, several heated tankers deliver glucose syrup to the mycoprotein production site at Billingham, UK, at strictly designated times. This timing is essential, as each tanker arrives just in time for the glucose to be fed into the fermenters. This approach is flawed, because it creates a situation where the fermenters rely on the punctuality of substrate delivery. Should a supply be delayed for any significant amount of time, production will have to be stalled, as only a few hours’ worth of sugar reserves are maintained on-site. Furthermore, the supply of the syrup using a heated container carries inherent risks and subsequent safety-related costs. Research into the suitability of alternative carbon sources which are more easily stored (e.g. dried sugars) may allow the storage of such a source on-site as a backup, preventing any issues with late syrup delivery. However, there is evidence to suggest that the use of a different sugar source can alter the amino acid content in Fusarium species. Anderson and Solomons (1984) compared total amino acid content in F. venenatum (F. graminearum in text) when grown on glucose and on ribose and found that the ribose-fed cultures showed a reduction in total amino acid content in comparison to those grown on glucose (394 mg/g and 494 mg/g, respectively). A fundamental selling point of Quorn™ mycoprotein is the high amount of non-animal-derived protein. Amino acid content

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is therefore a key consideration when investigating alternative carbon sources, because using a carbon source that would decrease the amount of protein would be undesirable. In addition, there are safety concerns that should be noted when considering alternative carbon sources. It is important to recall the intense safety testing that Quorn™ mycoprotein was subjected to before being sanctioned for sale. Permission was only granted after sufficient evidence that the production system did not result in the formation of mycotoxins. If an alternative carbon source is to be considered for the industrial process, it would be wise to exercise caution because studies of other fungal species suggest that mycotoxin production is influenced by the carbon substrate (Buchanan and Stahl 1984; Bouras and Strelkov 2010; Brzonkalik et al. 2011). Indeed, F. graminearum has shown increased trichothecene production when grown on certain carbon sources relative to levels produced using glucose (Jiao et al. 2008). Intriguingly, there was a strain-dependent effect with many of the carbon sources used, with the important exception of sucrose which showed significantly increased production of trichothecenes (relative to growth on glucose) independent of the strain used.

3.2.2

Nucleic Acid Levels

Another important consideration in the production of any single-cell protein product intended for human consumption is the nucleic acid content of the final product. As a core component in the structure of DNA and RNA, nucleic acids are abundant in all living organisms—particularly if they have a rapid growth rate. Yeasts and filamentous fungi have a relatively high nucleic acid content, approximately 10 and 8% of the dry weight, respectively (Kihlberg 1972). These levels are four- to fivefold greater than the average mammalian organ, and this can cause physiological complications if consumed in excess—a problem for SCP production (Scrimshaw 1975). Upon ingestion, purinic nucleic acids are ultimately metabolised to the relatively insoluble uric acid. In non-primate vertebrates, uric acid is then further converted into the soluble acid allantoin, which is the primary means of disposing of nitrogenous waste in such animals. As the allantoin conversion pathway does not exist in humans and other higher apes, uric acid must be excreted through the kidneys. If uric acid levels exceed the excretion capacity of the kidneys, the excess acid crystallises in joints and soft tissues, leading to gout and kidney stones (Sinskey and Tannenbaum 1975). Pyrimidinic nucleic acids are degraded to orotic acid, an excess of which causes liver damage. Knowing this, the sale of Quorn™ mycoprotein was approved by MAFF in 1985 on the condition that the nucleic acid content was reduced to within World Health Organization recommendations (Anderson et al. 1975). These recommendations state that for human consumption of SCP, ingestion of RNA should not exceed 2 g per day for adults. Under the modern mycoprotein fermentation conditions, F. venenatum biomass has an RNA content of 8–9% (w/w). Under the WHO guidelines, this would mean a maximum consumption of 20 g of mycoprotein per

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person per day (Trinci 1992). If Quorn™ mycoprotein was to be sold as a commercial product, it was therefore deemed economically unfeasible to retain RNA content this high. Accordingly, a method to reduce the RNA level in the fungal mycelia was developed. The harvested mycoprotein is subjected to heat treatment of 68  C for 15 min (Ward 1998). This process allows the endogenous RNases to remain active whilst halting growth and disrupting the activity of ribosomes. Cellular RNA is broken down into 50 nucleotides which are then able to diffuse out of the cell wall into the culture broth (Anderson and Solomons 1984). Crucially, the increased temperature inactivates proteases, limiting protein loss, and also renders the F. venenatum source organism non-viable (Finnigan et al. 2017). However, whilst RNA content is reduced below the consumption limit of 100 g of mycoprotein per day, the process is also thought to render the cell membranes leaky, thereby causing a drastic loss (ca. 33%) of dry biomass into the liquid (centrate) discharged from a subsequent centrifugation step (Solomons 1983; Finnigan et al. 2017). As a result, a significant proportion of potential mycoprotein is lost, resulting in less commercial product being available for sale. After the heat treatment, mycoprotein contains ca. 1% (w/w) RNA. This allows consumption of more than 100 g of mycoprotein per day to be well within the 2 g limit recommended by WHO, clearly a much more favourable scenario for a foodstuff.

3.2.3

Morphological Variants

It was decided early in the development of the process for mycoprotein production that continuous fermentation of F. venenatum would be used, because this was deemed a more economically viable process. Theoretically, the fermentations could proceed uninterrupted, with fungal mycelia being harvested throughout. However, in practice, the process must be terminated after ca. 1000 h (about 160 generations) after the initiation of continuous conditions (Quorn™ manufacturing specification, TJA Finnigan personal communication). This is as a consequence of the appearance of highly branched colonial mutants (often called C variants) in the fermentor (Fig. 3.4a–c) (Trinci 1992; Finnigan et al. 2017; TJA Finnigan personal communication). The C variants gained their name as they form compact, discrete, dense colonies on agar plates (Fig. 3.4d). This is not unusual in chemostat cultures of filamentous fungi, for example, it has been shown that morphological mutants arise in continuous cultures of Aspergillus nidulans and A. niger (Swift et al. 2000), as well as A. oryzae (Withers et al. 1994). In the case of F. venenatum, the mutations that produce the highly branched morphology are recessive and grant the mutants an increased specific growth rate compared to the parental strain (Wiebe et al. 1992; Simpson et al. 1998). Intriguingly though, Simpson et al. (1998) have shown that the highly branched phenotype of the C variants is not the characteristic that drives mutation but rather a consequence of a metabolic difference, assumed to have a selective advantage under continuous culture, which then produces a pleiotropic effect on hyphal morphology.

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Fig. 3.4 Different morphological variants of Fusarium venenatum as seen under the microscope, showing the differences in branching frequency and internodal length between the a and c variants. (a) Typical and desired morphology of F. venenatum (a variant). (b) Non-desired morphology of F. venenatum (c variant; including two inset examples). (c) A side-by-side comparison of a and c variants (right and left hand sides, respectively). (d) Growth plate (9 cm diameter) showing normal colony morphologies of F. venenatum along with a single compact, dense, white and sharply edged c variant colony (arrowed) following growth for ca. 48 h at 25  C

These mutants are similar in both chemical composition and nutritional quality as the wild-type culture (Trinci 1994). Yet they present a problem with one of Lord Rank’s main goals, being the achievement of a fungal product that emulated the structure of meat (Finnigan 2012). When C variants are introduced into the final product, instead of the expected production morphology, the meat analogue becomes crumbly rather than fibrous (TJA Finnigan and RI Johnson personal communication). Consequently, the quality of the final product cannot be maintained if the proportion of the C variant is allowed to increase in the fermenter. The solution by the Quorn™ company involves interrupting the fermentation process around 1000 h before the accumulation of the C variant has an adverse impact on the quality of the mycoprotein. The production process is then resumed several days later after cleaning and sterilisation of the relevant fermenter, leading to a loss of productivity (TJA Finnigan and RI Johnson personal communication). Prevention or delay in the appearance of colonial mutants would enhance productivity of the fermentation process and decrease the cost of production. Three strategies to reduce the appearance of C variants have been suggested (Trinci 1994). The first involves operating the fermentor at a lower dilution rate, to

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select against certain mutant varieties. However, this change was found to be offset by decreased biomass production within the fermentor, rendering the adjustment non-viable from an economic viewpoint. The second strategy involves a rotational programme of selection pressures within the fermentor, producing an environment where no single mutant would be favoured at all times. This solution also poses a problem; not only does it complicate the production procedure but may also lead to a change in the final product. Finally, it may be possible to isolate or generate sparsely branched strains of F. venenatum which are more stable than A3/5 with regard to mycelial morphology (Trinci 1994). Diploid strains of A3/5 used in laboratory-scale fermentations did not produce colonial mutants until 1957 and 2028 h after the onset of continuous flow (Naylor et al. 1999; Moore et al. 2011). Colonial mutants appeared to take ca. five- to sixfold more generations to appear in two diploid strains compared to the appearance of colonial mutants in haploid A3/5 (Wiebe 2002). The use of such strains within the fermentor could potentially greatly improve the efficiency of the mycoprotein production process, assuming no other unwanted effects.

3.2.4

Mycotoxin Production

The genus Fusarium is perhaps best known for comprising a large number of plantpathogenic species. For example, F. oxysporum f. sp. cubense is the causal organism of Panama disease on bananas, which has destroyed hectares of banana plantations in Latin America and increasingly worldwide (Xue et al. 2015), whilst F. graminearum and F. culmorum cause fusarium head blight, a devastating disease of wheat and barley responsible for billions of dollars of damage each year (Nganje et al. 2004). Linked to their activity as plant pathogens, Fusarium species are capable of producing a wide variety of mycotoxins, primarily fumonisins and trichothecenes, but also zearalenone and gibberellic acids. Of the trichothecenes, the most wellknown are diacetoxyscirpenol (DAS), nivalenol, deoxynivalenol (DON) and T-2 toxin (Desjardins 2006). F. graminearum, F. crookwellense, F. culmorum and F. pseudograminearum have been shown to consistently produce trichothecenes. A second major group of trichothecene-producing Fusarium includes F. sambucinum and the related species F. venenatum (Altomare et al. 1995). Certain strains of F. venenatum have been shown to produce the mycotoxins scirpentriol (SCIRP), 15-acetoxyscirpenol (15 AC-SCIRP), DAS, and 4-monoaceytoxyscirpenol (4-MAS) and other trace secondary metabolites under suitable in vitro growth conditions with severe nitrogen limitation and deregulation of carbon metabolism, even including the production strain A3/5 (Altomare et al. 1995; O’Donnell et al. 1998; Miller and MacKenzie 2000). However, fortunately the industrial conditions used for mycoprotein production (with excess nutrients present) do not induce mycotoxin production. Nevertheless, samples of Quorn™ mycoprotein are taken regularly from the production line to test for the presence of trichothecenes as well as other mycotoxins, the most important of which are DAS and DON (Fig. 3.5a, b). The presence of any mycotoxin is yet to be detected in any

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Fig. 3.5 Chemical structures of mycotoxins. (a) Diacetoxyscirpenol (DAS). (b) Deoxynivalenol (DON)

production run in the history of mycoprotein production. Indeed, recent genome sequence analysis of F. venenatum has shown that the fungus appears to be less well equipped for mycotoxin production when compared to F. graminearum. Fifteen secondary metabolite gene clusters identified in F. graminearum were either absent or possessed a highly diverged sequence in F. venenatum. Five of these were shown to have increased in planta expression in F. graminearum (King et al. 2018).

3.3

Future Biotechnology Research Challenges

Although favourable processes for the production of Quorn™ mycoprotein have been established, there is nevertheless the continued demand for improvement in the biotechnology of mycoprotein production, both from economic and environmental sustainability viewpoints. Various areas of future research may address these biotechnological challenges. First, it may be possible to implement strain improvement strategies to increase output by F. venenatum A3/5. There are several success stories on the use of strain improvement with filamentous fungi in biotechnology, for example, Aspergillus niger (citric acid; Haq et al. 2001; Kirimura et al. 1992), Penicillium chrysogenum (penicillin; Newbert et al. 1997) and Trichoderma reesei (cellulases; Peterson and Nevalainen 2012) among others. Due to current restrictions on the use of GM technologies in foodstuffs, such strain improvement will likely involve the use of classical methods for strain improvement for the immediate future. One particularly enticing prospect is that it might be possible to induce a sexual cycle in F. venenatum [which is currently only known as an asexual organism (Yoder and Christianson 1998)] and use this for commercial strain improvement, breeding in desired traits from other F. venenatum strains, as described elsewhere (Ashton and Dyer 2016). For example, sexual breeding has recently been used to produce new strains of Penicillium chrysogenum and P. roqueforti with improved characteristics for penicillin and blue cheese production, respectively (Böhm et al. 2013; Dyer and Kück 2017; PS Dyer, M Novordvorska, H Darbyshir, S Swilaiman unpublished results). Indeed,

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closely related Fusarium species such as F. graminearum and F. sambucinum display a sexual cycle (O’Donnell et al. 1998; Yoder and Christianson 1998) suggesting there is hope for such a strategy to be applied. Furthermore, mating-type genes, which are key regulators of fungal sexual reproduction (Dyer et al. 2016), have recently been identified from F. venenatum (King et al. 2018; author’s unpublished results). Another classical method, protoplast fusion, has also been applied in preliminary studies to make heterokaryons of F. venenatum (Yoder and Christianson 1998) and might offer another option for follow-up work. In the future it may even be possible to use new technologies such as CRISPRCAS methodologies for strain improvement purposes (Anon 2018). Indeed, CRISPR-CAS has recently been approved in the USA for use with the fungus Agaricus bisporus (Waltz 2016), although the European Court of Justice has recently ruled that CRISPR-CAS-modified organisms come under the GMO contained use regulations (Michalopoulos 2018). Such technologies might allow, for example, the directed deletion of elements involved with mycotoxin production and/or C variant evolution, thereby improving the characteristics of the production strain. Previous work has demonstrated that deletion of the gene for trichodiene synthetase via conventional GM procedures resulted in the loss of production of trichothecenes and related compounds in F. venenatum (Royer et al. 1999; Miller and MacKenzie 2000). A further area of research concerns reducing wastage in the production process. Dehydration of the fermentor ‘waste’ known as centrate produces a powdered ingredient rich in 50 nucleotides and with applications as a yeast-free flavour in foods with proven umami impact (Finnigan et al. 2017). Alternatively, research is also focussed on ways to return centrate for use in the fermentation, thereby reducing the effluent load and water use which will further enhance the sustainability of the process (Quorn Foods R&D, TJA Finnigan and RI Johnson personal communication). Another area of future biotechnology challenge includes producing mycoprotein tailored for amino acid requirements in different applications, for example, amino acid profiles suitable for canine and feline pet diets as an alternative to meat. In addition, it may be possible to manipulate the system in ways that compensate for the detrimental impacts on amino acid composition that have been reported during growth on alternative, cheaper carbon sources (described above).

4 Conclusions Quorn mycoprotein is an important example of how the successful application of fungal biotechnology can address problems faced by the global community. Despite being initially developed as a solution to the ‘impending’ protein famine in the midto-late twentieth century, Quorn mycoprotein has continued to prove the viability of SCP as an alternative to traditionally cultivated protein sources. Whilst no crisis occurred, the rapidly increasing population of the Earth may cause a similar situation to occur, and furthermore there are environmental concerns with meat production.

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Therefore, investments in SCP, be they fungal or otherwise, appear to be justifiable endeavours. It may be that in years to come, there will be a variety of food products from different SCP brands on supermarket shelves, and not just Quorn. Like other manufacturing processes, the fermentation of Fusarium venenatum to produce mycoprotein still has room for improvement, and certain issues are actively being investigated. Perhaps there are alternatives to the current production pairing of Fusarium venenatum and highly refined glucose that are better-suited to the production of mycoprotein. The vision of Lord Rank lives on. Acknowledgements We thank Gareth Edwards (an employee of RHM in the early 1970s) for providing information about the initial discovery and screening of the Quorn fungus. This chapter is dedicated to the memory of Dr. Geoff Robson who undertook pioneering physiological studies in F. venenatum in the 1990s at the University of Manchester and is sorely missed as a member of the mycological community.

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Brzonkalik K, Kerrling T, Syldatk C et al (2011) The influence of different nitrogen and carbon sources on mycotoxin production in Alternaria alternata. Int J Food Microbiol 147:120–126 Buchanan RI, Stahl HG (1984) Ability of various carbon sources to induce and support aflatoxin synthesis by Aspergillus parasiticus. J Food Saf 6:271–279 Coelho M, Monteyne AJ, Dirks ML et al (2018) Substituting meat/fish for mycoprotein for one week does not affect indices of metabolic health irrespective of dietary nucleotide load or serum uric acid concentrations in healthy young adults. Proc Nutr Soc 77(OCE4):E207 Denny A, Aisbitt B, Lunn J (2008) Mycoprotein and health. BNF Nutr Bull 33:298–310 Desjardins AE (2006) Fusarium mycotoxins: chemistry, genetics and biology. APS Press, Minnesota Dunlop MV, Kilroe SP, Bowtell JL et al (2017) Mycoprotein represents a bioavailable and insulinotropic non-animal-derived dietary protein source: a dose-response study. Br J Nutr 118:673–685 Dyer PS, Kück U (2017) Sex and the imperfect fungi. Microbiol Spectr 5. https://doi.org/10.1128/ microbiolspec.FUNK-0043-2017 Dyer PS, Inderbitzin P, Debuchy R (2016) Mating-type structure, function, regulation and evolution in the Pezizomycotina. In: Wendland J (ed) Growth, differentiation and sexuality, the mycota I, 3rd edn. Springer, Cham, pp 351–385 Edwards DG, Cummins JH (2010) The protein quality of mycoprotein. Proc Nutr Soc 69(OCE4): E331 Finnigan TJA (2012) Mycoprotein: origins, production and properties. In: Phillips GO, Williams PA (eds) Handbook of food proteins. Woodhead Publishing, Cambridge, pp 335–352 Finnigan TJA, Needham L, Abbott C (2017) Mycoprotein: a healthy new protein with a low environmental impact. In: Nadathur S, Wanasundara J, Scanlin L (eds) Sustainable protein sources. Academic Press, Amsterdam, pp 305–325 Fürst P, Stehle P (2004) What are the essential elements needed for the determination of amino acid requirements in humans? J Nutr 134:1558S–1565S Gibbens JC, Wilesmith JW, Sharpe CE et al (2001) Descriptive epidemiology of the 2001 foot-andmouth disease epidemic in Great Britain: the first five months. Vet Rec 149:729–743 Haq I, Samina K, Sikander A et al (2001) Mutation of Aspergillus niger of hyper production of citric acid from black strap molasses. World J Microbiol Biotechnol 17:35–37 Hülsen J, Hsieh K, Lu Y et al (2018) Simultaneous treatment and single cell protein production from Agri-industrial wastewaters using purple phototrophic bacteria or microalgae – a comparison. Bioresour Technol 254:214–223 Jalasutram V, Kataram S, Gandu B et al (2013) Single cell protein production from digested and undigested poultry litter by Candida utilis: optimisation of process parameters using response surface methodology. Clean Techn Environ Policy 15:265–273 Jiao F, Kawakami A, Nakajima T (2008) Effects of different carbon sources on tricothecene production and Tri gene expression by Fusarium graminearum in liquid culture. FEMS Microbiol Lett 285:212–219 Kihlberg R (1972) The microbe as a source of food. Annu Rev Microbiol 26:427–466 King R, Brown NA, Urban M et al (2018) Inter-genome comparison of the Quorn fungus Fusarium venenatum and the closely related plant infecting pathogen Fusarium graminearum. BMC Genomics 19:1–19 Kirimura K, Sarangbin S, Rugsaseel S et al (1992) Citric acid production of 2-deoxyglucoseresistant strains of Aspergillus niger. Appl Microbiol Biotechnol 36:573–577 Koivurinta J, Kurkela R, Koivistoinen P (1979) Uses of Pekilo, a microfungus biomass from Paecilomyces varioti in sausage and meat balls. Int J Food Sci Technol 14:561–570 Lam TT-Y, Zhou B, Wang J et al (2015) Dissemination, divergence and establishment of H7N9 influenza viruses in China. Nature 522:102–105 Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell, Oxford Marstrand PK (1981) Production of microbial protein: a study of the development and introduction of a new technology. Res Policy 10:148–171

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Solomons GL (1983) Single cell protein. Crit Rev Biotechnol 1:21–58 Swift RJ, Craig SH, Wiebe MG (2000) Evolution of Aspergillus niger and A. nidulans in glucoselimited chemostat cultures, as indicated by oscillations in the frequency of cycloheximide resistant and morphological mutants. Mycol Res 104:333–337 Thrane (2007) Fungal protein for food. In: Dijksterhuis J, Samson RA (eds) Food mycology: a multifaceted approach to fungi and food. CRC, Boca Raton, FL, pp 353–360 Trinci APJ (1992) Myco-protein: a twenty-year overnight success story. Mycol Res 96:1–13 Trinci APJ (1994) Evolution of the Quorn® myco-protein fungus, Fusarium graminearum A3/5. Microbiology 140:2181–2188 Ugalde U, Castrillo J (2002) Single cell proteins from fungi and yeasts. Appl Mycol Biotechnol 2:123–149 Waltz E (2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293 Ward PN (1998) Production of Food. US patent 5,739,030. 14 April 1998 Wiebe MG (2002) Myco-protein from Fusarium venenatum: as well-established product for human consumption. Appl Microbiol Biotechnol 58:421–427 Wiebe MG, Robson GD, Trinci APJ et al (1992) Characterization of morphological mutants generated spontaneously in glucose-limited, continuous flow cultures of Fusarium graminearum A3/5. Mycol Res 96:555–562 Wilesmith JW (1994) Bovine spongiform encephalopathy: epidemiological factors associated with the emergence of an important new animal pathogen in Great Britain. Semin Virol 5:179–187 Withers JM, Wiebe MG, Robson GD et al (1994) Development of morphological heterogeneity in glucose-limited chemostat cultures of Aspergillus oryzae. Mycol Res 98:95–100 Xue C, Penton R, Shen Z et al (2015) Manipulating the banana rhizosphere microbiome for biological control of Panama disease. Sci Rep 5:11124 Yadav JSS, Bezawada J, Ajila CM et al (2014) Mixed culture of Kluyveromyces marxianus and Candida krusei for single-cell protein production and organic load removal from whey. Bioresour Technol 164:119–127 Yoder WT, Christianson LM (1998) Species-specific primers resolve members of the Fusarium section Fusarium. Taxonomic status of the edible “Quorn” fungus reevaluated. Fungal Genet Biol 23:68–80 Yunus F-U-N, Nadeem M, Rashid F (2015) Single-cell protein production through microbial conversion of lignocellulosic residue (wheat bran) for animal feed. J Inst Brew 121:553–557

Chapter 4

The Current Biotechnological Status and Potential of Plant and Algal Biomass Degrading/Modifying Enzymes from Ascomycete Fungi Ronald P. de Vries, Aleksandrina Patyshakuliyeva, Sandra Garrigues, and Sheba Agarwal-Jans

1 Introduction Plant and algal biomass are main substrates for various industries and have become even more in the spotlight, with the global push toward a sustainable biobased society and economy. The main fraction of both types of biomass are their cell walls that mainly consist of various polysaccharides. Depending on the desired end product, processing the biomass requires modification of these polysaccharides or partial or complete degradation of them to their monomeric building blocks. Fungal enzymes play a critical role in these processes (Bischof et al. 2016; de Vries et al. 2016; Kruger et al. 2018; Mäkelä et al. 2014b). Fungi produce a high diversity of enzymes targeting these polysaccharides (de Vries et al. 2017; de Vries and Visser 2001; Rytioja et al. 2014; Vesth et al. 2018), making them highly suitable sources of industrial enzymes. In fact, fungal enzymes make up more than half of the enzymes currently used in industrial applications (Fig. 4.1), demonstrating that they are the enzyme source of choice for many applications. In this chapter, we will discuss the use of plant and algal biomass active enzymes from ascomycete fungi in relation to their current and potential applications. The plant biomass section is structured based on the type of application. However, due to the less well-established applications of algal biomass, this section is not structured based on application but rather by type of algae.

R. P. de Vries (*) · A. Patyshakuliyeva · S. Garrigues · S. Agarwal-Jans Fungal Physiology, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_4

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Fig. 4.1 Division of commercial enzymes used in biotechnological applications according to the organism they originate from. Based on AMFEP (2015) List of commercial enzymes. Association of manufacturers and formulators of enzyme products. http://www. amfep.org/list.html

2 Applications of Fungal Plant Biomass Degrading and Modifying Enzymes 2.1

Applications in Food and Feed Production

Fungi and fungal enzymes have been used in production of food for centuries, such as in fermentation, baking, and food modification (Table 4.1; Fig. 4.2) (Beppu 2000; Buchholz and Collins 2013). Enzymes produced by ascomycete fungi, specifically Aspergillus spp. and Trichoderma reesei, are used as food ingredients, additives, or processing aids (Table 4.2). Enzymatic processing of food has distinct advantages over traditional chemical-based technology, in that there is less waste and by-products, less energy consumption, and decreased environmental impact (James and Simpson 1996; Meyer 2010; Sharma et al. 2017). The food industry utilizes fungal enzymes for modification of products, rather than their full degradation. To this end, researchers have been able to engineer and produce very specific recombinant proteins/enzymes that cleanly carry out the required modifications, in large enough quantities to be used in industry.

2.1.1

Bread and Baking

Bread baking using baker’s yeast is one of the oldest biotechnological processes in the world. In modern times, the bread industry faces several challenges: how to decrease preparation time, increase the staling time of the product, and reduce flour variability, to ensure a product that is consistent in size, texture, and taste (Dahiya and Singh 2019). To this end, various combinations of enzymes from fungi are often employed and more importantly have replaced chemical additives. The effectiveness of these enzyme combinations is significant (Martinez Anaya and Jimenez 1997). The strains producing these enzymes have been genetically modified to improve the production yield and decrease costs.

Degradation of lipids, releasing fatty acids

Lipases

Applications Improvement of rheological properties of dough and bread Manufacture of lighter cream crackers, with an improved texture, palatability, and uniformity

(continued)

Reference Delcour et al. (1991), Michniewicz et al. (1991), de Queiroz Brito Cunha et al. (2018), Camacho and Aguilar (2003), Maat et al. (1992), Polizeli et al. (2005) Improvement of wine aroma Ganga et al. (1999) Used in release of fermentable sugars from starch, the Polizeli et al. (2005) carbohydrate storage in grain before fermentation Hydrolysis of arabinoxylans in barley cell wall, which results in decreased beer viscosity and muddiness Animal feed Treatment of fiber in grains to release nutrients Polizeli et al. (2005), Cafe et al. trapped inside macromolecules, producing a high (2002) energy, easily digestible food mixture for animals Prebiotics Production of xylo-oligosaccharides and Aachary and Prapulla (2011), arabinoxylooligosaccharides, by-products of xylan Kumar et al. (2018), Gullon et al. metabolism, for improvement of gut microbiota (2014), Courtin et al. (2008) Paper and pulp Improvement of pulp brightness and thickness, by Ferreira et al. (2016), Walia et al. production improving chemical extraction of lignin from pulp (2017), de Vries and Visser (2001) without compromising cellulose Baking Production of free fatty acids that increase strength of Monfort et al. (1999) gluten protein network Stabilization of gas cell structure in bread, resulting in Gerits et al. (2014) better loaf volume and crumb structure Paper and pulp Control of pitch Singh and Mukhopadhyay (2012), production Gutiérrez et al. (2009), Farrell et al. (1997) Biodiesel production High production yields and easier downstream Gog et al. (2012), Mohamed and Bornscheuer (2003), Adachi et al. purification steps (2011)

Function Industry Degradation of waterBaking insoluble (arabino)xylan to its soluble form by cleaving the main chain of the polysaccharide Wine production Beer production

Enzyme Xylanases

Table 4.1 Overview of the roles of fungal enzymes in industrial applications

4 The Current Biotechnological Status and Potential of Plant and Algal. . . 83

Function Degradation of starch by hydrolyzing α-1,4 glycosidic bonds, resulting in short-chain dextrins

Weakening of gluten structure by breaking down its peptide bonds

Hydrolysis of glycosidic bonds in pectic polymers

Enzyme α-Amylase

Proteases

Pectinase

Table 4.1 (continued)

Juice extraction

Beer production

Baking

Detergent industry

Pulp and paper production

Animal feed

Juice clarification

Industry Baking

Applications Increase in dough mobility, enhancing taste, crust color and toasting qualities of bread Release of fermentable sugars that increase yeast activity, resulting in a bigger bread loaf Removal of starch, thus preventing accumulation with pectins and proteins, removing haze Increase in energy derivation from feed for chickens and pigs, by degradation of starch in grain and grain by-products Production of starch with improved properties (e.g., lower viscosity, higher molecular weight) for paper coating and improvement of the writing quality of paper Used along with lipases, proteases, cellulases, and cutinases, to increase the efficiency of detergents Regulation of gluten structure in dough that improves dough viscoelastic properties, resulting in decreased mixing times, easier use with machinery, and better bread crumb quality Modification of proteins in grain for nutrition of yeast used in brewing Improvement of beer flavor, mouth feel, foam, and color Degradation of pectin that cross-links cellulose and hemicellulose, allowing action of cellulases. Enhancement in the pressing of pulp, resulting in increased juice yield, improved filterability, and reduction in viscosity Sharma et al. (2017), Ramadan (2019)

Polizeli et al. (2005)

Polizeli et al. (2005)

Belhaj et al. (2010), Kirk et al. (2002), Singh et al. (2016b) Deng et al. (2016)

de Souza and de Oliveira Magalhães (2010)

Gracia et al. (2003)

Dey and Banerjee (2014)

Reference Vandam and Hilde (1992), van der Maarel et al. (2002) Martinez Anaya and Jimenez (1997)

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Cellulases

Degradation of cellulose into glucose, through the combined action of exoglucanases, endoglucanases, and β-glucosidases

Kashyap et al. (2001), Garg et al. (2016), Villettaz (1993)

BeMiller (2019), Wang et al. (2013)

Kashyap et al. (2001)

(continued)

Hoondal et al. (2002), Murthy and Naidu (2011) Tea production Pretreatment of tea leaves, in extraction, and in treat- Uzuner (2019) ment of extract: breakdown of pectin in tea leaf cell wall Reduction of formation of foam on instant tea Hoondal et al. (2002) Paper and pulp Reduction of the amount of energy required to remove Rättö and Viikari (1996) production the bark from the wood Juice extraction and Extraction and clarification of fruit and vegetable Danalache et al. (2018) clarification juices, to produce nectar and purees Wine production Used at grape crushing stage Kashyap et al. (2001) Improvement of wine clarity after fermentation Villettaz (1993) Beer production Reduction of wort viscosity Ben Hmad and Gargouri (2017) Hydrolysis of glucan, improving filterability Ben Hmad and Gargouri (2017) Coffee production Removal of mucilaginous coat from coffee beans Hoondal et al. (2002) Tea production Pretreatment of tea leaves, extraction, and treatment Uzuner (2019) of extract Enhancement of green tea aroma, taste, flavor, and Uzuner (2019) cold-water solubility Biofuel and bioComplete hydrolysis of the lignocellulosic material Dilokpimol et al. (2016), Prasanna chemical production for bioethanol production et al. (2016), Araujo Silva et al. (2018)

Reduction of electrostatic repulsion that causes proteinaceous flocculants to aggregate, making them easier to remove Jams, preserves, and Production of commercial pectin as a by-product of jellies juice extraction and clarification, after treatment of fruit with pectinases Wine production Improvement of clarity as well as flavor and color intensity of wine post fermentation, via extraction of anthocyanins, tannins, and phenolic compounds Coffee production Removal of mucilaginous coat from coffee beans

Juice clarification

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Function Breakdown of glucose oligosaccharides into simple sugars

β-Glucan degradation

Cross-link of monomers, degradation of polymers, and ring cleavage of aromatic compounds

Degradation of phytic acid

Enzyme β-Glucosidase

β-Glucanase

Laccases

Phytases

Table 4.1 (continued)

Animal feed

Other industrial applications Leather processing

Juice clarification, wine production, baking Paper and pulp production Textile industry

Beer production

Bioethanol production Animal feed

Industry Wine production

Campos et al. (2001) Pazarlıoǧlu et al. (2005) Mäkelä et al. (2017)

Singh et al. (2016a)

Osma et al. (2010) Osma et al. (2010) Osma et al. (2010)

Singhania et al. (2013), Benoit et al. (2015) Ojha et al. (2019)

Reference Francis et al. (1998), Baffi et al. (2013)

Thanikaivelan et al. (2004), Singh et al. (2016b) Ojha et al. (2019), Singh and Satyanatayana (2014) Reduction in soil and water contamination by surplus Ojha et al. (2019); Singh and of phosphorus excretion by monogastric animals Satyanatayana (2014)

Pulp delignification process and elimination of lipophilic extractives known as pitch depositions Decolorization of the textile dye indigo Generation of the stoned-washed look of denim Production of chromatographic resins, coatings, and adhesives Use in mixtures during leather processing, along with proteases, amylases, and lipases Improvement in absorption/utilization of phosphorus

Applications Release of volatile compounds (like terpenes) bound to glycoside fractions in grape skin and juice, resulting in improvement of wine aroma Involvement in the final step of cellulose saccharification Breakdown of β-glucans found in barley and oats to improve feed digestibility Improvement of beer stability and shelf life Removal of chill haze by elimination of polyphenols Removal of undesirable phenolic compounds

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Feruloyl esterases

Removal of ferulic acid residues and cross-links from polysaccharides

α-Galactosidase Hydrolysis of terminal α-galactosyl moieties from glycolipids and glycoproteins β-Mannanases Degradation of mannan

(continued)

Gallage and Moller (2015), Singh et al. (2016b)

Singh et al. (2016b)

Dilokpimol et al. (2016)

Dilokpimol et al. (2016)

Dilokpimol et al. (2016)

Mikkelson et al. (2013)

Sachslehner et al. (2000), Chauhan et al. (2012)

Kim et al. (2006b), Wu et al. (2005)

Kim et al. (2006b), Wu et al. (2005)

Removal of raffinose and stachyose via pretreatment Katrolia et al. (2014) with α-galactosidase, to improve digestibility and nutritional uptake

Improvement of feed that results in better digestion and reduction in flatulence in pigs Improvement of feed, resulting in better growth/ immunity and higher egg production in chickens Coffee production Degradation of coffee mannan, in both partially purified and immobilized forms, as well as a soluble crude preparation, resulting in easy concentration of coffee bean extracts by evaporation Prebiotics Production of glycomannan, a by-product of mannan digestion, used as prebiotics that have beneficial effects on gut microbiota Animal feed Improvement of access of main chain degrading enzymes, resulting in better digestion and uptake of nutrients Biofuel and bioComplete hydrolysis of the lignocellulosic material chemical production for bioethanol production Pharmaceutical and Production of hydroxycinnamic acids during lignin industrial chemical depolymerization that are important in food and cosproduction metic industries due to their antioxidant and photoprotective properties Production of ferulic acid (antidiabetic, antiaging, and anticancer properties), which is important for pharmaceutical companies Production of ferulic acid as a precursor of vanillin

Animal feed

Animal feed

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Inulinase

Enzyme Tannase

Applications Decrease in binding of tea catechins to proteins in tea, reducing aggregation and precipitation during tea storage Reduction of bitterness, resulting in improved quality and taste and mouth feel of green teas Decrease of insoluble forms of tea cream that result from polyphenols, reducing tea turbidity Hydrolysis of the glyco- Production of useful Production of inulin-derived fructooligosaccharides sidic linkages in inulin to by-products for (FOS) and inulooligosaccharides (IOS), which are produce fructose, glucose, pharmaceutical also of great pharmacological value due to their and inulooligosaccharides industry health-promoting properties

Function Industry Catalysis of ester and Tea production disulfide bonds in hydrolyzable tannins or gallic acid esters, to release glucose or gallic acid

Table 4.1 (continued)

Chi et al. (2009)

Hoondal et al. (2002)

Chavez-Gonzalez et al. (2012)

Reference Ni et al. (2015)

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Fig. 4.2 Schematic presentation of the use of fungal enzymes for the conversion of plant biomass in various industrial areas

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Table 4.2 Overview of the industrial applications of plant biomass degrading enzymes from ascomycete fungi Enzyme type Cellulases

Enzymes Glucanases

β-Glucosidase

Hemicellulases

Cellobiohydrolases Xylanases

Mannanases

Fungal origin T. reesei T. harzianum A. oryzae A. niger Aspergillus sp. P. roqueforti Penicillium sp. Thermoascus aurantiacus N. crassa A. oryzae A. niger F. oxysporum P. roqueforti Penicillium sp. T. reesei T. harzianum N. crassa T. reesei A. niger Aspergillus sp. Penicillium sp. T. reesei T. harzianum Bispora sp. P. roqueforti N. crassa Talaromyces sp. T. reesei

Industrial applications Food industry Animal feed Textile industry Biofuel production Detergents

Biofuel production Pulp and paper industry Food industry Animal feed Textile industry

References Paramjeet et al. (2018) Kaur and Gupta Phutela (2017) Dagaris et al. (2009) Belghith et al. (2001) Singh et al. (2016a, b) Danalache et al. (2018)

Polizeli et al. (2005) Kim et al. (2006a, b) Rasmussen et al. (2006) Luo et al. (2009) Maitan-Alfenas et al. (2016) Paramjeet et al. (2018) Kaur and Gupta Phutela (2017) Dagaris et al. (2009) Araujo Silva et al. (2018) de Queiroz Brito Cunha et al. (2018) (continued)

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Table 4.2 (continued) Enzyme type Oxidoreductases

Enzymes Laccases

Glucose oxidases

Esterases

Feruloyl esterases (FAEs) Pectinases

Hydrolases

Lipases

Cutinases

Inulinases Amylases

Tannases Proteases

Fungal origin T. harzianum A. niger Aspergillus sp. Aspergillus sp. Penicillium sp.

Aspergillus sp. F. oxysporum P. chrysogenum A. niger Aspergillus sp. Penicillium sp. Bispora sp. T. harzianum Fusarium sp. Geotricum sp. Aspergillus sp. Candida sp. Penicillium sp. Fusarium sp. B. cinerea A. niger A. nidulans A. oryzae Penicillium sp. Aspergillus sp. Penicillium sp. Aspergillus niger Aspergillus sp. Penicillium sp. Aspergillus sp. Aspergillus sp. Penicillium sp. Fusarium sp.

Industrial applications Bioremediation Adhesives and coatings Pulp and paper industry Food industry Biofuel production Pharmaceutical applications Textile industry Leather industry Biofuel production Pulp and paper industry Pharmaceutical applications Food industry Animal feed Cosmetics Detergents Detergents Biofuel production Pharmaceutical applications Food industry Animal feed Pulp and paper industry Bioremediation Leather industry Textile industry

References Savitha et al. (2009) Campos et al. (2001) Osma et al. (2010) Pazarlıoǧlu et al. (2005) Singh et al. (2016b) Rodriguez Couto and Toca Herrera (2006) Dilokpimol et al. (2016) Singh et al. (2016b) Rättö and Viikari (1996) Polizeli et al. (2005) Sandri and da Silveira (2018) Chi et al. (2009) Singh et al. (2016b) Yang et al. (2017) Vandam and Hilde (1992) Ni et al. (2015) Adrio and Demain (2014) Chen et al. (2013)

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The presence of xylans and arabinoxylans has been shown to affect dough properties and bread quality (Delcour et al. 1991; Michniewicz et al. 1991). The correct balance of water extractable pentosans and water unextractable solids is critical in bread making to achieve the desired properties of the resulting loaf. Xylanases are enzymes that catalyze degradation of (arabino-)xylans and are commonly produced by filamentous fungi. Xylanases break down water-insoluble arabinoxylan to its soluble form by cleaving the main chain of the polysaccharide, which results in a redistribution of water to gluten. This facilitates better gluten coagulation, which improves the rheological properties of bread, such as the volume of the loaf, the uniformity of bread crumb structure, and shelf life (de Queiroz Brito Cunha et al. 2018). The stickiness of the dough is also reduced, ensuring that it does not stick to the machine parts (Camacho and Aguilar 2003; Maat et al. 1992). In bread making, xylanases act together with other enzymes (α-amylase, malting amylase, glucose oxidase, and protease) to improve the consistency, elasticity, and softness of the dough. For example, bread produced using baker’s yeast expressing recombinant lipase 2 from a Geotrichum species resulted in a higher loaf volume and a more uniform crumb structure than control (Monfort et al. 1999). Lipases act on lipids in flour, releasing fatty acids that partition to the gluten protein network, increasing its strength, which allows for better dough making. Fatty acids also stabilize the gas cell structure while baking, resulting in better bread load volume and crumb structure (Gerits et al. 2014). Addition of α-amylase affects the properties of dough, such as structure, as well as dough viscosity, ensuring ease of handling (Vandam and Hilde 1992). α-Amylases degrade starch by hydrolyzing α-1,4 glycosidic bonds, resulting in short-chain dextrins, which in bread baking enhances taste, crust color, and toasting qualities of the resulting bread (van der Maarel et al. 2002). α-Amylases (e.g., from Aspergillus oryzae) act on damaged starch, reducing its ability to immobilize water, thus increasing dough mobility, resulting in better dough handling. In addition, the enhanced production of fermentable sugars via amylase activity increases yeast growth and thus its ability to produce carbon dioxide, leading to larger loaves (Martinez Anaya and Jimenez 1997). The appropriate concentration of this enzyme ensures the proper rising of dough; e.g., the combined expression of Aspergillus nidulans endoxylanase and A. oryzae α-amylase showed a significant improvement in loaf volume and density, possibly due to a synergistic interaction (Monfort et al. 1996). Proteases from fungi (e.g., Aspergillus usamii) have been shown to improve the viscoelastic properties of dough, resulting in decreased mixing times, easier use with machinery, and better bread crumb quality. They do this by weakening the gluten structure, and hence softening it, via breaking down its peptide bonds, and they are used primarily to regulate gluten structure in dough (Deng et al. 2016). Biscuits are also subject to various treatments with fungal enzymes. Xylanase, for example, is used in biscuit dough and results in lighter cream crackers, with an improved texture, palatability, and uniformity (Polizeli et al. 2005). Similarly, the addition of proteolytic enzymes as well as other hemicellulases also improves these qualities of biscuits, crackers, and waffles.

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Juice Clarification

Natural juice produced from the edible portion of fruits, generally extracted by pressure or other mechanical means, is turbid, bitter, and viscous and has a tendency to develop haze due to a high concentration of pectin. Pectic substances make up about 0.5–4% of ripe fruit, and these are released in the liquid phase as cloudy particles when fruit tissue is ground, making the resulting juice viscous and pulpy (Kashyap et al. 2001). Other contributors to juice cloudiness are cellulose, starch, proteins, tannins, and lignins. Commercial production of fruit and vegetable juices requires extraction, clearing, and stabilization to ensure a consistent and palatable product. To this end, macerating enzymes are used to process fruit juice for less pulp, haze, and consistency, although standardization of this process can be difficult due to the broad variability in the specific quantities of pectin, hemicellulose, and cellulose each fruit has (Grassin and Fauquembergue 1996). Therefore, it is important to choose the right enzyme combination depending on the fruit composition and the desired final product. Juice clarification and extraction are carried out using a combination of pectinases, xylanases, mannanases, cellulases, and amylases, resulting in improved yield, stabilization of fruit pulp, reduction of viscosity, and clearing of juice (Polizeli et al. 2005). Pectins are polymers of D-galacturonic acid, which are commonly found in cell walls of plants. To process pectin in juices, pectinases from A. niger are commonly used, which have been produced either in submerged or solid-state cultivation (Sandri and da Silveira 2018; Sandri et al. 2011). Pectinases are a complex family of enzymes that hydrolyze glycosidic bonds in pectic polymers and can be broadly divided into esterases, which catalyze the de-esterification of pectin by the removal of methoxyl and acetyl esters, and depolymerases, which consists of different groups depending if they act on pectin, polygalacturonic acid, or rhamnogalacturonan. Depolymerases can be either hydrolases or lyases, breaking the glycosidic bond by two different mechanisms, introducing water across the oxygen bridge or lysis by trans-elimination, respectively. In the fruit juice industry, acidic pectinases are used in extraction and clarification of sparkling clear juices from apples, pears, and grapes, as well as the cloudy juices, sourced from prunes, citrus fruits, and tomatoes (Sharma et al. 2017). Pectinases can be used in both extraction and clarification of juice, resulting in enhanced coloring and concentrated juice flavor. During mechanical extraction, fruits with a high level of pectin are treated with pectinases, degrading the pectin that cross-links cellulose and hemicellulose, allowing (hemi-)cellulases to act on these substrates. This improves pressing of pulp and results in an increase in the juice yield, improved filterability, and reduction in viscosity, as has been shown in grape, peach, pear, plum, and apricot (Ramadan 2019). Pectinases also aid with juice clarification by degrading the negative charge carrying pectin, that forms a coat around the positively charged proteins, causing them to repel each other. The degradation of pectin reduces the electrostatic repulsion, causing the flocculants to aggregate, making them easier to remove (Kashyap et al. 2001).

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Cellulases are enzymes that degrade cellulose into glucose, through the combined action of exoglucanases, endoglucanases, and β-glucosidases. These enzymes are used to extract and clarify fruit and vegetable juices, to produce nectar and purees (Danalache et al. 2018). Xylanases and other hemicellulases, e.g., from Aspergillus sp. and Trichoderma sp., are also used in combination with cellulases for clarifying juices. Other potential contributors to turbidity in juices are starch and arabinans (side chains from pectin). These make juices difficult to filter, foul membranes, promote gelling after juice concentration, and contribute to post-concentration haze (Dey and Banerjee 2014). Starch can be removed from juice at the same time as pectin, using amylases, which prevent starch molecules from accumulating with pectins and proteins, thus eliminating haze.

2.1.3

Modification of Pectin in Jams

Pectin is used to generate water-soluble galacturonoglycan preparations of varying methyl ester contents and degrees of neutralization that are capable of forming gels (BeMiller 2019). It is this property of pectins that is harnessed to form spreadable gels, like jams, jellies, marmalade, and preserves. High methoxyl pectins are used to make jams, either by rapid gelation (to prevent settling of fruit pieces) or slower gelation (in preparation of jellies that allow for the escape of air bubbles) (BeMiller 2019). Commercial pectin is obtained as a by-product of juice extraction and clarification, after the treatment of fruit with pectinases. Pectin acetylesterase is an enzyme that catalyzes the transacylation reaction between pectin molecules, which was shown to be feasible to make fruit jam with a reduced sugar content (Wang et al. 2013).

2.1.4

Production of Wine/Beer/Beverages

The production of wine is similar to fruit juice, in that grapes have a high pectin content that makes them difficult to press. Enzymes are used for wine clarification, color extraction, and protein stabilization (Uzuner 2019). Enzymatic treatment also reduces gelling and hazing of grape juice as wine is being prepared. Enzymes from fungi like A. niger, Penicillium notatum, or Botrytis cinerea, including pectinases, cellulases and hemicellulases, are used during all three stages of wine production: the crushing stage, the free run juice stage prior to fermentation, and after fermentation is complete (Kashyap et al. 2001). At the crushing stage, commercial enzyme preparations are used to macerate the berries and increase the juice yield, reducing also the pressing time (Villettaz 1993). Because the maceration step releases colloidal substances into the juice, the second stage is a clarification step, where suspended particles settle along with undesirable microorganisms. Addition of pectinases after wine fermentation increases clarity (Kashyap et al. 2001).

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Some enzyme preparations have been reported to increase the color intensity/ stability and flavor of wines. For example, treatment with pectinases post fermentation increases the extraction of anthocyanins and tannins, phenolic compounds that increase the flavor and color intensity of wine (Garg et al. 2016). A xylanase (XlnA) from A. nidulans was used to make wine with a more pronounced aroma (Ganga et al. 1999). β-Glucosidase is an enzyme that breaks down glucose oligosaccharides into D-glucose. In wine making, volatile compounds like terpenes are bound to glycoside fractions derived from grape skin and juice. These are released when these fractions are enzymatically treated (Francis et al. 1998). For example, β-glucosidase from Aureobasidium pullulans has been shown to improve wine aroma (Baffi et al. 2013). An important aspect of red wine making is color extraction. Addition of enzyme cocktails with a broad range of activities, including hemicellulases, cellulases, and pectinases, during the extraction process, give the best results (Villettaz 1993). The most important enzymes for malting and brewing of beer are amylases, proteases, peptidases, β-glucanases, and xylanases. These enzymes have several roles in the production of beer and malted liquor, primarily in fermentation, control of viscosity, and chill proofing (protection of beer clarity/brightness at low temperatures). The first stage of fermentation requires the release of fermentable sugars from starch, the carbohydrate storage in grain, which is achieved by adding amylases, xylanases, and β-glucanases. During mashing and primary fermentation, cellulases are added to reduce the viscosity of wort and hydrolyze glucan, which improves the filterability (Ben Hmad and Gargouri 2017). In addition, proteins in grains are essential for nutrition of the yeast used in brewing, as well as for flavor, mouth feel, foam, and color, and are therefore modified using proteases and peptidases. Xylanases are also used to hydrolyze arabinoxylans that are released when the cell wall of barley is hydrolyzed, which results in decreased beer viscosity and muddiness (Polizeli et al. 2005). Laccases are enzymes found in fungi, plants, insects, bacteria, and archaea that belong to the blue multicopper oxidases. Laccases cross-link monomers, degrade polymers, and carry out ring cleavage of aromatic compounds (Shraddha et al. 2011). In food industry, laccases are used to eliminate undesirable phenolic compounds, including for baking, juice processing, and wine stabilization. In beer making in particular, laccases not only improve beer stability but also increase its shelf life (Osma et al. 2010). Chill haze occurs when proteins are precipitated at low temperatures, stimulated by small quantities of naturally occurring proanthocyanidins polyphenols. Addition of laccase at the end of the brewing process removes this haze effect, by removing the polyphenols.

2.1.5

Animal Feed

Feed ingredients for pigs and poultry can contain indigestible components, because the animal does not produce the proper endogenous enzymes to digest them. To ensure maximum uptake and efficiency, immunological health of the animals, and a

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good feed conversion ratio (the ratio of the weight of feed consumed to the weight gained by the animal), enzymes are used to increase the availability of essential nutrients. Treatment with enzymes either produces nutrients by hydrolysis or liberates nutrients blocked by these fibers, increasing their absorption (Hoondal et al. 2002). In addition, these enzyme cocktails can reduce unwanted residues in excreta and thus protect the environment (Polizeli et al. 2005). Usually these enzymes complement what is already present in the gut of the animals. There are four main categories of enzymes that are used in the feed sector: enzymes targeting phytates, enzymes targeting viscous cereals like triticum, enzymes targeting nonviscous cereals like maize and jowar, and enzymes targeting noncereals/legumes (Ojha et al. 2019). Combinations of xylanases with other enzymes like glucanases, pectinases, cellulases, proteases, amylases, phytase, galactosidases, and lipases help with processing grain to highly digestible feed for animals. Fiber is a major component of plant-based material. Xylanases are used in the treatment of fiber in animal feed, acting on arabinoxylan and its by-products, found in grains (Polizeli et al. 2005). In its soluble form, arabinoxylan can increase the viscosity of ingested feed, leading to mobility issues and less efficient uptake of nutrients. The addition of xylanase can help to produce a high energy, better digestible food mixture, by releasing nutrients trapped inside macromolecules and improving the access of digestive enzymes to their substrate. A commercial preparation, Avizyme, consisting of xylanase, protease, and amylase, was added to poultry feed, and it was shown that birds raised on this feed obtained higher net energy (Cafe et al. 2002). Along with xylanase, β-glucanase is the other major fiber-digesting enzyme used for feed production, which acts on β-glucans found in barley and oats. Taken together, these enzymes act on the cell walls and release nutrients from grain endosperm and the aleurone layer of cells. Starch in grain and grain by-products is degraded by α-amylases, increasing the energy derivation from feed for chickens and pigs. This results in an improved pork/chicken and egg production (Ojha et al. 2019). For example, an α-amylase-treated corn/soybean diet improved the nutritional digestibility of broilers, as well as their performance (Gracia et al. 2003). Phytic acid is a form of organic phosphorus found in plant-derived food like cereals, legumes, soybean, and others. It is not available to nonruminants such as pigs, poultry, and humans, because their gastrointestinal tracts lack phytase. Phytic acid chelates important metal ions that are required for digestive enzymes to work, thus inhibiting their activity. Phytases added to feed prior to animal consumption improve the absorption/utilization of phosphorus and also decrease the contamination of soil and water by the surplus of phosphorus excretion by monogastric animals (Ojha et al. 2019; Singh and Satyanatayana 2014). Nonruminant animals lack α-galactosidase, which hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins (Ademark et al. 2001). In order to process the high amount of soybean meal that is present in feed, pretreatment with α-galactosidase to remove raffinose and stachyose can improve digestibility and nutritional uptake (Katrolia et al. 2014). Meal supplements high in mannan can result in indigestion and sticky droppings in chickens and flatulence in pigs, because pigs cannot digest mannans or manno-

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oligosaccharides. When the high mannan containing feed is treated with β-mannanases, broiler chickens showed improved body weight, and pigs showed reduced flatulence (Kim et al. 2006b; Wu et al. 2005). The use of mannanases in feed has shown improved utilization of nutrients, higher egg production, better growth and immunity, as well as improved digestibility and reduced uric acid in poultry. Similar effects were also seen in fish and goats (Wu et al. 2005). Feruloyl esterases are responsible for removing ferulic acid residues and crosslinks from polysaccharides. Feruloylation in foraging feed can inhibit the ruminant digestive system. Addition of feruloyl esterases (in an enzyme cocktail) improves the access of main chain degrading enzymes, resulting in better digestion and uptake of nutrients (Dilokpimol et al. 2016).

2.1.6

Tea and Coffee

The polysaccharide fraction of green coffee beans consists of arabinogalactan, mannan, and cellulose, the presence of which complicates processing the beans. These compounds, in particular mannans, increase the viscosity of coffee, impede its industrial processing, and therefore need to be processed using enzymes, many of which are derived from fungi. For example, mannanases extracted from Sclerotium rolfsii were shown to effectively degrade coffee mannan, in both partially purified and immobilized form, as well as a soluble crude preparation (Sachslehner et al. 2000). As a result of this treatment, coffee bean extracts can be easily concentrated using evaporation (Chauhan et al. 2012). The fermentation of coffee is accelerated using pectinase treatment, which removes the mucilaginous coat from the coffee bean, which could be helped along by cellulases and hemicellulases (Hoondal et al. 2002). For example, Robusta coffee fermentation was shown to be improved using pectinases derived from A. niger (Murthy and Naidu 2011). The degradation of mucilage improves the quality of the coffee bean. The processing of green tea requires the usage of cellulases, hemicellulases, as well as pectinases, during pretreatment of tea leaves, extraction, and treatment of extract (Uzuner 2019). Enzymatic treatment of tea enhances the aroma, taste, flavor, and cold-water solubility of green tea. The formation of tea haze as well as cream formation are the biggest issues encountered during tea processing, causing discoloration and precipitation of substances. Tannase, derived from Aspergillus sp., is the most common enzyme used in tea processing. Tannase is a key enzyme that catalyzes ester and disulfide bonds in hydrolyzable tannins or gallic acid esters, to release glucose or gallic acid (Ni et al. 2015). The hydrolytic action of tannase decreases tea catechins binding to proteins in tea, reducing aggregation and precipitation during tea storage. Tannase has also been shown to reduce bitterness and result in improved quality and taste and mouth feel of green teas (Chavez-Gonzalez et al. 2012). Insoluble forms of tea cream that result from polyphenols can be reduced by addition of tannases, reducing tea turbidity. The formation of foam on instant tea can be reduced by the addition of alkaline pectinases, which break down pectin present in the cell wall of tea leaves (Hoondal et al. 2002). This also results in

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change in color of tea during processing, resulting in the characteristic tea aroma. In particular cellulase together with laccase has been shown to increase the quality of black tea (Murugesan et al. 2002). Interestingly, crude enzyme from Aspergillus sp. was more effective in improving tea quality than purified pectinase enzyme, and this was because the crude extract contained a combination of, e.g., cellulases, hemicellulases, pectinases, and proteinases (Garg et al. 2016).

2.1.7

Prebiotics

Some by-products of fungal enzymes are beneficial as prebiotics in functional foods for both human and animal consumption. Xylo-oligosaccharides are by-products of xylan metabolism, released during xylanase treatment, and possess prebiotic effects. Xylo-oligosaccharides encourage the growth of beneficial bacteria in the human gut, such as bifidobacteria, while restricting harmful bacteria (Aachary and Prapulla 2011). They are added to foods (e.g., desserts, confectionary products, breakfast cereals, chocolate), as well as beverages (e.g., soymilk, coffee, tea, alcoholic beverages) and milk products (e.g., powdered milk, instant milk, and ice cream) (Kumar et al. 2018). Another example of a prebiotic compound is a by-product of wheat bran processing, arabinoxylo-oligosaccharides, which have been shown to stimulate bifidobacterial growth in human fecal samples (Gullon et al. 2014). The same effect has been shown in chickens that are on feed that is supplemented with these oligosaccharides (Courtin et al. 2008). Dietary fiber comprises of soluble oligosaccharides, as well as polysaccharides that cannot be digested in the mammalian small intestine. Glucomannans are structural polymers in plant cell walls and carbohydrate storage compounds in seeds. Linear glucomannans are used as thickening agents in food and beverages. These can also be used as prebiotics that have beneficial effects on gut microbiota and human health. Konjac flour glucomannan in particular has been shown to produce manno-oligosaccharides at the industrial level (van Zyl et al. 2010). Traditionally, the breakdown of mannan to manno-oligosaccharides is done by a bacterial endo-β-mannanase but can also be efficiently done by mannanases from T. reesei (Mikkelson et al. 2013).

2.2

Applications in Paper and Pulp Production

The pulp and paper industry is one of the largest industries in the world. Nowadays, bleached Kraft pulp is the most important pulp obtained for paper production. However, this industry uses a wide variety of chemicals that pose a risk to the environment due to the possible formation of dioxins and other toxic compounds (Polizeli et al. 2005). The first stage of the pulp production process involves chemical wood pretreatment and the subsequent degradation of the recalcitrant lignin. After this, the colored pulp obtained must be bleached in order to be suitable

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for paper production (Mette Nissen et al. 1992). The bleaching process traditionally involves the utilization of many different chemicals, such as chlorine dioxide, sodium hydroxide, hydrogen peroxide, and chlorine peroxide, with the disadvantages of high gas emission rates and the high economic costs associated with the process (Polizeli et al. 2005). In this context, plant biomass degrading enzymes from fungi have been implemented as environmentally friendly alternatives in the pulp pretreatment and bleaching process (Pérez et al. 2002). The use of lignocellulolytic enzymes, particularly xylanases, decreases the amount of chemicals needed to obtain good quality pulp for paper production (Pérez et al. 2002). Cellulose degradation is of great concern during the pulping process, as it affects the final quality of the paper (Walia et al. 2014). Cellulose-free xylanases from filamentous ascomycetes, mainly produced by the genus Aspergillus (de Vries 2003; Maitan-Alfenas et al. 2016), are applied for the removal of lignin linked to xylan with little or no detrimental effect to the pulp, since xylanases have no cellulolytic activity (Ferreira et al. 2016; Walia et al. 2014). Pretreatments with fungal xylanases improve chemical extraction of lignin from pulp, minimizing the amount of chemicals needed and reducing economic and environmental costs (Walia et al. 2017; de Vries and Visser 2001). In addition, xylanases have been reported to help increase pulp brightness and thickness (Savitha et al. 2009). Ligninolytic enzymes, e.g., laccases, play a role in the pulp delignification process and the elimination of lipophilic extractives known as pitch depositions, although to a lesser degree than lipases (Singh et al. 2016a). Lipases are essential in the paper and pulp industry for the control of pitch (Singh and Mukhopadhyay 2012), which is normally generated from lipophilic extractives containing alkanes, fatty acids, resin acids, sterols, triglycerides, and waxes, and represent a serious problem in the paper industry because pitch deposits are usually associated with lower production yields and quality of the final product (Gutiérrez et al. 2009). Fungal lipases are of great interest due to their stability, selectivity, and broad specificity and are widely applied in the paper industry (Singh and Mukhopadhyay 2012). The main lipase-producing ascomycete fungi are from the genera Geotrichum, Aspergillus, Candida, and Penicillium. On the other hand, another alternative for the (bio)control of pitch is the application of some pitch-metabolizing fungi, such as the ascomycete Ophiostoma piliferum, directly to the pitch deposits as a pretreatment to improve pulping efficiency (Farrell et al. 1997). Fungal pectinases, on the other hand, are more often applied in the food industry (see Sect. 2.1.2), but they also play a role in pulp and paper industry, since they can be added during the debarking process, reducing the amount of energy required to remove the bark from the wood (Rättö and Viikari 1996). α-Amylases, predominantly from Aspergillus (A. niger) and Penicillium, are also applied in the paper industry to produce starch with improved properties (e.g., lower viscosity, higher molecular weight) for paper coating and to improve the writing quality of the paper (de Souza and de Oliveira Magalhães 2010).

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Applications in Production of Biofuels and Biochemicals

Fossil fuels are the most widely used energy sources worldwide. However, these have many drawbacks, since they are nonrenewable and are associated with environmental and health risks (Voloshin et al. 2016). Biofuels are sustainable and safer energy resources that have become an alternative to replace the rapid depletion of fossil fuels. Biofuels, such as bioethanol or biomethanol, can be produced from lignocellulosic biomass obtained principally from forest and agricultural crop residues, including forest woody feedstocks, corn stovers, rapeseed residues, sugarcane bagasse, and fruit peels. However, also several waste streams can be used for bioethanol production, such as paper waste, household food and kitchen waste, or wine and coffee residues (Jeihanipour and Bashiri 2015). Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin. Its complex and compact structure makes lignocellulose extremely resistant to fragmentation. Thus, the conversion of lignocellulosic biomass into biofuel is an economically challenging task, since many pretreatments are required in order to transform lignocellulosic material into fermentable mono- and oligosaccharides, increasing the costs associated with biofuel generation (Araújo et al. 2017). Lignocellulose-degrading fungi have received much attention due to the fact that fungal pretreatments during industrial biofuel production imply low energy consumption and little or no impact to the environment (Paramjeet et al. 2018). Many filamentous ascomycetes, specially from the genera Aspergillus, Penicillium, and Trichoderma, but also Fusarium and Neurospora (Ferreira et al. 2016) naturally produce extracellular enzymes that are able to degrade lignocellulose, which include cellulases (endoglucanases, cellobiohydrolases, and β-glucosidases), hemicellulases (e.g., endoxylanases, β-xylosidases, α-L-arabinofuranosidases) (Araujo Silva et al. 2018), oxidoreductases (e.g., laccases, glucose and/or galactose oxidases, lytic polysaccharide monooxygenases) (Mäkelä et al. 2017; Johansen 2016), and the so-called accessory enzymes feruloyl esterases (FAEs), which are necessary for complete hydrolysis of the lignocellulosic material for bioethanol production (Dilokpimol et al. 2016). FAEs are widely produced by ascomycete fungi (Dilokpimol et al. 2016). A. niger, A. oryzae, Penicillium roqueforti, T. reesei, and Trichoderma harzianum have been reported to secrete high amounts of different hemicellulases and cellulases, including β-glucosidases (Paramjeet et al. 2018). These last enzymes, however, represent a bottleneck in bioethanol production since they are involved in the final step of cellulose saccharification and get inhibited by glucose, their final product (Singhania et al. 2013). For this reason, many efforts are currently being made in order to optimize the production yield of cellulases in filamentous ascomycetes (Prasanna et al. 2016; Araujo Silva et al. 2018). Approaches that are used for this are the use of genetically modified fungi to produce higher amounts of more effective cellulases (Murray et al. 2004; Kovács et al. 2008) and the generation of mixed fungal populations to improve saccharification efficiency (Benoit et al. 2015). In addition, plant pathogenic ascomycetes, such as Botrytis cinerea or Fusarium sp., have been

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recognized as very efficient lignocellulolytic fungi, becoming excellent sources for plant-degrading enzymes for biofuel production (Mäkelä et al. 2014a). Biodiesel also represents an ecological alternative to fossil fuels. Biodiesel can be chemically or enzymatically synthesized from plant and algal oils by transesterification reactions. However, enzymatic synthesis is less energy-consuming and generates fewer undesired by-products, being a promising alternative to chemical synthesis (Szczęsna Antczak et al. 2009). In this context, fungal lipases have attracted the attention in the last decades for biodiesel production, since their application results in high production yields and easier downstream purification steps (Gog et al. 2012). The main problems regarding the application of lipases for biodiesel generation are their sensitivity to glycerol, which is the main side product of the transesterification reaction, and their production costs (Gog et al. 2012). For this reason, different approaches are used to optimize the enzymatic activity, stability and shelf life of lipases, such as immobilization (Mohamed and Bornscheuer 2003). Currently, the major lipase-producer ascomycete is Candida antarctica, but other species (e.g., A. oryzae) are being genetically modified to produce high amounts of different cocktails of lipases for biodiesel production (Adachi et al. 2011). Some commercial lipases used for biodiesel production have been reviewed previously (Gog et al. 2012). Apart from biofuel generation, plant cell wall degrading enzymes from ascomycetes can also be used for the production of pharmaceutically and industrially relevant chemicals with different applications. For instance, FAEs release considerable amounts of hydroxycinnamic acids, including ferulic acid, during lignin depolymerization (Dilokpimol et al. 2016). These compounds are very interesting in food and cosmetic industries due to their antioxidant and photoprotective properties. In addition, antidiabetic, antiaging, and anticancer properties have been associated with ferulic acid, which makes this compound very interesting for pharmaceutical companies (Singh et al. 2016b). Moreover, ferulic acid acts as a precursor for the synthesis of vanillin, one of the principal and most demanded flavor and aroma compounds in the market (Gallage and Moller 2015; Singh et al. 2016b). Storage polysaccharides from plant biomass can also be degraded by fungal enzymes for multiple applications. Inulin, for example, can be hydrolyzed by fungal inulinases to produce glucose and fructose, which are mainly used in food industry, but can also be used for bioethanol production. Inulin-derived fructooligosaccharides (FOS) and inulooligosaccharides (IOS) are also of great pharmacological value due to their health-promoting properties (Chi et al. 2009). Another example is lactic acid. Soluble sugars obtained from lignocellulosic material by fungal enzymes can also be used for lactic acid production through direct yeast, bacterial, or fungal fermentation (Castillo Martinez et al. 2013). Lactic acid has been traditionally used in food industry, but it is also used as a precursor of many industrially and economically relevant compounds, such as propylene glycol (Castillo Martinez et al. 2013) for plastic production, among other applications (Datta and Henry 2006).

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Other Applications

Fungal plant biomass degrading enzymes from ascomycetes can be additionally applied in many different fields, including bioremediation, textile, and pharmaceutical industry, and some examples of this are presented here. Fungal hydrolases, laccases, lyases, and peroxidases have been proposed for their application in bioremediation due to their wide capacity to degrade a wide range of organic contaminants and xenobiotics (Singh et al. 2016b; Yang et al. 2017). Oxidoreductases such as polyphenol oxidases and laccases are also used to synthesize 3,4-dihydroxylphenyl alanine and actinocin, respectively, both with important pharmaceutical applications (Singh et al. 2016b; Mäkelä et al. 2017). In the textile industry, laccases can be used for the decolorization of the textile dye indigo (Campos et al. 2001), as well as for the generation of the stoned-washed look of denim (Pazarlıoǧlu et al. 2005). The latter application also uses fungal cellulases to generate this stoned-washed look in an environmentally friendly manner (Belghith et al. 2001). Laccases can also be applied for the production of chromatographic resins, coatings, and adhesives, among other industrial applications (Mäkelä et al. 2017). In leather industry, laccases, together with fungal proteases, amylases, and lipases, are applied alone or in mixtures during leather processing (Thanikaivelan et al. 2004; Singh et al. 2016b). Additionally, in detergent industries, fungal amylases, lipases, proteases, cellulases, and cutinases are used as additives to increase the efficiency of the detergents (Belhaj et al. 2010; Kirk et al. 2002; Singh et al. 2016b).

3 Applications of Fungal Algal Biomass Degrading and Modifying Enzymes Seaweeds include red (Rhodophyceae), green (Chlorophyceae), and brown (Phaeophyceae) algae. They have a unique chemical composition and represent a source of highly valuable components, such as proteins, polysaccharides, carotenoids, vitamins, minerals, and sterols (Fernand et al. 2017). This composition of seaweeds makes their utilization as food, feed, and medicinal additives attractive. Recent developments in seaweed biorefineries have focused on seaweed polysaccharides as a potential source for biofuel production (Masarin et al. 2016; Bruhn et al. 2011; Adams et al. 2011). In addition, seaweeds are used to control eutrophication in coastal waters, for example, when they are incorporated into mariculture systems (Wu et al. 2015). The use of seaweeds as raw materials in various industrial applications or as a remediation agent for eutrophication leads to an increased amount of seaweed waste. Therefore, it is critical to recycle seaweed waste to improve conditions of marine environments and preserve them. Seaweed biomass degrading enzymes play a crucial role in extracting high-value components from seaweeds as well as in processing seaweed waste. Microorganisms, particularly derived from marine environments, are capable of producing seaweed-specific enzymes that have

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wide applications in food, pharmaceutical, and cosmetic industries (Balabanova et al. 2018). Agarases, alginate lyases, and carrageenases are among the most promising industrial enzymes currently. The majority of seaweed-specific enzymes have been reported from bacteria such as Pseudomonas sp., Bacillus sp., Vibrio sp., Agarivorans sp., Thalassomonas sp., and Alteromonas sp., while studies on fungal algal biomass degrading and modifying enzymes are limited (Li et al. 2011; Harshvardhan et al. 2013; Zhu et al. 2018b; Yoon et al. 2017; Zhang et al. 2018; Jouanneau et al. 2010) (Table 4.3). Fungal species that have abilities to produce algae-specific enzymes with interest in industry have been mainly identified from marine habitats (Furbino et al. 2018; Solis et al. 2010; Schaumann and Weide 1990; Hifney et al. 2018; Rodriguez-Jasso et al. 2010). Certain polysaccharides commonly found in terrestrial plant biomass, cellulose and hemicellulose, also constitute an important part of seaweed biomass. In addition to these polysaccharides, seaweeds also contain specific polysaccharides, such as agar and carrageenan found in red seaweeds; alginate, laminarin, and fucoidan in brown seaweeds; and ulvan in green seaweeds. The enzymatic mechanisms to degrade and modify these seaweed polysaccharides are poorly understood compared to plant polysaccharides. This section collates current knowledge on the fungal enzymes needed for the conversion of seaweed-specific polysaccharides into valuable products (Fig. 4.3).

3.1

Applications of Red Seaweed Degrading and Modifying Enzymes

Agar is a well-known polymer synthesized by red seaweeds such as Gracilaria and Gelidium and is degraded by agarases classified into α-agarases (E.C. 3.2.1.158) and β-agarases (E.C. 3.2.1.81). α-Agarases belong to glycoside hydrolase (GH) family GH96 of the Carbohydrate-Active Enzyme (CAZy) database (www.cazy.org) and cleave α-1,3 linkages in agarose, while β-agarases hydrolyze β-1,4 linkages in agarose and belong to GH16, 50, 86, and 118 families (Ohta et al. 2005; Wang et al. 2006). Agarases have wide applications in food, cosmetics, and nutraceuticals industries (Chi et al. 2012; Jahromi and Barzkar 2018). They are used to produce oligosaccharides from agar that possess antioxidant activities and moisturizing and whitening effects on human skin (Kim et al. 2017). Agar-derived oligosaccharides are able to inhibit bacterial growth, slow down starch degradation, and improve food quality (Giordano et al. 2006). Moreover, agarases are also commonly used in microbiological, cytological, and physiological research. So far, only a few studies have shown agarase activities produced by fungi (Gomaa et al. 2015, 2017; Furbino et al. 2018; Fawzy et al. 2018). Agarases producing fungal strains have been isolated from seawater (Cladosporium sp. and Phoma lingam) (Solis et al. 2010) and different types of seaweeds (Cladosporium sp., Doratomyces sp., Penicillium chrysogenum, Penicillium citrinum, Penicillium sp., Aspergillus flavus, Curvularia lunata, Dendryphiella arenaria, etc.) (Furbino et al. 2018).

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Table 4.3 Overview of the algal biomass degrading enzymes from ascomycete fungi Enzyme type Lyases

Hydrolase

Enzymes Glucuronan lyase Ulvan lyase Alginate lyase

Laminarinase

Fucoidanase

Carrageenase

Agarase

Fungal origin T. reesei P. longicolla C. intermedia A. cruciatus D. salina D. arenaria A. niger E. chevalieri S. chartarum A. nidulans A. niger P. claviforme T. viride D. salina A. japonicas R. miehei P. rolfsii T. amestolkiae Mucor sp. A. niger P. purpurogenum Fusarium sp. E. chevalieri D. arenaria E. chevalieri A. nidulans C. funicola S. chartarum A. ochraceus A. terreus Phoma sp. P. chrysogenum Penicillium sp. B. bassiana Pseudogymnoascus sp. Doratomyces sp. Cladosporium sp. P. lingam Doratomyces sp. P. chrysogenum Penicillium sp. A. flavus C. lunata D. arenaria Acrophialophora sp.

Polymeric substrate Ulvan

References Delattre et al. (2006)

Ulvan Alginate

Li et al. (2017) Schaumann and Weide (1990) Wainwright (1980) Wainwright and SherbrockCox (1981)

Laminaran

Lee et al. (2014) Grant and Rhodes (1992) Kulminskaya et al. (2001) Chaari and Chaabouni (2018) Méndez-Líter et al. (2018)

Fucoidan

Rodriguez-Jasso et al. (2010) Hifney et al. (2018)

Carrageenan

Solis et al. (2010) Furbino et al. (2018)

Agar

Solis et al. (2010) Furbino et al. (2018) Gomaa et al. (2015, 2017) Fawzy et al. (2018)

(continued)

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Table 4.3 (continued) Enzyme type

Enzymes

Fungal origin

Polymeric substrate

References

Acremonium sp. A. terreus C. pruinosum C. salinae C. lunata D. arenaria L. thalassiae M. trigonosporus S. brevicaulis S. candida S. rostrata S. racemosum

Carrageenases are enzymes involved in the degradation of carrageenan obtained from different red seaweeds, for instance, Kappaphycus, Eucheuma, and Chondrus (Rhein-Knudsen et al. 2015). They are classified in three types, κ- (EC 3.2.1.83, GH16), ι- (EC 3.2.1.157, GH82), and λ- (EC 3.2.1.162) carrageenases, which hydrolyze β-1,4 glycosidic linkages of carrageenan and release oligosaccharides with various biological activities (Chauhan and Saxena 2016). It has been demonstrated that carrageenan-derived oligosaccharides have antitumor, anti-inflammation, antiviral, anticoagulation, antioxidant and immunomodulatory activities, and potential biomedical and pharmaceutical applications (Haijin et al. 2003; Talarico and Damonte 2007; Zhu et al. 2018a). Carrageenases are also used in detergent industries (Chauhan and Saxena 2016), where they are added to detergents to remove gum containing stains. The application of carrageenases in combination with other enzymes for the removal of thickeners and excess dye after textile printing reduces time and use of energy and water (Chauhan and Saxena 2016). Commercially available carrageenases are currently mainly produced by bacterial strains. Limited knowledge is available about fungal strains that have abilities to produce carrageenases. For instance, seawater-derived fungi (Aspergillus ochraceus, Aspergillus terreus, and Phoma sp.) (Solis et al. 2010) and fungal strains isolated from seaweeds (Penicillium chrysogenum, Penicillium sp., B. bassiana, Pseudogymnoascus sp., and Doratomyces sp.) were reported as a potential sources of carrageenases (Furbino et al. 2018).

3.2

Applications of Brown Seaweed Degrading and Modifying Enzymes

Brown seaweed polysaccharides including alginate, fucoidan, and laminarin have a wide variety of potential applications due to their broad range of biological properties. These beneficial activities are related to the chemical composition and structure of these

Fig. 4.3 Schematic presentation of the use of fungal enzymes for the conversion of algal biomass in various industrial areas

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polysaccharides. Alginate is present in most brown seaweed species, but the amounts of alginate vary. Commercially, alginates are mainly produced in Laminaria hyperborea, Laminaria digitata, Laminaria japonica, Ascophyllum nodosum, Macrocystis pyrifera, and Sargassum spp. (Rhein-Knudsen et al. 2015). Depolymerization of alginate is catalyzed by alginate lyases (EC 4.2.2.3), which hydrolyze alginate by the β-elimination reaction to cleave the glycosidic bond (Li et al. 2011). These enzymes belong to seven polysaccharide lyase (PL) families (PL5, 6, 7, 14, 15, 17, 18) of the CAZy database. Alginate and alginate-derived oligosaccharides produced by alginate lyases play an important role in food, feed, pharmaceutical, nutraceutical, and biofuel industries. They have gelling, emulsifying, and film-forming properties (Gomaa et al. 2018a, b). Furthermore, oligoalginates produced by alginate lyases have potential applications in protection against pathogens and as therapeutic agents such as anticoagulants and tumor inhibitors (An et al. 2009). These oligosaccharides also have applications in the field of agriculture as growth promoters for plants (Hu et al. 2004). Monomers released from alginate degradation by alginate lyases are essential for bioethanol production in biofuel applications (Takeda et al. 2011; Lee and Lee 2016). In addition, alginate lyases are important for the determination of alginate composition by using alginate lyase fingerprinting and enzymatic assay and for the preparation of protoplasts of brown algae (Inoue et al. 2011). Most of the characterized alginate lyases originate from alginate-degrading bacteria (Cantarel et al. 2009), but alginate lyases were also found in several fungi (Corollospora intermedia, Asteromyces cruciatus, Dendryphiella salina, Dendryphiella arenaria, A. niger, Eurotium chevalieri, Stachybotrys chartarum, and A. nidulans) that were isolated from seaweeds (Schaumann and Weide 1990; Wainwright 1980; Wainwright and Sherbrock-Cox 1981). However, there has been relatively little research reported on these enzymes, and the genes encoding these enzymes have not yet been sequenced or cloned. Fucoidan is another complex polysaccharide found in various species of brown seaweeds such as Undaria, Macrocystis, Laminaria, Sargassum, etc. Many studies have shown that fucoidans possess antioxidant, antiviral, anticoagulant, anticancer, and anti-inflammatory activities and can act as an immunomodulator (Chevolot et al. 1999; Zhuang et al. 1995; Zhao et al. 2018). Despite these numerous beneficial activities, fucoidan molecules have high molecular weight, are structurally different, and have a viscous nature, which inhibits their pharmaceutical and therapeutic applications. Therefore, oligosaccharides with low molecular mass derived from fucoidan have a large potential for applications (Silchenko et al. 2013; Holtkamp et al. 2009; Berteau and Mulloy 2003). Fucoidanases (EC 3.2.1._) are key enzymes to hydrolyze the complex structure of fucoidan and produce fuco-oligosaccharides (Silchenko et al. 2013; Holtkamp et al. 2009) and are also an important tool to structurally characterize fucoidan. Only few characterized fucoidanases (GH107) have been reported, and these were isolated from marine bacteria (Silchenko et al. 2013; Colin et al. 2006; Silchenko et al. 2018). Fucoidanases have been also found in marine fungi, and it has been demonstrated that Mucor sp., A. niger, Penicillium purpurogenum, Fusarium sp., E. chevalieri, D. arenaria, E. chevalieri, A. nidulans, C. funicola, and S. chartarum have the potential to produce fucoidanase (RodriguezJasso et al. 2010; Hifney et al. 2018). Furthermore, fungal treatment of the brown

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seaweed Cystoseira trinodis increased the antioxidant properties of fucoidan through the generation of fucoidan with a lower molecular weight and looser structure (Hifney et al. 2018). Besides unique polysaccharides such as alginate and fucoidan, brown seaweeds (Laminaria, Saccharina, Ascophyllum, Fucus, and Undaria species) produce the storage polysaccharide laminarin which is a β-glucan with β-1,3- and β-1,6-linkages (Garcia-Vaquero et al. 2017). Laminarinases are enzymes that catalyze the hydrolysis of laminarin and include endo-β-1,3-1,4 glucanase (EC 3.2.1.6; GH3, 16, 26), β-1,3-glucanase (EC 3.2.1.39, GH16, 17, 55, 64, 81), β-1,6-glucanase (EC 3.2.1.75, GH5, 13, 30), pullalanase (EC 3.2.41, GH13), exo-β-1,3/1,6-glucanase, and endo-β-1,4-glucanase (GH131) (Balabanova et al. 2018). β-1,3-glucanases have a key role in the efficient degradation of laminarin. Laminarin and its derivatives were reported to exhibit antioxidant, anticancer, anti-apoptotic, and immune-stimulating activities, have a protective function against pathogens, and have potential for bioethanol production (Kim et al. 2006a; Yin et al. 2014; Kadam et al. 2015; Harris et al. 2014). Therefore, laminarinases are promising biocatalysts in the degradation of laminarin for various applications, such as generation of biochemicals and bioenergy. Laminarinases with activity against laminarin were isolated from several ascomycete fungi such as A. niger, Penicillium claviforme, Trichoderma viride, D. salina, Aspergillus japonicus, Rhizomucor miehei, and Penicillium rolfsii (Lee et al. 2014; Grant and Rhodes 1992; Kulminskaya et al. 2001). Laminarinase from P. rolfsii was purified and characterized (Lee et al. 2014; Chaari and Chaabouni 2018). Based on the enzymatic hydrolysis activity to produce fermentable sugars, this laminarinase has potential for bioethanol production. In addition, the acidophilic and broad pH stability as well as good thermostability of the purified enzyme has advantages for feed, food, pharmaceutical, biochemical, and biofuel industries (Lee et al. 2014). Another efficient laminarinase for saccharification of laminarin has been purified and characterized from Talaromyces amestolkiae (Méndez-Líter et al. 2018).

3.3

Applications of Green Seaweed Degrading and Modifying Enzymes

Ulvan is the most abundant carbohydrate synthesized by green seaweeds (Ulva and Enteromorpha) (Lahaye and Robic 2007). Ulvan and oligosaccharides released from it have recently gained attention for a variety of applications in food, feed, and chemical industries. They also have antioxidant, anticoagulant, immunomodulator, antiviral, and antitumoral activities that are interesting for pharmaceutical and nutraceutical applications (Qi et al. 2005; Mao et al. 2006; Leiro et al. 2007; Ivanova et al. 1994; Kaeffer et al. 1999). In addition, it has been demonstrated that ulvan has plant defense and plant resistance activities with a potential to be used in agricultural applications (Jaulneau et al. 2010; Paulert et al. 2010). Furthermore, biomaterials such as ion exchanger hydrogels, nanofibers, membranes, and microparticles can be

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built using ulvan polymers (Toskas et al. 2011; Alves et al. 2012; Morelli and Chiellini 2010). Despite these promising applications of ulvan and its derivatives, identification and characterization of enzymes involved in ulvan degradation and release of oligo- and monosaccharides have been poorly investigated, in contrast to the enzymes that degrade agars and carrageenan from red seaweeds and alginates from brown seaweeds. Enzymes that have been identified to have ulvanolytic activities are ulvan lyase (EC 4.2.2._, PL24, 25, and 28), β-glucuronidase (EC 3.2.1.31, GH1, 2, 30, 79, 154), β-1,4-glucuronan lyase (EC 4.2.2.14, PL14, 20), and unsaturated β-glucuronyl hydrolase (EC 3.2.1._, GH105) (Nyvall Collen et al. 2011; Quemener et al. 1997; Delattre et al. 2006; Nyvall-Collén et al. 2014; Kopel et al. 2016). There are only a few reports on ulvanolytic enzymes from fungi. An extracellular glucuronan lyase was isolated and purified from Trichoderma sp. GL2 (Delattre et al. 2006). It was active toward ulvan producing various low molecular weight ulvans (Delattre et al. 2006). Another study identified an ulvan lyase (PL24) encoding gene in Phomopsis longicolla, which is a seed-borne fungus causing Phomopsis seed decay in soybean (Li et al. 2017). However, the role of ulvan lyases in P. longicolla is unknown.

4 Conclusions and Future Prospects This chapter provided an overview of the applications of fungal enzymes naturally involved in the degradation of plant and algal biomass. This presents one of the best examples of the implementation of a highly efficient and diverse biological system, degradation of biomass by fungi for their growth and reproduction, into humandesigned processes. The long history and subsequent broadening of these applications highlight their importance for human society, which is likely to grow even further as we are moving to a biobased renewable society. Implementation of plant biomass conversion using fungal enzymes has already occurred in many industrial sectors (Fig. 4.2), while the conversion of algal biomass is more in its infancy, in part due to the highly diverse cell wall structure and composition of the different algae. The availability with an ever-growing number of fungal genomes (Grigoriev et al. 2014) has supplied us with a collection of (putative) enzymes that could never have been foreseen in the pre-genomics era, and this number will continue to grow exponentially. While traditionally only well-studied industrial fungi (e.g., A. niger, T. reesei) were considered as enzyme suppliers, now also other fungi have shown high potential as enzyme sources (Espagne et al. 2008; Peng et al. 2017). This resource can be mined not only for novel enzymes or activities but also for similar enzymes with more advantageous properties. It has already been shown that orthologous enzymes from related fungi can have strongly different properties with respect to proteolytic (de Vries et al. 1997) or temperature stability (Culleton et al. 2014). In addition to the discovery of novel enzymes, another issue to be solved is the production of these enzymes. Current production yields are still hampering their

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application in some industrial processes. In this context, the biotechnological production in higher amounts of enzymes with improved physicochemical properties will increase the future perspectives for their application in these and other unexpected industrial processes yet to be exploited.

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Part II

Developments in Key Enabling Technologies

Chapter 5

Genetic Transformation of Filamentous Fungi: Achievements and Challenges Alexander Lichius, Dubraska Moreno Ruiz, and Susanne Zeilinger

1 Introduction Genetic transformation represents a form of horizontal gene transfer and is defined as a process by which exogenous genetic material is taken up into a cell. Transformation of bacteria was initially reported by Frederick Griffith 90 years ago when he described that a non-virulent strain of Streptococcus pneumoniae could be made virulent by a ‘transforming principle’ derived from heat-killed virulent strains (Griffith 1928). The ‘transforming principle’ was identified in 1944 by Oswald Avery, Colin MacLeod and Maclyn McCarty as DNA that had been isolated from a virulent S. pneumoniae strain (Avery et al. 1944). In the late 1970s, a reliable method for the stable transformation of Saccharomyces cerevisiae spheroplasts was presented (Beggs 1978; Hinnen et al. 1978), thereby opening the molecular era in yeast genetics and establishing budding yeast as a leading eukaryotic model organism (Mitrikeski 2013). However, already in 1973, Mishra and Tatum reported transformation of the filamentous fungus Neurospora crassa (Mishra et al. 1973). Nowadays, all major groups of fungi can be transformed, and the genetic manipulation of these organisms is of great importance not only for research purposes but also for their applications in biotechnology. In this chapter, we focus on five filamentous fungi comprising the model organism N. crassa and four species with applications in industry or agriculture, i.e. Aspergillus niger, Aspergillus oryzae, Trichoderma reesei and Trichoderma atroviride. A. niger and A. oryzae are both commercial key producers of enzymes, while A. oryzae is as well an important workhorse in Japanese fermentation industry for the production of the traditional Japanese fermented foods sake, soy sauce and miso (Cairns et al. 2018; Machida et al. 2008). Both species are listed as generally recognized as safe (GRAS) organisms by the Food and Drug Administration (FDA) A. Lichius · D. M. Ruiz · S. Zeilinger (*) Department of Microbiology, University of Innsbruck, Innsbruck, Austria e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_5

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of the United States due to their long and safe use (Schuster et al. 2002; Sewalt et al. 2016). Besides the two mentioned Aspergillus spp., T. reesei is the second major fungal system for industrial enzyme production, especially cellulases, and it is as well classified as GRAS (Paloheimo et al. 2016). In contrast to T. reesei, which only has weak mycoparasitic abilities, the strong mycoparasite T. atroviride is applied in plant disease control as fungal biocontrol agent against a wide range of economically important plant pathogens (Druzhinina et al. 2011). Generating detailed insights into gene function in these filamentous fungi is advantageous to allow their further development into robust cell factories. This functional genomics approach as well as the generation of improved strains with targeted genetic manipulations however requires effective and reliable transformation methods.

2 Prerequisites for the Genetic Transformation of Fungi Six major prerequisites need to be fulfilled to successfully transform fungal cells: (1) high amounts of pure transforming DNA, (2) highly competent cells of a suitable recipient strain that allow passage of exogenous DNA through the cell wall matrix, (3) a mechanism that delivers the transforming DNA across the plasma membrane into the cytoplasm, (4) a mechanism that safely traffics the DNA through the cytoplasm and across the nuclear envelop, (5) a mechanism that expresses the transforming DNA integrated into the fungal genome or maintained, for example, in an autonomously replicating plasmid, and (6) a recovery and selection procedure to grow and isolate positive and stable transformants.

2.1

Transforming DNA

With modern molecular cloning procedures, such as PCR-based DNA recombination and straightforward DNA synthesis, is the generation of transforming DNA fragments or larger gene replacement cassettes not very challenging anymore. Highly efficient in vitro cloning systems, such as the NEBuilder® HiFi DNA Assembly Cloning Kit, allow the seamless recombination of multiple DNA fragments in one reaction, provided unique 20–25-bp-long homologous overlaps between adjacent fragments are present. The included DNA ligase seals nicks at the recombination sites resulting in a fully closed DNA molecule that is immediately available for downstream applications. In case of a plasmid, this allows the immediate generation of diagnostic PCR amplicons to confirm full assembly and sent critical regions for sequence verification without the need to process the molecule through Escherichia coli beforehand. Once DNA sequencing has confirmed its correctness, the preparation of large amounts of transforming DNA (in the range of up to 10 μg/transformation) by PCR or classic plasmid preparation from E. coli is somewhat trivial. DNA purity is important and conveniently secured by the use of

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commercial DNA clean up kits or an even simpler PEG/MgCl2-based size-selective DNA precipitation procedure that avoids any yield loss (Lis 1980; Paithankar and Prasad 1991).

2.2 2.2.1

Generation of Competent Cells Natural Competency

The fungal cell wall presents a natural barrier for the uptake of exogenous genetic material, and thus its permeation is the first critical step in the transformation procedure. In bacteria, naturally competent cells harbour genes that encode the machinery to import DNA as means of horizontal gene transfer (Solomon and Grossman 1996), a phenomenon that is highly dependent on the prevalent environmental conditions. Similarly, natural transformation of budding yeast occurs under starvation conditions. For instance, when nongrowing S. cerevisiae cells start to metabolise sugars in the absence of any other nutrients, added plasmid DNA is taken up alongside (Nevoigt et al. 2000). This, however, requires more than 25-times higher DNA concentrations than sufficient for achieving high transformation efficiencies with artificial transformation approaches. Interestingly, 40 years ago, Mishra speculated that N. crassa possesses a ‘physiological preparedness or competence for DNA uptake and transformation’ as he observed a linear dependence of DNA uptake by a young (30 h) culture on the amount of DNA added to the growth medium, which was doubled by the presence of CaCl2 (Mishra 1979). However, the mechanisms remained unclear, and natural competency based on active import of ‘naked’ DNA from the environment has, to our knowledge, not yet been experimentally confirmed for filamentous fungi.

2.2.2

Generation of Competent Cells by Washing and Chemical Treatment

Due to practical reasons, it is common practice to make fungal cells highly competent by increasing cell wall permeability through chemical and/or enzymatic pretreatments. Physical methods that weaken or remove the cell wall, such as grinding or sonication, do also exist and are occasionally mentioned in the literature but, to our knowledge, are not frequently used in the fungal research community. Some filamentous fungal species, such as N. crassa or T. reesei, produce vegetative spores (macroconidia) that at a very young age (harvested from 5- to 20-day-old cultures) require only a few simple washing steps in sorbitol solution to become sufficiently competent for electroporation (Margolin et al. 1997; Navarro-Sampedro et al. 2002; Schuster et al. 2012). In other species, the cell wall presents a significant but not preclusive barrier to transforming DNA and becomes sufficiently weak by

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cultivation in the presence of mono- or bivalent cations, such as Li+ (Ito et al. 1983; Ramon and Fonzi 2009) or Ca2+ (Bruschi et al. 1987).

2.2.3

Generation of Competent Cells with Cell Wall Degrading Enzymes

In many filamentous fungal species, however, the cell wall presents such an impenetrable barrier that the generation of sphero- or protoplasts often seem to be the only possibility to acquire sufficiently competent cells for electro- or PEG-mediated transformation. Spheroplasts are cells whose cell wall has been partially lysed, for example, with the snail stomach enzyme β-glucuronidase commercially denominated as Glusulase® (Eddy and Willianson 1957; Hutchison and Hartwell 1967), whereas protoplasts are completely wall-less cells generated with more potent enzyme mixes from fungi, such as the lysing enzymes from T. harzianum (Kitamoto et al. 1988) sold under the trade name Glucanex®. Alternatively, the lysing enzyme mix from Aspergillus (Viscozyme®) or more elaborate combinations of the two plus hemicellulases and chitinases from other fugal species (de Bekker et al. 2009; Patil and Jadhav 2015) have been used. Since the production of the frequently used Novozyme 234 lytic enzyme mix has been discontinued, β-D-glucanase was recommended as a suitable replacement (Jung et al. 2000) and is commercially available as the winemaking enzyme VinoTaste Pro (Novozymes) (Chiang et al. 2013). If required for practical reasons, it is possible to maintain sphero- or protoplasts for at least 1 day at 4
C and perform the transformation the next day. Interestingly, protoplasts of A. oryzae displayed a higher transformation efficiency after this treatment compared to the batch of cells that was electroporated immediately after preparation (Ozeki et al. 1998).

2.2.4

Natural Factors That Influence Protoplasting

Natural factors, such as the age and developmental stage of conidial germlings, or the degree of spore pigmentation, influence protoplasting and consequently transformation efficiencies. For instance, best results were obtained with protoplasts prepared from conidial germlings of A. nidulans incubated for 4.5 h, whereas transformation efficiencies were much lower with protoplasts prepared from shorter or longer incubated cells (Koukaki et al. 2003). In the strongly pigmented A. niger, protoplasting was significantly aggravated due to the high melanin content of the cell wall (Bos and Slakhorst 1981); however, this was alleviated by choosing hyphae instead of conidia as inoculum for preparative liquid cultures (Cho et al. 2012). With respect to protoplast sensitivity, inorganic salts, such 0.6–0.7 M sodium chloride or 1.2 M magnesium sulphate, or other sugars than sorbitol, such as 0.8 M mannitol or 0.6 M sucrose, proofed to be better osmotic stabilisers during protoplast preparation, but also during protoplast regeneration post transformation (reviewed in Fincham 1989). A comprehensive overview on protoplast preparation parameters in numerous filamentous fungal species has recently been provided elsewhere (Li et al. 2017).

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As rightly stated by Li and Tang in their review, ‘There is no universal transformation method that can be applied to different fungal strains. Preparation of protoplasts can hardly be standardized. Part of the difficulties comes from our limited knowledge of cell wall hydrolases. Development of an optimized protoplast-mediated transformation (PMT) method for fungi still requires significant effort’. It is safe to assume that the more effort is invested in the generation of protoplasts, the higher the resulting transformation efficiency will be. This, however, is overall quite laborious and requires the purchase and preceding testing of expensive enzymes, as well as the osmotic stabilisation and careful handling of the wall-less cells.

2.2.5

Generation of Competent Cells by Natural Swelling

An intermediate approach between the application of cell wall weakening chemicals and partial or complete enzymatic lysis of the cell wall is the utilisation of spore swelling procedures. The incubation of conidia in nutrient-rich liquid media and at elevated temperatures for several hours—depending on the species at up to 35
C and for up to 16 h under vigorous shaking—forces rapid isotropic cell expansion. Every doubling of the cell diameter causes an eightfold increase of the cell volume and the halving of the surface-to-volume ratio. This leads to the natural thinning of the cell wall crucial for enabling high-efficiency passage of exogenous DNA. The addition of cell wall weakening agents during the final hours of swelling incubation weakens the cell wall further and increases transformation efficiencies (Chakraborty and Kapoor 1990; Ozeki et al. 1994). This obviously adds costs for enzymes and represents a transition towards spheroplast generation; however, this does not add further protocol steps and might help with the preparation of competent cells for ‘difficult’ species. Furthermore, longer periods of swelling incubation can be conveniently conducted overnight, and temperatures may be adjusted to have perfectly sized spores ready the next morning. How cell size influences transformation efficiency by electroporation is discussed in Sect. 3.2.

2.2.6

Generation of Competent Cells from Conidial Germlings

The extension of swelling incubation beyond the point of germ tube emergence is another possibility to prepare competent cells. Young germlings can either be used directly for electrotransformation (Ozeki et al. 1994), which, however, due to the increased sensitivity of the germ tube tip in comparison to non-germinated cells, might require adjustment of the electric settings used for swollen cells, or they are used as source material for the preparation of sphero- or protoplasts (Kothe and Free 1996; Nargang and Nargang 1996). As there is some confusion with respect to the terminology of ‘swollen’ and ‘germinated’ cells in the literature, we recommend to define ‘germination’ as the point of cell symmetry breaking, i.e. first appearance of a nascent germ tube from the spherical cell, and to classify cell preparations according

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to the morphological status of 80% of the population, in order to ensure reproducibility of applied protocols.

2.3

Delivery Mechanism into the Cytoplasm

Once the cell wall matrix is sufficiently weakened, the plasma membrane is the last barrier before the DNA enters the cell. Methods for intracellular DNA delivery comprise viral vectors; chemical-mediated delivery, such as liposomes or polyethylene glycol (PEG); direct insertion via biolistic or magnetic particles; Agrobacteriummediated transfer; and physical membrane poration (Meacham et al. 2014). PEG addition in the presence of Ca2+ or Mg2+ (Ito et al. 1983; Liu and Friesen 2012) and electroporation (Chakraborty et al. 1991) are the two most frequently used techniques to transiently enhance plasma membrane penetrability of fungal cells and to facilitate the entry of DNA molecules. Noteworthy is that the precise function of PEG during the transformation process still remains obscure. The widely accepted view that PEG helps to deliver the DNA across the lipid bilayer by promoting protoplast adhesion and trapping of DNA between the touching membranes has been challenged by findings showing that protoplast fusion is not involved in the process of DNA uptake. The findings by Kuwano et al. (2008) indicate that PEG functions after the delivery of the DNA into the cell and rather suggest that divalent cations play the fundamental role as signalling molecules in a pathway that mediates DNA passage across the lipid bilayer (Kuwano et al. 2008). This comes close to a mechanism of natural competence in filamentous fungi, as already hypothesised by Mishra in 1979 (Mishra 1979). However, given the considerable number of known Ca2+- and Mg2+-dependent cellular processes (Romani 2011; Tisi et al. 2016), it will be a major challenge to identify the molecular components that act as DNA (co) transports in such a pathway. In any case, the addition of carrier DNA, such as purified single-strand DNA or salmon sperm dsDNA, is a frequently applied aid to increase the efficiency of DNA uptake by up to a factor of 100 (Gietz and Schiestl 2007; Schiestl and Gietz 1989). In contrast, the mode of action of electroporation is much better understood. The micro- to millisecond exposure of the plasma membrane to an applied electric field elevates the transmembrane voltage resulting in conformational changes between lipid and protein molecules (Förster and Neumann 1989; Neumann 1989). These structural rearrangements within the lipid bilayer lead to the transient formation of membrane pores through which transforming DNA enters the cell by passive diffusion (Weaver 2003). This induced permeability is reversible as long as the duration and magnitude of the electric field does not overstep the critical limit for cell survival. Hence, electroporation settings need to be carefully balanced to allow efficient transfer of the transforming DNA into the cytoplasm while at the same time ensuring complete and timely resealing of the pores before cell leakage occurs (Kotnik et al. 2012). Electroporation is highly versatile and can be applied to introduce different

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types of small molecules into both intact fungal cells and sphero- or protoplasts. For further details on the biophysics of electroporation, refer to Sect. 3.2.

2.4

Traffic Through the Cytoplasm and into the Nucleus

Once the transforming DNA has successfully crossed the cell wall and plasma membrane, the cytoplasm is the next significant obstacle on its way to the nucleus. ‘Naked’ DNA does not persist longer than 50–60 min in the cytoplasm before it becomes degraded by nucleases (Lechardeur et al. 1999). Therefore, immediately upon internalisation, naked DNA rapidly forms large DNA-protein complexes comprised of up to several hundred different proteins (Badding et al. 2012). These include DNA-binding proteins, transcription factors, chaperones, dynein and kinesin as well as β-importins and function to protect the DNA from degradation, mediate its transport along the cytoskeleton towards the nuclear envelop and finally regulate its transport through the nuclear pore complex (NPC) (Bai et al. 2017). Noteworthy is the fact that due to its highly viscous environment, only DNA fragments smaller than 2 kb can diffuse through the cytoplasm in a reasonable physiological time frame, whereas larger DNA fragments depend on active transport (Lukacs et al. 2000). Furthermore, the amount of transforming DNA molecules that ultimately enter the nucleus through the NPC is only 1–10% of those that reached the cytoplasm in the first place (James and Giorgio 2000; Tseng et al. 1997). Hence, in the interest of efficient transformation, the transforming DNA should be kept as small as possible to facilitate its transit to and into the nucleus. Additionally, the application of highstarting molarities of transforming DNA and the provision of sufficient recovery time for the cells after transformation are simple experimental means to counteract the inefficiencies of both trafficking processes. A so far underappreciated approach is the uninterrupted delivery of transforming DNA from the cytoplasm across the nuclear envelope through the application of ultrashort (nanosecond) electric pulses of high intensity (up to 300 kVcm1). These have been found to interact with subcellular structures without affecting the plasma membrane in mammalian cells (Schoenbach et al. 2004). Hence, it seems long overdue to test the application of millisecond and nanosecond pulse combinations to facilitate the non-stop delivery of DNA molecules directly into the nuclei of filamentous fungi. Agrobacterium-mediated transformation (AMT) employs the nuclear localisation signals (NLS) encoded in the two virulence proteins VirE2 and VirD2 to traffick A. tumefaciens T-DNA through the cytoplasm and into plant nuclei (Ziemienowicz 2001). VirE2 binds the DNA, protects it from degradation and facilitates its transport along microtubules to the nucleus, whereas VirD2 transports the T-DNA complex across the nuclear envelope (reviewed in Nester 2015). Because the host range of A. tumefaciens comprises yeasts, algae, plants and even human cells, it is likely that a similar, if not the same, shuttling mechanism operates during AMT of filamentous fungi.

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Integration and Expression of the Transformed Genetic Information

Three basic approaches allow the transient or stable expression of transformed genetic information in filamentous fungi: (1) expression from autonomously replicating plasmids (ARPs) that persist for shorter or longer periods of time in the cytoplasm, (2) expression after random integration into the genome and (3) expression after targeted integration at a defined locus.

2.5.1

Autonomously Replicating Plasmids

In S. cerevisiae and other yeasts, a variety of ARPs are available to generate stable transformants which often permit higher transformation and expression efficiencies than obtained following chromosomal integration of linearised DNA (Dhar et al. 2012; Liachko and Dunham 2014; Nakamura et al. 2018). For filamentous fungi, so far, only one reliable ARP, ARp1 from A. nidulans containing the AMA1 plasmid replicating sequence (Gems et al. 1991), and two transformation enhancers (ANS1 and MATE) (Aleksenko and Clutterbuck 1997) have been identified and successfully employed to maintain high-level gene expression from extrachromosomal loci in numerous other species, including A. fumigatus and T. reesei (Kubodera et al. 2002), A. niger (Sarkari et al. 2017) and A. oryzae (Katayama et al. 2018). In contrast to yeast ARPs, Arp1 is mitotically unstable in filamentous fungi (Verdoes et al. 1994). While for one experimental setting this may pose an undesirable disadvantage, for others, it’s a valuable tool for the construction of expression systems which demand the timely loss of the ARP (Sarkari et al. 2017). Interestingly, the initial evidence of ARPs in the model fungus N. crassa (Grant et al. 1984; Hughes et al. 1983; Paietta and Marzluf 1985) has to our knowledge not yet been successfully confirmed.

2.5.2

Random or Targeted Integration into the Genome

In most cases, the transforming DNA integrates into the genome by recombination with the chromosome. Random integration of genetic information happens by non-homologous end joining (NHEJ) repair during which the transforming DNA becomes ligated into spontaneous double-strand breaks (DSBs) in the fungal genome (Chang et al. 2017). Because NHEJ is the major repair mechanisms of DSBs in many eukaryotes, it occurs with a much higher efficiency compared to targeted events and is thus the technically ‘easiest’ and probably quickest way to generate stable transformants. However, the downside is that due to the lack of control where and how often the DNA integrates, unwanted disruptions in the genome frequently occur. Hence, special care needs to be taken with the selection and phenotypic validation of resulting transformants.

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Fig. 5.1 Illustration of the fate of the transforming DNA and the potential effects of Cas9/sgRNAmediated site-specific DNA DSBs. Repair of DSBs and integration of the transforming DNA can occur via NHEJ or HR. In case of NHEJ, the transforming DNA integrates ectopically, i.e. at a random genomic location (light grey), and leaves the target locus (red within dark grey genomic region) intact. Repair of DNA DSBs via NHEJ is error-prone and may also result in small insertions or deletions (indels; green) at the break site thereby resulting in gene inactivation. In case of HR, the homologous sequences in the transforming DNA mediate homology-based recombination resulting in insertion of the marker gene (blue) at the intended target locus (dark grey). Defined gene corrections may as well be achieved by CRISPR-Cas9-mediated editing via incorporation of a donor DNA template being homologous to the target locus and containing the desired edits

The targeted integration of transforming DNA at a specific locus occurs via homologous recombination (HR) (Kaniecki et al. 2018; Li and Heyer 2008). Although random integration of transforming DNA may still occur despite the presence of homologous DNA regions, in general, longer stretches of homology between the transforming and the genomic DNA promote HR. For this reason, sequences homologous to the 50 and 30 regions of the desired integration site are usually employed to flank the transforming DNA. For most fungi, 0.5–1 kb stretches of homology are sufficient; in general, however, longer regions of homology promote HR. Figure 5.1 schematically shows the key differences between NHEJ and HR. HR is standardly used for the genetic modification across all filamentous fungal species and was instrumental for generating the large gene deletion strain collections of S. cerevisiae (Saccharomyces Genome Deletion Project: http://www-sequence. stanford.edu/group/yeast_deletion_project/deletions3.html) (Giaever et al. 2002; Giaever and Nislow 2014) and N. crassa (Neurospora Functional Genomics Project: http://www.fgsc.net/ncrassa.html) (Collopy et al. 2010; Colot et al. 2006; Dunlap et al. 2007; Park et al. 2011). However, in contrast to yeasts, which use HR as default DSB repair mechanism, the natural frequency of HR is limited to no more than 20% in filamentous fungi, rendering the targeted integration of transforming DNA highly inefficient. Hence, the use of Ku70 or Ku80 deletion strains as recipients, which lack regulatory key

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components of the NHEJ pathway whose absence boosts HR frequency to almost 100%, was the decisive step to allow (high-throughput) knockout procedures in several filamentous fungi, including N. crassa, Aspergillus spp. and Trichoderma spp. (Catalano et al. 2011; da Silva Ferreira et al. 2006; Guangtao et al. 2009; Nayak et al. 2006; Ninomiya et al. 2004; Takahashi et al. 2006). Notably, HR efficiency is affected by the target locus and the topology of the transforming DNA, with linear fragments—in contrast to circular plasmid DNA—significantly increasing the recombination frequency in several fungi (Martín 2015).

2.6 2.6.1

Recovery, Selection and Isolation of Transformants Transformant Recovery

Due to the significant chemical, enzymatic and/or physical stresses the cells experience during preparation and transformation, a recovery phase in a rich medium without selection pressure is—in most cases—an essential part of the procedure. It not only allows rebuilding the weakened or removed cell wall but also provides the necessary time to traffick the transforming DNA into the nucleus (Sect. 2.4) and integrate it into the genome (Sect. 2.5). Obviously, the efficiency of both processes directly determines the final yield of correct transformants. For obvious reasons, protoplast recovery requires more attention than the recovery of intact cells. Nevertheless, elaborate recovery protocols can be found for both approaches and slightly vary depending on the fungal species. Two major recovery methods are frequently applied: (1) recovery in a liquid medium with subsequent plating on selection medium and (2) recovery in top agar without selection pressure that is poured onto bottom agar with appropriate selection pressure. In the first case, used liquid media range from minimal to complex but usually contain a rich carbon and nitrogen source. Incubation conditions are set to the optimal growth temperature of the respective species and last between 1 and 4 h under gentle shaking, although protocols that employ overnight incubation at room temperature also do exist. After this recovery time, the cells are either pelleted and washed beforehand or directly plated onto selection medium. In the latter case, the selective chemical slowly diffuses from the bottom agar into the top agar allowing several hours of recovery before the selection pressure steadily increases. Incubation usually occurs at the optimal growth temperature of the species. As for the transformation method itself, there is no universal recovery protocol for filamentous fungi, and various combinations of these two basic approaches are described in the literature. Hence, individual testing and optimisation of existing protocols are usually required.

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Transformant Selection with Resistance and Nutritional Marker Genes

Due to the low efficiency of transformation, cells that have successfully integrated the transforming DNA need to be separated from those that have not. This selection is generally based on the use of selective markers that suppress or do not permit the growth of non-transformants. Selection markers currently available for filamentous fungal species are summarised in Table 5.1. Dominant markers are genes encoding proteins mediating resistance against chemical drugs, such as the widely used hph gene. hph originates from E. coli and encodes a hygromycin B phosphotransferase able to detoxify the aminoglycoside antibiotic hygromycin B (Gritz and Davies 1983). Other dominant marker genes currently available for filamentous fungi are bar (phosphinothricin/glufosinate resistance) and nat1 (nourseothricin resistance). The second frequently used selection method is based on nutritional genes. This requires the availability of appropriate auxotrophic mutants with a specific deficiency in amino acid, uracil or nitrogen metabolism. Resistance and nutritional markers may be combined to simultaneously select for different genetic modifications, such as knockout complementation or the expression of two different possibly genetically modified target genes. In addition, some nutritional marker such as pyr4/ pyrG, amdS and niaD are bidirectional markers as they allow two-way selection. In the case of pyr4, gene inactivation leads to uridine/uracil auxotrophy but 5-fluoroorotic acid resistance thereby opening up the possibility to screen either for gain or loss of the marker gene (Dave et al. 2015). As the number of available selection marker genes is limited and hence hampers consecutive genetic manipulation, marker recycling is of interest. Respective methods commonly are based on the bacteriophage-derived cre/loxP system involving the excision of the marker gene by the site-specific Cre recombinase when flanked by loxP repeats which, however, requires expression of the recombinase in the target organism (Sauer 1987).

2.6.3

Transformant Isolation

The occurrence of a particular integration type is highly variable and depends on the species transformed, the transformation method applied and the DNA used (linear or circular). The HR success rates differ significantly between species and strains used and can be very high, as, for example, ~80% in A. nidulans (Yelton et al. 1984), or very low with no more than 2–5% in T. reesei (Mach et al. 1995; Seiboth et al. 1992) or N. crassa (Case 1986). For fungal species that form uninucleated conidia, such as Aspergillus spp. or T. atroviride, homokaryon purification of transformants is simply achieved by isolating individual monosporic colonies, whereas for species with predominantly multinucleate conidia, such as N. crassa or T. virens, at least three rounds of homokaryon purification plating will be necessary to achieve a high probability of the stochastic loss of non-transformed nuclei (Fincham 1989). In

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Table 5.1 Nutritional and resistance markers commonly used in Aspergillus, Neurospora and Trichoderma studies Gene Encoded function Nutritional markers am Glutamate dehydrogenase

Species

References

N. crassa

amdS

Acetamidase

A. niger A. oryzae N. crassa T. reesei T. atroviride

argB

Ornithine carbamoyltransferase

adeA

A. oryzae

Jin et al. (2004)

A. oryzae N. crassa

hxk-1 niaD

Phosphoribosylaminoimidazole Succinocarboxamide synthase Phosphoribosylaminoimidazole carboxylase 5-Aminolevulinate Imidazoleglycerol-phosphate dehydratase Hexokinase Nitrate reductase

A. niger A. oryzae N. crassa T. reesei A. oryzae

Kinsey and Rambosek (1984) Kelly and Hynes (1985) Gomi et al. (1992) Yamashiro et al. (1992) Penttilä et al. (1987) Reithner et al. (2007) Buxton et al. (1985) Gomi et al. (1987) Weiss et al. (1985) Penttilä et al. (1987) Jin et al. (2004)

nic-1

Nicotinic acid

pan-2

3-Methyl-2-oxobutanoate hydroxymethyltransferase OMP decarboxylase/orotidine-5-Phosphate decarboxylase

Elrod et al. (2000) Ebbole and Sachs (1990) Guangtao et al. (2010) Unkles et al. (1989) Campbell et al. (1989) Akins and Lambowitz (1985) Low and Jedd (2008)

adeB hemA his-3

pyr4 / pyrG/ura3

qa-2 sC trp-1/trpC

5-Dehydroquinate hydrolase ATP sulfurylase Trifunctional enzyme of tryptophan biosynthesis

ura5

Orotidine-5-monophosphate pyrophosphorylase Resistance markers bar Phosphinothricin acetyltransferase

Benomyl

Mutated β-tubulin

T. reesei A. oryzae A. niger N. crassa N. crassa A. oryzae A. niger N. crassa T. reesei N. crassa A. oryzae N. crassa A. niger T. reesei

N. crassa A. niger N. crassa

de Ruiter-Jacobs et al. (1989) Goosen et al. (1987) Turner et al. (1997) Gruber et al. (1990) Case et al. (1979) Yamada et al. (1997) Schechtman and Yanofsky (1983) Goosen et al. (1989) Bergès and Barreau (1991) Avalos et al. (1989) Ahuja and Punekar (2008) Orbach et al. (1986) (continued)

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Table 5.1 (continued) Gene ble

Encoded function Phleomycin-binding protein

Species A. oryzae N. crassa T. atroviride

hph

Hygromycin B phosphotransferase

G418

Geneticin resistance

A. niger N. crassa T. reesei T. atroviride A. niger T. atroviride

nat1

Nourseothricin acetyltransferase 1

oliC

Oligomycin-resistant mitochondrial ATP synthase Pyrithiamine resistance

ptrA

N. crassa T. atroviride A. niger

References Cheevadhanarak et al. (1991) Austin et al. (1990) Cardoza et al. (2006) Punt et al. (1987) Staben et al. (1989) Mach et al. (1994) de Groot et al. (1998) Rambosek and Leach (1987) Gruber et al. (2012) Smith and Smith (2007) Atanasova et al. (2018) Ward et al. (1988)

A. oryzae

Kubodera et al. (2000)

certain fungi, such as Trichoderma spp., genetic and phenotypic stability can be significantly affected by the transformation procedure and selection marker used (Cardoza et al. 2006) and needs to be especially considered during subculturing in the absence of selection pressure.

3 Technical Approaches for the Genetic Transformation of Fungi Essentially all major transformation approaches initially established in yeasts (Gietz and Woods 2001; Kawai et al. 2010) have been applied to filamentous fungi (Li et al. 2017; Ruiz-Diez 2002). Species-specific optimisation of standard protocols, however, is usually required in order to achieve efficiencies with the generally more challenging transformation of filamentous fungi close to those known from yeasts. Protocol adaptation is to some extent alleviated by the more complex morphology of filamentous fungi in comparison to the unicellular yeasts. The multicellularity of filamentous fungi provides different cell types—including macroconidia, microconidia (usually uninucleate) and various types of mycelial cells, most notably subapical hyphal tip cells—whose different characteristics render one or the other transformation approach more or less convenient. Ultimately, it comes down to the physiological and genetic makeup of the particular filamentous fungal strain which transformation method will be most successful. Genetic defects in cell wall or plasma membrane biosynthesis, for instance, will most likely influence the procedure, negatively or positively. In the

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following, currently popular approaches are presented with a focus on the five filamentous model species featured in this review.

3.1

PEG/CaCl2-Mediated Protoplast Transformation

Protoplasting, initially pioneered with S. cerevisiae in its reduced form of spheroplasting by Hutchison and Hartwell in 1967 (Hutchison and Hartwell 1967), was for the first time applied in a filamentous fungus by Case et al. in 1979 in order to prepare competent cells of N. crassa for subsequent PEG/CaCl2-mediated transformation (Case et al. 1979). The review by Fincham (1989) provides an excellent summary of the methodological state-of-the-art at the time and an overview of the more than 20 filamentous fungal species that were successfully transformed using this method over the following decade (Fincham 1989). Since then, the general procedure of PEG/CaCl2-mediated protoplast transformation remained unchanged and follows six major steps: (1) the osmotic stabilisation of protoplasts, most commonly through the addition of 0.8–1.2 M sorbitol in the suspending buffer/medium; (2) the exposure of the cells to up to 10 μg pure transforming DNA for 10–30 min on wet ice, followed by (3) the addition of a high concentration of PEG (40–45% v/v with a molecular weight range between 3000 and 8000 gmol1) along with (4) 10–50 mM CaCl2; (5) 10–30 min incubation of the mix at room temperature to allow DNA uptake, and finally followed by (6) recovery incubation and plating on appropriate selection medium. Minor changes in order to adapt the basic protocol to species-specific requirements or to simplify the overall approach and improve transformation efficiencies are possible on any of these major steps. Comprehensive reviews with protocol details applicable to various fungal species have been previously provided (Li et al. 2017; Liu and Friesen 2012).

3.2

Electrotransformation

Electrotransformation can be applied to dormant (Navarro-Sampedro et al. 2002), swollen or germinated conidia (Chakraborty et al. 1991; Sánchez and Aguirre 1996; Schuster et al. 2012) and to sphero- or protoplasts (Kothe and Free 1996). Provided competent cells are readily prepared, electrotransformation offers a simple and quick method and is thus widely applied to numerous filamentous fungal species. Electrotransformation uses an electric field to induce the charge separation across the plasma membrane and elevate the resting transmembrane voltage (TMV). This elevated or induced TMV leads to the reversible formation of membrane pores through which DNA can enter the cell. The term electroporation was coined by Neumann in 1982 after the successful introduction of genetic information into mouse myeloma cells upon the application of pulsed electric fields (Neumann et al. 1982). To allow efficient pore formation yet prevent irreversible damage to the cells, the

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pulse shape (exponential decay or square wave pulse), the pulse strength and pulse duration are essential (Chang 1992). The first electrotransformation of a filamentous fungus succeeded with N. crassa in 1990 using exponential decay pulses with electric field strengths of 9–12.5 kVcm1 and durations between 5 and 20 ms (Chakraborty and Kapoor 1990). This basic protocol is still being used today, albeit with numerous species and lab-specific adaptations.

3.2.1

Basic Biophysics of Electroporation

There are different theoretical models to explain the mechanism of electroporation and its potential to introduce non-permeable molecules into the cell (Escoffre et al. 2009). The model mostly accepted today is the electromechanical model that states that the pores are rapidly created in response to the elevated TMV (Weaver and Chizmadzhev 1996). The increase of the TMV above a certain threshold (critical TMV) leads to the formation of metastable hydrophobic pores within the bilayer which become stabilised through the inflow of water molecules. This in turn reorientates the lipid molecules along the ‘water wire’ to form an energetically more stable hydrophilic pore (Kotnik et al. 2012). The critical TMV to induce pore formation depends on the applied field strength E and the cell size and is position-dependent with respect at which angle the cell surface is hit by the electric field lines. This relationship is described by the steadystate Schwan’s equation which assumes a single spherical cell with a non-conductive plasma membrane (Schwan 1957) (Fig. 5.2): ΔΦ ¼

3 E r jcos δj 2

ð5:1Þ

In this equation, ΔΦ is the induced TMV in [V], 32 represents the geometrical factor of a perfect sphere, E is the external electric field strength in [Vcm1], r is the cell radius in [cm] and δ is the polar angle measured from the centre of the cell relative to the direction of the electric field (Kotnik and Miklavčič 2000). The equation shows that the induced TMV is maximal at the cell poles where the electric field runs in parallel to the polar plane (δ ¼ 0
and 180
) and hits the membrane tangent in a right angle, and equals zero at the equator, where the electric field hits in a right angle (δ ¼ 90
and 360
) but runs parallel to the plasma membrane tangent. This theoretical prediction has been experimentally proven by using voltagesensitive fluorescent dyes (Escoffre et al. 2009; Hibino et al. 1993), and extended models have been developed to apply this function to spheroid or irregularly shaped cells (Pucihar et al. 2006; Valič et al. 2003). Because the intensity of the induced TMV gradually drops from the poles towards the equator following a cosinus function, pore formation can occur with an efficiency of more than 50% only on 2/3 of the cell surface area, precisely in a 60
angle region around the polar plane. Furthermore, as the relationship between E and cell size is inverse, smaller cells require a higher field strength than larger cells to achieve

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Fig. 5.2 Steady-state Schwan’s equation allows determining the induced TMV gradient established along the plasma membrane of a spherical cell (green circle) during exposure to an electric field E generated between two electrodes (grey rods). The applied voltage leads to charge separation along the plasma membrane (/+), elevating the TMV. The induced TMV, however, depends on the polar angle δ between the electric field direction and the polar plane of the cell and is maximal (100%) at the cell poles (yellow dots) and drops by 50% two-thirds along the way to the equator. Hence, only 2/3 of the cell surface (blue area) experience more than half of the critical TMV intensity required for efficient pore formation. Please refer to the text for further details

the same induced TMV. This effectively means that larger cells are easier to electroporate. In addition to cell wall thinning, this is another reason why spore swelling improves the efficiency of electrotransformation. Furthermore, swelling leads to the formation of a nearly perfect spherical cell, permitting the practical application of Schwan’s equation in the first place.

3.2.2

Determining the Critical TMV for Successful Electroporation

Knowing the average radius of the cells to be electroporated and using Eq. 5.1, it is possible to deduce the maximal induced TMV required for successful transformation from previous experiments. For example, when swollen T. atroviride cells with an

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average cell radius of 2.75 μm are subjected to a field strength of 9 kVcm1, the achieved maximal TMV is 3.7 V, which is sufficient to channel plasmid DNA into the cells (Hackl 2019; in prep.). In comparison, freshly harvested macroconidia of N. crassa have an average radius of 3.25 μm (Raju and Griffith 2004); hence, they experience a maximum TMV of 3.6 V when electroporated with an E of 7.5 kVcm1 (Navarro-Sampedro et al. 2002). Assuming that plasma membrane biophysics are similar in both species, this suggests that the critical TMV for the successful electroporation of ascomycetes is in the range of 3.5–4 V. Comparing electroporation protocols from different labs working with filamentous fungi shows that the applied maximum TMV varies between 3.6 and 7.3 V. Thus, determining the average cell radius of one’s own cells allows to set electroporation conditions to meet these criteria as a starting point for protocol optimisation. We applied this calculation and established spore swelling regimes to determine and adjust the average cell sizes for different Trichoderma species. We found that doubling of the cell diameter of 4- to 6-day-old, freshly harvested spores by incubation at 28–30
C and 300 rpm for 4–6 h along with setting the applied field strengths to result a maximum TMV of 4 V allowed efficient electrotransformation of T. atroviride, T. virens, T. asperellum and T. harzianum (Lichius, A. and Hackl, L. et al., unpublished). A second important factor for the optimisation of electrotransformation protocols is the duration of the electric pulse, expressed as the time constant τ of the resistorcapacitor discharge circuit used to generate the exponential decay pulse (Chang 1992; Reberšek and Miklavčič 2011): τ ¼RC

ð5:2Þ

As Eq. 5.2 shows, Tau measured in [ms] is the product of the circuit resistance R in Kiloohms [kΩ] and the capacitance C measured in Microfarad [μF]. In simple terms, τ is the time in which the applied voltage has decayed to about 37% of its initial peak value (Reberšek and Miklavčič 2011). Hence, by adjusting resistance and/or capacitance, τ can be set to a desired value, which typically ranges between 5 and 20 ms for filamentous fungi. Notably, due to the additional resistance introduced by the cuvette and cell suspension buffer, the finally achieved time constant is always shorter than the theoretic value. Many electroporators offer a test function to determine the maximal possible time constant as well as the achieved peak voltage for each particular setup. Both values are important indicators to the quality of the delivered pulse. In summary, it comes down to individual testing and optimisation. The precise quantification of electroporation efficiencies by determining the number of positive transformants per μg DNA (Sánchez and Aguirre 1996) is for understandable reasons rarely done. Nevertheless, the theoretical insights into the biophysics of electroporation provide a valuable starting point for optimising existing procedures and establishing this technique in other filamentous fungal species not yet efficiently accessible to electrotransformation.

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Agrobacterium-Mediated Transformation (AMT)

Virulent strains of the alpha-proteobacterium A. tumefaciens bear a >200 kb tumourinducing plasmid. This Ti plasmid contains the transfer DNA (T-DNA) region that becomes transferred into plant cells and randomly integrates into the plant nuclear genome during infection with the bacterium. Based on the finding that exogenous DNA inserted between the borders of the T-DNA is co-transferred during interkingdom conjugation, A. tumefaciens has been developed into a vehicle for introducing foreign genes into plants (Schell and van Montagu 1997). In 1998, de Groot and co-authors reported that Agrobacterium can also efficiently transfer its T-DNA to filamentous fungi (de Groot et al. 1998). Equivalent to the application of AMT for genetic engineering of plants, a binary vector system is used for fungal transformation. This consists of a small shuttle plasmid containing the T-DNA that, after assembly in E. coli, is transformed into an A. tumefaciens helper strain. Cocultivation of the transformed A. tumefaciens helper strain with fungal protoplasts, conidia or hyphal tissue on a medium containing acetosyringone, a plantderived chemical wounding signal, induces the bacteria’s vir genes. These are contained on a second plasmid and encode the T-DNA transfer system, thereby resulting in transfer of the T-DNA region of the plasmid into the fungal genome (de Groot et al. 1998).

3.3.1

The Pros and Cons of AMT

One major advantage of AMT is the possibility of directly transforming conidia, which in several fungi are uninucleate, thereby facilitating the otherwise tedious process of homokaryon purification from the heterokaryotic transformants normally obtained after PEG-mediated transformation of multinucleate protoplasts. Other benefits are that AMT typically leads to single-copy integration of the T-DNA into the fungal genome (de Groot et al. 1998) and that transfer of large DNA fragments of up to 75 kb is possible as shown for Fusarium oxysporum and Aspergillus awamori (Takken et al. 2004). In addition, based on the fact that NHEJ proteins are required for the integration of the T-DNA into the host genome (van Attikum et al. 2001), the use of NHEJ-deficient mutants forces the T-DNA to integrate into the fungal genome by HR thereby resulting in enhanced gene knockout frequencies. Major limitations of AMT on the other hand are the requirement of binary vector preparation, which can be tedious, and the necessity of optimising the A. tumefaciens cocultivation parameters for each fungal species. The latter have a major effect on transformation frequency and include the type of fungal material (spores, tissue or protoplasts), the biomass ratio of bacterial to fungal cells, the temperature and duration of the cocultivation and the concentration of acetosyringone (Li et al. 2017). In addition, although sequence-independent integration of the transformed DNA into the fungal genomes has been described for several fungi (de Groot et al. 1998), more recent studies found that T-DNA integration is biased towards promoters and AT-rich

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regions due to specific DNA-bending properties (Blaise et al. 2007; Choi et al. 2007).

3.3.2

The Application Range of AMT in Filamentous Fungi

AMT has been applied successfully to many fungal species, mainly Ascomycetes, but also Basidiomycetes and Zygomycetes, and it turned out to be especially useful for transformation of fungi that are not at all or only with very low efficiency amenable to other transformation methods (Frandsen 2011; Michielse et al. 2005). In their initial publication, De Groot and co-authors (de Groot et al. 1998) reported the successful AMT of conidia or hyphal tissues of several filamentous fungi including N. crassa, A. niger and T. reesei using hygromycin resistance as selection marker. The transformation frequencies varied from species to species and for N. crassa and T. reesei were reported to be similar to optimal naked DNA transfer methods, while AMT of A. niger seemed less efficient. Comparisons of transformation efficiencies, however, should be regarded with caution as the number of transformants per μg DNA normally is given as measure for the PEG-mediated protoplast transformation approach, which cannot be used in the case of AMT, where the DNA is directly transferred between bacterial and fungal cells. Based on the publication of de Groot et al. (1998), we developed a modified AMT protocol for gene disruption in T. atroviride strain P1. This approach employs an overlay-based procedure to overcome the lack of formation of distinct single fungal colonies due to background growth on hygromycin-containing selection medium and resulted in stable gene deletion mutants; the vast majority of which harboured single-copy integration of the transferred T-DNA (Zeilinger 2004). For T. atroviride strain T11, stable transformants with randomly integrated T-DNA were only obtained when hygromycin B resistance was used as selection marker, while strains transformed to phleomycin resistance were highly unstable (Cardoza et al. 2006). Using the hph gene for generating T. atroviride strain T23 mutants with improved dichlorvos-degradation ability by AMT-mediated random mutagenesis resulted in 110 positive transformants with high mitotic stability. Two transformants with increased degradation ability were further analysed and confirmed to have singlecopy T-DNA insertions (Sun et al. 2009). Interestingly, transformation efficiencies were similar for all three T. atroviride strains with about 50 transformants per 107 conidia. Similarly, AMT was successfully used in various T. reesei strains including CBS 383.78, QM9414, Rut C30 and ZU-02 as a tool for random insertional mutagenesis (Gao et al. 2018; Zhong et al. 2007), for transforming the fungus with heterologous genes (de Groot et al. 1998; Long et al. 2018) and for homologous recombination (Zhang and Xia 2017). While most studies on AMT of fungi relied on antibiotic resistance markers, uracil auxotrophic recipient strains and pyrG as a biochemical marker are an option for AMT as well. Using this selection system, 1060 transformants per 106 spores could be obtained in the Japanese A. oryzae strain RIB40 that successfully expressed

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the DsRed fluorescent reporter gene under control of the endogenous amyB promoter (Nguyen et al. 2017).

3.4

Biolistic Transformation

Biolistic transformation, also known as particle bombardment, is based on the bombardment of cells with nucleic acid-coated tungsten or gold particles that pierce cell walls and membranes and enter intact cells without killing them. This approach was first reported in 1987 for the delivery of RNA or DNA into plant cells to circumvent the limited host range of A. tumefaciens and the regeneration problems of protoplast transformation in plants (Klein et al. 1987). Biolistic transformation has been as well successfully developed for the introduction of DNA into fungal spores or hyphae of several species including those that are difficult to culture or from which protoplasts are hard to prepare (reviewed in Li et al. 2017). This transformation method requires a special particle delivery equipment, such as the often used PDS-1000/He system sold by Bio-Rad Laboratories in which the DNA-coated particles are accelerated at high velocity under vacuum onto an agar plate containing the target cells, and that is positioned at a specified target distance in the bombardment chamber (Te’o and Nevalainen 2015). After bombardment, the plate is covered by an overlay medium supplemented with the appropriate selection chemical and incubated until transformants appear. In general, transformation frequencies are in the range of 10–100 transformants/μg DNA for filamentous fungi, whereby various factors, such as cell type and cell density, instrumental settings (particle type and size, vacuum and pressure levels, target distance) and the procedure used for coating the particles with DNA, affect the outcome. Furthermore, the osmotic composition of the medium during bombardment was reported to impact transformation yield in some cases with osmotic support having a positive influence on the efficient transformation of fungal cells (Sanford et al. 1993). Although biolistic transformation is simple and suitable for various cell types—even for the transformation of mitochondria and chloroplasts (Johnston et al. 1988)—it usually is only considered for fungi in which other transformation approaches have failed and is thus not very commonly used in these organisms, probably due to the special and expensive equipment needed. Nevertheless, the model N. crassa was the first filamentous fungus having been biolistically transformed. Armaleo et al. introduced a plasmid carrying the wild-type qa-2 gene into a N. crassa qa-2-deficient recipient strain, thereby restoring the catabolic competency for quinic acid by stable integration into the genome at both homologous and non-homologous sites (Armaleo et al. 1990). While no publications on the application of particle bombardment to A. niger or A. oryzae are available, attempts for the transformation of A. nidulans and A. giganteus have been made. Bombardment of A. nidulans conidia with DNA-coated tungsten particles of 0.5 μm diameter on sorbitol-containing media resulted in ~80 transformants/μg DNA (Fungaro et al. 1995), whereas biolistic transformation of germinated conidia of

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A. giganteus with 0.6 μm diameter tungsten particles without osmotic stabilisation turned out to be inappropriate, as only very few transformants were gained (Meyer et al. 2003). The first Trichoderma strains subject to biolistic transformation were T. atroviride P1 (formerly named T. harzianum) and T. virens G41 (formerly named Gliocladium virens) (Lorito et al. 1993). In this study, Trichoderma conidia were bombarded with DNA-coated tungsten particles of 1.07 μm diameter at a target distance of 6 cm. The yield turned out to be proportional to the conidial density with the highest rates of transformants emerging with 107 conidia per plate. Direct comparison with transformation of the same strains using the protoplast-mediated approach revealed significantly higher transformation rates and a higher genetic stability of the transformants upon particle bombardment (Lorito et al. 1993). Several years later, biolistic transformation of industrially relevant T. reesei strains was described for the first time (Hazell et al. 2000; Te’o et al. 2002). In both studies, intact conidia were transformed to hygromycin resistance using either the ‘standard’ PDS-1000/He equipment or a seven-barrel Hepta Adaptor system. With yields of up to 40 transformants/μg plasmid DNA, these studies resulted in considerably lower numbers than those previously reported for T. atroviride and T. virens but are probably attributed to the different settings and parameters used for T. reesei, such as tungsten particle diameters of 1.1 and 0.7 μm, respectively, and target distances of either 3 or 6 cm, making a direct comparison impossible.

3.5

Transformation Using Shock Waves

Shock wave-mediated transformation of filamentous fungi is a recent development first published by Magana-Ortiz and co-authors in 2013 (Magana-Ortiz et al. 2013). The mechanical waves that result from the sudden release of energy in a limited space cause a transient increase of cell membrane permeability allowing the uptake of macromolecules such as DNA (Lauer et al. 1997). The exposure of spore suspensions of the four test fungi (A. niger, T. reesei, F. oxysporum, Phanerochaete chrysosporium) in the presence of plasmids harbouring the hph gene to underwater shock waves resulted in the formation of hygromycin-resistant colonies (Magana-Ortiz et al. 2013). As expected, the transformation frequency per μg of DNA differed for the four tested fungi, which may be due to differences in cell wall composition, and in general was very low. This was explained by the authors to be due to the damage of a high percentage of DNA during the procedure, especially when 300 or more shock waves were employed, because conidial viability was only reduced by one order of magnitude by the treatment. When comparing the number of obtained transformants per number of utilised cells, however, the transformation frequency was several orders of magnitude higher compared with published protocols. For A. niger, transformation frequencies of 1.6  104 to 2.7  104 per 107 conidia were achieved, which was 280 and 5400 times higher than with protoplast-mediated transformation and AMT, respectively. Shock wave transformation of T. reesei resulted in 8  104 to 12  104

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transformants per 107 conidia which is an up to 100-fold improvement compared to AMT, a 500-fold increase compared to protoplast-mediated transformation and an increase of four orders of magnitude compared with the biolistic method (MaganaOrtiz et al. 2013). In their article, the authors mentioned several advantages of shock wave transformation such as the direct use of fungal spores as recipient cells, the high reproducibility and ease of use of the method including the fact that the same shock wave frequency, energy, voltage and number can be used to transform different fungal species. Until now, the shock wave approach has not yet been employed as a routine for additional fungal species which may be due to the fact that shock wave sources and instruments are expensive and not among the equipment of standard microbiology or molecular biology laboratories.

4 Strategies to Facilitate and Further Improve Fungal Transformations Independent of the chosen method, the genetic transformation of filamentous fungi generally comprises a complex sequence of experimental steps. Hence, any means to simplify and shorten the protocol allows a quicker and in many cases more efficient performance of the approach.

4.1

Stock-Keeping of Competent Cells

A significant mitigation is the cryo-stockage of competent cells since (1) the often laborious preparation of competent cells, such as protoplasts, can be batch-processed to provide a huge number of cell aliquots for future experiments saving otherwise repeatedly required preparations; (2) this furthermore ensures consistently competent cells and allows to compare yields obtained with different DNAs or recipient strains and (3) allows to spontaneously perform a transformation without having to wait for several days of pre-cultivation. The simplest option is to shock-freeze freshly harvested and washed (1 M sorbitol) conidia in liquid nitrogen and store the cells at 80
C, as, for example, standardly used for N. crassa (NavarroSampedro et al. 2002). Next best option would be to use a swelling procedure (Sect. 2.2) and store the swollen yet ungerminated cells after shock-freezing in liquid nitrogen in 50% glycerol at 80
C. The same is possible with germinated cells; however, the recovery rate of viable cells is usually somewhat decreased. Similarly, sphero- or protoplasted cells can be stored at ultra-low temperatures. This approach has already been used by Volmer and Yanofsky in 1986, who found that cryo-stored spheroplasts of N. crassa remained viable for at least 6 months (Vollmer and Yanofsky 1986). It also has been described early on that certain additives, such as

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DMSO, heparin, PEG, MOPS or CaCl2, can extend viability of sensitive cells, such as germlings or protoplasts, after even longer storage times (Nargang and Nargang 1996). Instead of shock-freezing in liquid nitrogen, controlled freezing of cell aliquots in tubes in isopropanol at 80
C, for instance, using a ‘Mr. Frosty’ freezing container (Nalgene), can increase regeneration efficiency by up to 40%, as reported for A. niger (de Bekker et al. 2009; de Bekker 2011). Unfortunately, for Trichoderma spp., a method for protoplast cryo-storage that maintains reasonable viability has so far not been described. Penttilä et al. reported the drastic reduction of transformation efficiency of cryo-stored protoplasts of T. reesei and recommended to prepare them freshly before every experiment (Penttilä et al. 1987). A reevaluation of this approach seems timely. Whatever the preparation method and cell type used, it is generally advisable to use up cryo-stored competent cells within 2 years after their preparation and by all means avoid thawing/refreezing cycles by preparing small aliquots of 50–100 μL suitable for one transformation each in order to maintain viability and genetic stability.

4.2

Improving Homologous Recombination

Due to the interest in decreasing non-homologous recombination for, e.g. the generation of single gene deletion knockout strains, mutants defective in the NHEJ pathway were generated for several fungal species. NHEJ involves several proteins including the Ku70/Ku80 complex, which binds to the broken DNA ends, and the DNA ligase IV-Xrcc4 complex, which seals the break (Dudásová et al. 2004). Deletion of either mus-51 or mus-52, which code for the Ku70 and Ku80 proteins, significantly increased the gene targeting frequency in N. crassa (Ninomiya et al. 2004), and deletion of the DNA ligase IV-encoding gene mus-53 even led to a 100% gene targeting efficiency (Ishibashi et al. 2006). Similar results were obtained for Aspergillus spp. (Krappmann et al. 2006; Nayak et al. 2006; Takahashi et al. 2006). In A. niger, deletion of the ku70/kusA gene resulted in an increase in HR efficiency to above 90% (Meyer et al. 2007), while a 100% efficiency could be obtained in ku70/ ku80 double deletion mutants (Zhang et al. 2011). In T. reesei, a ku70 deletion mutant showed an increase in the gene replacement efficiency of up to 95% (Guangtao et al. 2009), and deletion of mus53 enhanced the HR frequency to levels between 60 and 100% using homologous flanking regions of 500 to 1500 bp (Steiger et al. 2011). For T. atroviride, several attempts to generate ku70-deficient mutants failed, while a Δku80 strain could be obtained (S. Gruber, S. Zeilinger, unpublished). This, however, showed enhanced sensitivity to oxidative stress, UV radiation and the DNA-damaging agent phleomycin (S. Zeilinger, R. Segreto, unpublished), rendering it highly impractical as recipient strain. Similar side effects resulting from NHEJ deficiency were reported for N. crassa, T. reesei and A. niger where respective deletion strains were more sensitive to chemicals such as phleomycin, bleomycin and methyl/ethyl methanesulfonate, as well as to X-ray and UV exposure compared to the wild types (Guangtao et al. 2009; Ninomiya et al. 2004; Pel et al. 2007). To

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compensate for the higher mutation rates, it is advisable to eliminate the Δku70/ku80 deletions in resulting transformants through backcrossing with the wild type. This, however, is only possible if the sexual cycle of the used species is known and experimentally accessible in a reasonable time. This does apply to N. crassa (Colot et al. 2006) and since recently—with some restrictions—to T. reesei (Linke et al. 2015); however, outcrossing is so far no practical option for T. atroviride, A. niger and A. oryzae.

4.3

Pharmacological Inhibition of NHEJ

An alternative approach to the genetic inhibition of NHEJ is its pharmacological suppression. The major advantages of this approach are that it can be performed independent of the strain background and that the inhibitory effect lasts only as long as the suppressing chemical is present. As mentioned above already, besides Ku70 and Ku80 homologs, which bind to and recruit the catalytic DNA-dependent protein kinase subunits (DNA-PKcs) to the DSB ends (Davis et al. 2014), DNA ligases are other crucial enzymes responsible for sealing DSBs during NHEJ (Ellenberger and Tomkinson 2008). SCR7 (short for Sathees Chukkurumbal Raghavan 7) is a small molecule (5,6-bis(benzylideneamino)-2-mercapto-pyrimidin-4-ol) that specifically inhibits DNA ligase IV-dependent NHEJ (Srivastava et al. 2012) and thus forces the cell to repair DSBs by HR. Notably, in contrast to the originally reported structure of SCR7(A), the active compound is in fact the derivative structure SCR7 pyrazine (2,3-dihydro-6,7-diphenyl-2-thioxo-4(1H)-pteridinone, also referred to as SCR7-G/-X) (Greco et al. 2016; TOCRIS 2018). Interestingly, SCR7 pyrazine not only suppresses NHEJ when applied at high concentrations (50–200 μM) but has also been shown to efficiently enhance CRISPR/Cas9-mediated homology-directed repair (see Sect. 4.5) up to 19-fold when used at low concentrations (1 μM) (Maruyama et al. 2015), however most likely via different modes of actions. Qin et al. (2017) used 50 μM SCR7 (pyrazine) to facilitate targeted transformation of an N. crassa wild-type strain by HR (Qin et al. 2017), and we now routinely apply SCR7 pyrazine during the recovery phase of electrotransformed Trichoderma cells with very good results (Lichius et al., unpublished).

4.4

Split-Marker Systems to Increase the Chances of Targeted Integration

Due to the low frequency of HR in the majority of filamentous model fungi, the screening for transformants with the desired targeted integration among the majority of those that bear the transforming DNA ectopically frequently resembles a search for a needle in a haystack. One possibility to overcome the time-consuming

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screening procedure is to use the split-marker approach, which was initially developed for S. cerevisiae (Fairhead et al. 1996). This approach is based on the generation and transformation of two partially overlapping DNA fragments by PCR, each of which bears part of a selectable marker gene followed by sequence stretches homologous to the target locus. The formation of a functional selection marker requires an additional crossing over event (plus the two usual ones) in order to integrate the transforming DNA at the target locus (Fig. 5.1). Thereby, the frequency of correctly targeted deletion constructs is increased because only transformants in which the two overlapping marker fragments have successfully recombined will grow on selection medium (Catlett et al. 2003). In general, resistance as well as auxotrophic genes can be used as split markers (Kück and Hoff 2010); however, it has to be considered that none of the two marker fragments alone is able to confer resistance or prototrophy. The split-marker approach was successfully applied in various fungi including N. crassa (Colot et al. 2006), A. niger (Arentshorst et al. 2015) and T. reesei (Derntl et al. 2015). We recently successfully used this technique for the generation of gene deletion mutants in T. atroviride, employing the hph gene as split marker which was flanked by ~1500 bp regions mediating integration at the target locus (Atanasova et al. 2018). Compared to PEG-mediated transformation of protoplasts with a complete gene deletion cassette, the split-marker approach resulted in a considerably lower number of transformants, of which however the majority showed the desired gene deletion event.

4.5

Genome Editing Tools

Despite enormous methodological progress in constructing gene replacement cassettes and the usefulness of NHEJ-deficient strains, mutant generation in fungi remains laborious due to inefficient homologous recombination on which classic genetic manipulation techniques are often based on (Meyer et al. 2016; Shapiro et al. 2018). Besides the attempt to introduce precise genetic changes to specific locations in the genome in order to analyse genotype-phenotype relationships in functional genomic studies and to generate strains with desired phenotypes, random integration can be applied for mutagenesis. Restriction enzyme-mediated integration (REMI) represents a method for generating insertional mutants in fungi, whereby the transforming DNA, usually a plasmid, is integrating randomly in a non-homologous manner into the chromosome by the aid of a restriction enzyme. The enzyme-digested transforming DNA is transformed along with the respective restriction enzyme using protoplast- or electroporation-mediated transformation which is suggested to result in the generation of complementary ends of the transforming and the genomic DNA that are then ligated in vivo to generate a non-homologous integration event (Olmedo-Monfil et al. 2004). The gene/DNA region mutated by the integration subsequently can be identified by recovery of the transforming plasmid plus flanking sequences (Riggle and Kumamoto 1998).

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Applications of REMI mutagenesis include the generation of T. atroviride mutants with improved capability of degrading the pesticide dichlorvos (Tang et al. 2009), the generation of A. oryzae strains with improved heterologous protein production (Yaver et al. 2000) and the generation of morphological mutants in A. niger by promoter-tagged REMI, where a plasmid containing a strong transcriptional promoter was used to activate gene transcription (Shuster and Bindel Connelley 1999). A variant of the REMI approach, named SEMI gene targeting, has been described for T. reesei (Ouedraogo et al. 2016). SEMI stands for I-SceI enzyme-mediated integration and is based on the expression of the S. cerevisiae I-SceI meganuclease resulting in a DSB at a predetermined site in a strain with an engineered I-SceI restriction site in its genome, which stimulates transformation frequency as well as homologous recombination. By combining SEMI with NHEJ deficiency, homologous recombination efficiencies of 90–100% could be obtained in T. reesei (Ouedraogo et al. 2016). Further, recent genome editing technologies allow to introduce genome modifications at specific target loci. These tools are based on site-specific DNA DSBs that are brought about by engineered endonucleases and their repair by the cells’ intrinsic mechanisms. The kind of repair mechanism used is crucial for the outcome of genome editing. The error-prone NHEJ pathway thereby results in point mutations, small deletions or frameshifts that can lead to a complete loss of gene function (Fig. 5.1). Homology-directed repair, on the other hand, requires a repair template homologous to the target site. By supplying an exogenous repair template with desired modifications, base changes or gene replacement at the target locus can be achieved (Hille and Charpentier 2016; Zhang et al. 2017). The most commonly used endonucleases for genome editing are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-associated protein 9 (Cas9). While ZNFs and TALENs are first- and second-generation genome editing tools, respectively, that are not routinely used in filamentous fungi, CRISPR-Cas9 is regarded as the third-generation tool that recently started its triumphal march also in the fungal research field (Shi et al. 2017). Components of the CRISPR-Cas9 System and Its Variants CRISPR, which stands for clustered regularly interspaced short palindromic repeats, together with the Cas proteins, constitutes an adaptive immune system in prokaryotes that preserves memories of prior infections by integrating short segments of foreign DNA, so-called spacers, at the CRISPR locus (Sternberg et al. 2016). The Cas9 protein originates from bacterial type II CRISPR-Cas systems and is an endonuclease that, guided by a specific RNA sequence, introduces site-specific DSBs into DNA targets (Doudna and Charpentier 2014). For genome editing purposes, a simple two-component system with a single guide RNA (sgRNA) that determines the DNA target site and that binds Cas9 is used. Alterations in the guide sequence of the sgRNA direct Cas9 to any DNA target sequence of interest given that a protospacer adjacent motif (PAM), a 2–6 base pair sequence adjacent to the DNA sequence targeted by Cas9, is present in the DNA. These few requirements make CRISPR-Cas an easy-to-use and cost-effective system suitable for the precise and efficient targeting, editing, modification, regulation and marking of genomic loci.

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CRISPR-Cas9 is not limited to manipulations at single loci, but by simultaneously expressing multiple guide RNAs, the system even allows multiplexed genome editing thereby leading to the generation of multiple gene knockouts in a single experiment (Doudna and Charpentier 2014). Beyond gene editing, nucleasedead Cas9 (dCas9), a catalytically deactivated Cas9 variant unable to cleave DNA, can be used for regulating the expression of a target gene by fusing dCas9 to a transcriptional repressor or activator (Doudna and Charpentier 2014). The method has also been developed towards epigenome engineering in mammalian cells by fusing dCas9 with various DNA effectors or histone methylases and acetylases for introducing site-specific DNA methylation or histone modifications (Hilton et al. 2015; Laufer and Singh 2015). Tagging of dCas9 with fluorescent proteins (FPs) can furthermore be used as an alternative to fluorescence in situ hybridisation (FISH) for the detection of desired genomic loci to which the dCas9-FP hybrid is directed by a respective sgRNA (Chen et al. 2013; Xu and Qi 2018). The CRISPR-Cas9 method has turned out as promising tool also for synthetic biology approaches such as directed evolution of biomolecules. A multiplex CRISPR-Cas9 system named CRISPRm that uses multiple ribozyme-protected single guide RNAs has been presented for industrial S. cerevisiae strains thereby allowing to integrate DNA libraries into the yeast genome for engineering proteins for improved metabolic activity (Ryan and Cate 2014). CRISPR-Cas-Mediated Engineering of Filamentous Fungi Altogether, the CRISPR-Cas system offers a plenitude of possibilities for fungal genetic manipulation as it is relatively simple to use, cost-effective and multiplexable. The possibility to simultaneously target multiple genomic loci is a big advantage especially with fungi for which in most cases only a limited set of functional selection markers is available (see Sect. 2.6). Guide RNA design and hence targeting are highly flexible with the exception that a PAM motif is required that could limit target site selection. What however remains a prerequisite is the efficient delivery of the CRISPR-Cas system into the target cells; hence, its use remains limited to organisms for which effective transformation tools exist. Delivery methods include the transformation of plasmids or ribonucleoprotein particles (RNPs) that comprise pre-assembled Cas9sgRNA complexes, as well as delivery of the genetically encoded components via AMT (Shapiro et al. 2018). CRISPR-Cas technology has been applied for genetic engineering of various filamentous fungi including industrially relevant species as well as pathogens (Nodvig et al. 2015; Shi et al. 2017). In the model species N. crassa, both Cas9 and sgRNA constructs were introduced into fungal conidia by electroporation together with donor plasmids in order to replace the endogenous clr-2 with the β-tubulin promoter. Direct comparison of the CRISPR-Cas9 system and usage of a mus-51-deficient strain revealed similar homologous recombination efficiencies (Matsu-Ura et al. 2015). In 2015, CRISPR-Cas was as well applied to T. reesei using a two-step approach. First, a cas9-expressing strain was generated which was then transformed with in vitro transcribed sgRNAs (Liu et al. 2015). In this study, the ura5 gene was targeted, and all transformants analysed showed the desired frameshift mutations leading to gene inactivation. In addition, by co-transforming

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sgRNA targeting the lae1 gene with a DNA fragment containing a selectable marker and ~600-bp 50 and 30 flanking regions of lae1, Δlae1 mutants could be generated by HR with an almost 100% frequency. The authors further reported successful Cas9mediated simultaneous HR of multiple genes with frequencies of 45% for double and 4% for triple recombination events (Liu et al. 2015). Based on this study, we aimed at transferring the CRISPR-Cas9 system established in T. reesei to T. atroviride. However, when using the cas9-expressing plasmids and the protocol kindly provided by Gen Zou together with sgRNAs targeted to T. atroviride ura5, all transformants, although emerging on selective media, had unaltered ura5 gene sequences (A. Resnyak, S. Zeilinger, unpublished). While the Trichoderma studies applied in vitro transcribed sgRNAs, a ribozymebased strategy for the release of the sgRNA was successfully implemented in six different Aspergillus species including A. niger as well as A. brasiliensis, a species that has not previously been genetically engineered (Nodvig et al. 2015). This system was based on a single plasmid that contains the genes encoding codon-optimised Cas9 and a single sgRNA flanked by ribozymes together with the AMA1 sequence responsible for supporting plasmid replication but ensuring that the plasmid is rapidly lost without selection to minimise putative off-target effects. Applying this system for targeting the conidial pigmentation-related gene albA of A. niger resulted in indel mutations ranging from 7 to 83 base pairs at the Cas9 cleavage site indicating errorprone repair by NHEJ. Comparative experiments in addition showed that the efficiency of mutagenesis in the different Aspergillus species varied considerably. Reasons for this variation were suggested to include different expressions of cas9 and sgRNA genes, different propagation stabilities of the AMA1-based plasmid, Cas9 codon optimisation not being optimal for all tested species and differences in the efficiency of accurate NHEJ repair in the individual species (Nodvig et al. 2015). Recently, the same group reported on several improvements of their CRISPR-Cas9 system such as the use of a polymerase III promoter for sgRNA production and the development of CRISPR-tRNA vectors that allow the splicing of multiple different sgRNAs out of a common transcript thereby paving the way for multiplex experiments. In addition, single-stranded oligonucleotides were directly used to efficiently introduce specific point mutations and gene deletions in Aspergilli showing that there is no need for complex dsDNA gene targeting substrates (Nodvig et al. 2018). Hitherto published applications of the CRISPR-Cas technology in A. niger are focused on metabolic engineering such as generating a strain for the efficient production of galactaric acid by elimination of its catabolism and developing a toolbox for the generation of strains carrying heterologous expression cassettes at a defined genetic locus (Kuivanen et al. 2016; Sarkari et al. 2017). In A. oryzae, the CRISPR-Cas system was successfully implemented for gene inactivation not only in the sequenced strain RIB40 but also in industrial strains used for the production of sake and soy sauce, although with quite low efficiency of only 10–20% (Katayama et al. 2016; Nakamura et al. 2017). CRISPR-Cas Pitfalls and Future As indicated by the mentioned studies, CRISPR-Cas technology bears a great potential for researchers working with filamentous fungi. However, it also bears several pitfalls. These include the fact that

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CRISPR-Cas9-mediated mutations are less defined than those originating from gene replacement approaches, the heterogeneity of fungal genomes within a species jeopardising the use of common sgRNAs and the requirement of PAM sites that may limit comprehensive applicability (Idnurm and Meyer 2018; Krappmann 2017; Peng et al. 2016). In no time, CRISPR-Cas has gained enormous importance in genome editing, and a recent analysis revealed that its use in fungal systems is on an exponential rise (Idnurm and Meyer 2018). The prospects of this system for filamentous fungi comprise the efficient generation of genome-wide single gene deletion libraries for species with sequenced genomes; the activation of silent gene clusters for, e.g. secondary metabolite production by using dCas9 variants in the process of searching for novel bioactive substances; the high-throughput genetic engineering by multiplex approaches; and the generation of minimal genomes for industrial fungal cell factories (Idnurm and Meyer 2018; Krappmann 2017; Peng et al. 2016).

5 Concluding Remarks and Outlook Several techniques are well established for fungal transformation nowadays and are constantly being improved and successfully adapted to an increasing number of species. Genome sequencing combined with genome manipulation has been used to design fungal strains for the production of homologous and heterologous proteins as well as metabolites. In this way, the development of fungal genetic transformation over the last 30 years contributed to groundbreaking discoveries and applications in fundamental research, industry and pharmacology. Even though considerable information regarding theoretical and practical aspects of fungal transformation methods are widely available, the genetic manipulation of many fungi still faces major challenges and is all but trivial. Some of these obstacles may be overcome by a better understanding of fungal biology, especially through deeper insights into the molecular details that underlie transformation processes but also their impact at the organismic level, such as pleiotropic ‘off-target’ effects of single gene deletion. Despite the triumphant procession of genome editing tools such as CRISPRCas9, their application to filamentous fungi is still at an early stage. Only in combination with efficient transformation approaches will these tools contribute to the further exploitation of these powerful organisms. Acknowledgements We would like to thank our former BSc/MSc students Markus Delitz and Linda Salzmann for providing valuable background data on electroporation for this manuscript. The Tyrolean Science Fund (Tiroler Wissenschaftförderung, TWF; grant TWF-256524 to AL) and the Austrian Science Fund (FWF; grant P28248-B22 to SZ) are acknowledged for funding.

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Chapter 6

Bottlenecks and Future Outlooks for High-Throughput Technologies for Filamentous Fungi Kyle Rothschild-Mancinelli, Susanne M. Germann, and Mikael R. Andersen

1 Introduction 1.1

Why Do We Need HTP Technologies for Filamentous Fungi

In an age of efficiency, the ability to parallely execute many processes has allowed us to make advances at a speed dwarfing previous rates. This need for parallel activities has spread across society from farming to engineering to biology. Our previous work in a low-throughput lab bench scale, while still valuable in some aspects, has outlived its use as the main driver of bio-industry and advances in genetic engineering and synthetic biology. As an example, one could consider the case of wanting to knock out 150 genes. If a researcher were to do this traditionally, it would take both a lot of time as well as having 150(+) tubes, risking many mistakes as the same process gets repeated over and over. But with two 96-well plates, the same 150 knockouts could easily be run in parallel along with ample room for controls. This would take the same time as one two-tube run, yet 148 more results would be achieved at the same time, with substantial limitation of the risk of mistakes from repetitive processes. (See Fig. 6.1 for a conceptual sketch of molecular biology). While significant progress has been made developing and validating highthroughput processes for bacteria, yeast, and some mammalian cell lines, there has been a significant time lag before their adaptation to filamentous fungi (Alberto et al. 2009; Baryshnikova et al. 2010; Brouzes et al. 2009; Jai et al. 2018). Filamentous fungi are important because of their overwhelming use and massive future potential in industry; for example, nearly all of the world’s citric acid is produced by the

K. Rothschild-Mancinelli · S. M. Germann · M. R. Andersen (*) Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_6

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Fig. 6.1 The general molecular biology workflow starting from DNA synthesis ending in product screening. (A) Here, DNA is shown amplified after PCR has been performed. (B) Next, the amplified DNA is assembled into vectors or plasmids. (C) The plasmids are transformed into the host cells and grown up. (D) The correct transformants are finally assayed for their production capabilities

fungus Aspergillus niger alone due to its 92% theoretical max yield production capabilities (Karaffa and Kubicek 2019). In addition to the importance of filamentous fungi in the industrial sector, they are also dangerous pathogens, and cause thousands of deaths a year (Brown et al. 2012). There are many reasons why filamentous fungi have lagged behind the rest of their sister host organisms, which will be outlined later in the chapter. It is important to recognize the need for the development of high-throughput processes, the current work that is being done to set up these processes, and what the future holds once we have a validated functional filamentous fungal high-throughput process in place.

1.1.1

Why Are High-Throughput Technologies Important for Academic Use?

While academic work may be narrow in scope, considerable amount of labour is required to achieve results. For example, the prospect of knocking out and looking at the effects of one 10-gene secondary metabolite cluster in fungi can require up to 3,628,800 (10 factorial) different parallel knockouts to best understand its effects. To perform such an array of experiments without errors is neither feasible nor humanly possible. Most examples following the design-build-test-analyze cycle (Fig. 6.2) will suffer from similar problems. There are many examples of high-throughput technologies being developed and applied in academic settings. One of the largest uses of high-throughput technologies in academia is in the sequencing and assembly of DNA parts and vectors. The push to sequence the human genome by Craig Venter set off a chain reaction of exploration into high-throughput sequencing and assembly of these reads, and to this day, it still remains a gold standard for the testing of new sequencing techniques (Wheeler et al. 2008; Rothberg et al. 2011; Pendleton et al. 2015). One of the newest sequencing technologies uses ultra-long reads (up to 882 kb) to assemble the human genome at above a 99.8% accuracy (Jai et al. 2018). There has already been some progress toward screening filamentous fungi using high-throughput technologies. These include the work done in France on nanoliterscale droplet-based fungal separation and screening, which is important for

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Fig. 6.2 The design-buildtest-analyze (DBTA) cycle which is a critical part of experimental science in both industry and academia. First, the experiment/strain/ DNA is designed, then the designs are built and transformed, then the new strains are tested, and finally the results are analyzed, which leads to the next round of the cycle. This cycle and its individual iterations are critical to highthroughput processes staying stable and organized

quantification and detection of secreted enzymes without large amounts (milliliter to liter scale) of growth media (Beneyton et al. 2016, 2017). This kind of development and growth of high-throughput techniques in fungi is essential for the progression of the field.

2 Filamentous Fungal Molecular Biology: Where Are We Now Despite slower than the more mature systems developed for E. coli and S. cerevisiae, filamentous fungi have experienced great growth in building massively paralleled molecular processes such as mutagenesis development, basic screening methods (Beneyton et al. 2016) and the beginnings of single-spore flow cytometry and sorting as shown with Aspergillus nidulans (Delgado-Ramos et al. 2014). Additionally, various knockout libraries have been built using semi-high-throughput methods (e.g. in Neurospora crassa), which have started to bridge the gap between low-throughput genetic manipulation and high-throughput screening (Colot et al. 2006).

2.1

Morphological Challenges

Aside from some slight genetic differences, it is mainly the morphology that sets filamentous fungi apart from other cellular chassis for high-throughput automation. Filamentous fungi can grow as pellets, or as filaments, and make spores that are very

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Fig. 6.3 Issues encountered with picking fungal colonies. (a) This photo shows a plate of three fungal colonies. Note four things: first, the colonies have grown together; second, the irregular shaping of the colonies; thirdly, the fuzziness of the mycelia at the edge of the colonies; and lastly, the discoloration due to sporulation of the colonies. (b) This is a close-up representation of one of the colored “dots” in part (a). Each conidiophore is made up of multiple strings of spores that are easily dislodged from the string and airborne

hardy and easily airborne (Fig. 6.3). Some issues concerning filamentous fungi have not been tackled with other organisms that have been adapted to high-throughput screening, mainly their morphological differences from other chassis. These include (but are not limited to): Larger more spread out plated colonies with easily airborne spores—making colony picking hard due to identification and contamination risks Nonhomogenous growth in liquid media due to mycelia—making liquid handling hard due to filamentous fungal pellets clogging pipette tips The potential for heterokaryons—making screening hard due to difficulty in clearly isolating an isogenic strain Above all else is the constant concern of contamination. Filamentous fungi are common lab contaminants, and that has sown fear into many lab managers, making people apprehensive of the risk of exposing their robotics to fungi. While it should be emphasized that the risk of contamination is not null, if procedures are carefully set in place to clean the equipment and lab spaces after working with filamentous fungi, then contamination should be mostly negated. However, this is something that needs to be addressed. Later in this chapter, processes of sterilization pre- and postexperiment will be discussed in further detail.

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Colony Picking

If we take the three categories previously listed (colony picking, liquid handling, and screening), all have advanced in a similar rate (Beneyton et al. 2016). Even tough, it may appear that liquid handling may have made the most progress towards a defined process, a counterargument could be made that colony picking is most advanced and can be performed largely using existing equipment. Although sporulation is an issue with contamination of both the samples and equipment when picking with a machine, it can be overcome by picking before sporulation (although this is not a fix for heterokaryons). This requires having a defined fungal spot on agar that can be detected by imaging software. Additionally, various robots currently have picking heads dedicated to fungi and filamentous yeast (Qpix, Molecular Devices). This means that we are at the stage of at least being able to pick isolated fungal colonies from plates. There are still issues of colonies growing together, and the size of the colonies, when trying to scale down from a single petri dish to something like a 48-well large cultivation tray (see Fig. 6.3), but these are minor issues compared with the first stage of making fungal plating and picking a reality.

2.1.2

Liquid Handling

Liquid handling for filamentous fungi has its own inherent challenges. While spore suspensions are easy to work with and require no changes to what works with other organisms, once the cells start to grow, then the issues begin. An inherent trait of filamentous fungi is filamentous growth. These filaments or mycelia cause the fungi to grow in varying sized clumps in liquid, which make pipetting nearly impossible depending on the size and stickiness of the clumps. Yet even if the liquid can be taken up in the pipette tips, it makes getting a truly representative sample a challenge in small volumes as there could be a clump in one sample and not in the other. Additionally, even while growing in liquid, sporulation is still a frequent occurrence, which can lead to contamination issues within the robot. Finally, consistently growing various filamentous fungi in microtiter plates at 96-well volumes as a workable downscaling of reflecting large industrial platforms has yet to be completely documented.

2.1.3

Screening

Screening may be regarded by some as the easiest step to perform, but it could also be the hardest depending on which assay you want to implement. Traditional screening methods such as spore color identification and color-based enzyme assays can become very difficult for a variety of reasons including the potential for a spore contamination event. There is a need to develop a very sophisticated pipeline to visually distinguish vast numbers of colors (some of which may be growing into

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each other), and then score them as well as track on the plate so that each different colony is located. The gold standard in the screening might be sequencing, but there are at least two drawbacks to this. First is cost. Sequencing (be it Sanger or nextgeneration sequencing) is expensive on a large scale, which might be acceptable for companies with a budget dedicated to it, but it is not sustainable in academia where funds are more limited.

2.1.4

Heterokaryons

Another issue that filamentous fungi have, which is not present in other organisms already adapted to high-throughput technologies, is potential formation of heterokaryons which occurs when multiple nuclei of different genotypes are present in one fungus (Roper et al. 2011). This presents a clear problem for screening and isolating a single genotype because when carrying over a colony into successive experiments, it needs to have only one genotype or else the results are essentially the same as those arising from a contaminated experiment. Of course, there is one advantage of analyzing heterokaryons and not avoiding them completely, that is, they can be used to identify essential genes via knockout approaches (Arentshorst et al. 2012).

3 State of the Art 3.1

State-of-the-Art Current High-Throughput Technologies for Other Organisms

In both academia and industry, high-throughput platforms have become increasingly vital to stay competitive within fast and robust data acquisition, strain development, and production environments. Accordingly, applications using high-throughput processes to replace laborious tube-based or single-plate formats are consistently taking over. Especially with regard to cultivation, screening, and liquid handling, there are some very convenient and robust new methods available. However, most of these methods are designed for single cells growing in suspension, such as yeast, bacteria, and mammalian cells. Screening of large numbers of organisms is a big bottleneck in the development and implementation of high-throughput processes, but in certain organisms, there has been significant progress made. One of the most prominent high-throughput tools for strain development available in yeast is collections for genome-wide screens of various kinds, such as the yeast deletion collection (YKO) (Giaever and Nislow 2014) and spinoff technologies, such as synthetic genetic array (Baryshnikova et al. 2010; Kuzmin et al. 2014) and HIPHOP chemogenomics (Lee et al. 2014). These libraries allow the user to screen hundreds or more yeast

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cultures at a time and accurately output the data so that statistical packages can be applied for visualization. One of the most impressive current high-throughput data processes using mammalian cells is the Human Protein Atlas (HPA). It is a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarraybased immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level (Uhlen et al. 2010, 2015). This initiative is supremely important for cell factories and screening purposes because one of the many screens for protein engineering is cellular localization, and being able to detect in a precise way, the single-cell localization of proteins in vast numbers of samples is applicable across organismal hosts. In continuing to develop tools in mammalian cells that are applicable in other organisms, data sets from Uhlen 2015 and 2010 (mentioned above) were used in the cloning of >60,000 unique human gene fragments into expression vectors by novel solid-phase cloning (SPC) schemes where streptavidin-coated paramagnetic beads were used as solid support, allowing for automated assemblies of single and multiple DNA parts (Lundqvist et al. 2015). Furthermore, the current commercial highthroughput/high-content screening robotic systems are highly popular in medical research as well since they can be readily programmed to perform and analyze robotics-optimized cytogenetic assays (Repin et al. 2017). To assemble DNA is a time-consuming and labor-intensive process that creates bottlenecks regardless of the organism but is still necessary to build integration vectors. The lessons learned from developing these three high-throughput processes can be applied to how novel screening methods should be developed for fungi. Additionally, when the time comes to adapt the same or similar processes to fungi, the ground work will have already been laid making it easier. As screening is so variable, new high-throughput methods to replace traditional screening assays (such as manual documentation of numbers, colors, or other outputs) are becoming more and more popular, such as multiparameter fluorescenceactivated cell sorting (FACS) for antibody selection which replaces traditional yeast-display screening assays (Schröter et al. 2018) or the 96-well plate-PCHPLC (96-PH) for the rapid identification of high-yield L-valine strains (Huang et al. 2018). These are important because they allow parts of traditional screening methods (such as fluorescence) to be integrated into high-throughput processes. The addition of sorting takes the next step to automate isolation of positive targets from the negative, which would be difficult to do mistake-free by hand in a highthroughput environment. Also, companies are on the forefront of using highthroughput flow cytometry (HTFC) platforms for small-molecule high-throughput screening (HTS), structure-activity relationship (SAR) and phenotypic screening, and antibody screening (Ding et al. 2018). Lastly, sequencing is an important step in any design-build-test-analyze cycle with DNA parts. Next-generation sequencing (NGS) platforms have tremendously increased the amount of sequence data available (Hui 2012). Furthermore, the scale of the number of samples handled together has increased from single preparations to

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96-plex or 384-plex sequencing setups. The new challenges are the bioinformatics and statistical analysis of the acquired data, such as 16S ribosomal RNA (rRNA) and gene high-throughput sequencing (HTS) data to e.g. identify the members of microbiome populations (Ju and Zhang 2015). High-throughput automated fermentation systems are also becoming more and more relevant for clone selection and bioprocess optimization (Velez-Suberbie et al. 2018). The main challenge in adapting any of these HTP methods to filamentous fungi is the lack of resources in a relatively small scientific community, with major stakeholders mainly interested in how to tackle undesired fungal infestations and infections (Meyer et al. 2016). From the technical point of view, the phenotype of the filamentous fungus is a major issue. The apical growth of the hyphae after spore germination forming an expanding branched mycelia network (Brand and Gow 2009) creates severe difficulties when trying to adapt assays that, for example, measure growth by measuring optical density (OD). Spore dispersal causing cross contamination represents an added challenge in strain development. These phenotypic hallmarks make it difficult to implement the known multi-well high-throughput methods directly on filamentous fungi, and major design changes are needed to make liquid handling and fungal strain cultivation to work (Alberto et al. 2009; Beneyton et al. 2016).

3.1.1

High-Throughput Versus High-Output

When designing an experiment for high-throughput screening, it is important to consider the desired output. This differentiation between throughput and output is critical to truly understand the concept of a high-throughput approach. Highthroughput refers to the movement of a large number of samples through a process (the number may increase as a result of the process, but the starting point must also be high), whereas high-output is when there are a large number of data pointing at the end of the process, yet it may have started with a small sample size (e.g., screening transformations may result in 5x or more increase in output). Ideally, a lower number of input samples will create a relevant output result, leading to the desired strain/measurement/data. Therefore, screen design and educated guesses become highly relevant in order to avoid unproportionally high-sample numbers or even worse, a large fraction of the experimental samples yielding irrelevant results.

3.1.2

High-Throughput Screening (HTS) Versus High-Content Screening (HCS)

Understanding how to utilize high-throughput and high-content screens effectively can change the course of a project in terms of both the quantity of samples run and quality of data analyzed. High-throughput screening methods are usually automated versions of assays that can be as varied as compound detection in media to

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organismal phenotypic changes (Schröter et al. 2018; Beneyton et al. 2016). This is in contrast to high-content screening which has a focus in using image analysis and microscopy to detect intracellular compounds and other changes in cellular expression phenotypes, which is particularly applicable in therapeutics studies addressing the cellular localization and target areas of proteins (Uhlen et al. 2015). In drug-discovery studies, the development and implementation of high-throughput and high-content assays have been crucial for the discovery of therapeutics. Whereas high-throughput screening (HTS) of small molecules allows the rapid interrogation of the effects of up to hundreds of thousands of small molecules in a variety of in vitro and cell-based assays, high-content screening (HCS) approaches might sacrifice some of these high-throughput capabilities in return for biological and phenotypic complexity in the assay endpoints used (Varma et al. 2011).

4 Data Management and Analysis in High-Throughput Processes The new term for this type of data resulting from high-throughput operations is “big data.” Although this term is slightly misleading (the data itself is not big, but rather the amount of data produced is), the data management and analysis in highthroughput processes run akin to their sister workflows in a lower-throughput setting. Many might disagree with this as tools have had to be adjusted to handle big data workflows, but to think of them as different will only further isolate these low- and high-throughput workflows from one another instead of letting them build on each other. There are some differences, mainly when working with any highthroughput process producing data. The first thing that is apparent is the vast output, which at first glance can turn people away. The first few times, this can seem like a daunting task to sort through it all, but with a little adjustment in the mind-set, it is actually easy to sort. This is what can be referred to as “the big number hump.” The easiest way to get over “the big number hump” is to not think of the data as individual data points but to subcategorize the data into smaller chunks (even if one smaller chunk may contain everything from technical replicates to biological replicates to biologically distinct samples). By subcategorizing the data, a 10,000 data point table can be reduced to only 10 or 20 data points, which is much more manageable than 10,000 data points. Now that “the big number hump” is no longer an issue with the output, we can begin to define the data. There is a wide array of statistical packages that already exist that can handle massive data sets and make them workable such as excel, JMP, and R (some of these are open source like R, and some of them are proprietary such as Microsoft Excel and JMP), as well as programming languages (e.g., Python and Perl) which have extensive libraries for statistics. Depending on preference, these packages can be used either from a terminal or in a user interface. In addition to these programs, the data handling language SQL allows users to manipulate and build

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tables out of existing tables and produce only the most needed data sets. This can then be further manipulated in a stats package where the final graph or chart can be produced. In the instance where gene annotations or alignments are needed, there are various databases and resources available. The most well-known and used of which is NCBI (https://www.ncbi.nlm.nih.gov) and FungiDB (Basenko et al. 2018). These all offer basic BLAST (Basic Local Alignment Search Tool) options, as well as a host of metadata about your query (Altschul et al. 1990). These tools are perfect for understanding what gene/protein/transcript you may have and how similar it is to all the other uploaded ones out there (e.g., to build phylogenetic trees). Additionally, they all have other more specific functions, but they are all bound by the same two limitations: (1) they are hosted online, so an Internet connection must be available, and (2) for high-throughput metadata analysis using these systems, a great amount of time and computing power is at risk of being used. Similarly, to get around this, a local database can be built using downloaded information that is specific to your needs, thus eliminating searching through the entire NCBI/FungiDB databases. To populate these databases, you might need annotation data. There are, among others, three databases available for this that are considered the best: IPR, KEGG, and GO. These allow you to search based on IDs and get protein description.

4.1

Nonnumerical Data Types

There are more types of data than just straight numerical inputs such as sequencing results, color, names, pictures, etc. Even though these data types are not exclusive to fungi, they are worth addressing because fungi have some requirements that can make straight adaptation of preexisting nonnumerical pipelines harder. First, we can gather sequencing results and names in one group. These are because the existing pipelines to handle such data types in non-fungal organisms are easily adaptable. From desktop programs such as CLC Main Workbench and cloud-based services such as Benchling (www.benchling.com), importing files produced from sequencing results is the same. The other obstacle that is currently being overcome is the multiple chromosome conundrum where inputting multiple chromosomes for one organism can be achieved as both separate and individual entities. There is already progress on this in mammalian and yeast cells, so there is no reason why it could not be adapted to filamentous fungi. Names can be handled in different ways. The first is through a barcoded tracking system where a digital map of the first plate is made and that is given a barcode, and subsequent work and transfer of the organisms in that plate are tracked and names are carried over. Another way to deal with this is to assign numbers to the individual names and turn it into a numerical data type for tracking. Both systems require advanced tracking data for which many software packages are already available in addition to homebuilt LIMS systems.

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Color represents both a similar and unique challenge to filamentous fungi because they can adapt several different colors at once through the mycelia and the spores and released into the medium. For many experiments, the resulting spore color is of vital importance to demonstrate successful genetic engineering, but sometimes the mycelial color is the target data. This means that a functional system must be able to handle color without being manually recorded. This is still a gaping hole that needs to be fixed in the future. Related to color is photo documentation. While there exists great software integration of cameras into various pieces of high-throughput technologies (QPIX, Molecular Devices), in a high-throughput format, 96/384/1536 photos from each run are not a viable option for both storage and data representation. What is needed is an automatic way to convert photo data into numerical or character data types. Almost all photo data can be converted into one of these types. Numerical data can include number of colonies per well, size of colonies, number of contaminants, ratios, etc. Character data can include yes/no questions, color, colony shape, and species name. Yet with large and spread out colonies, filamentous fungi can make photo documentation tough. When programs are set for millimeter-scale differences for bacteria and yeast, fungi act on a centimeter scale, which is not insurmountable but still a notable challenge.

4.2

Reproducibility, Pipeline Development, and Database Fractionation

Reproducibility is the essence of good science. The achievement of this goal becomes exponentially more difficult as data grow and morph into big data, but the biggest block to this is not as some argue, the difference between having to handle 10 versus 10,000 data points, but rather nonstandardized pipelines and databases. In Aspergillus niger, for example, to go from a gene name to the data that require storing in a database, one would need to go through two other databases first. The multitudes and reproduction of data and databases are major challenges facing the future of high-throughput/big data pipeline development and reproducibility. There are many different databases of varying access that are needed to be sorted through sometimes just to find one answer. This is frustrating and impossible to upscale. The way people are getting around this replication and fractionation of data across multiple databases is by implementing local LIMS (Laboratory Information Management System) within their own groups or companies. In this system, you import the information you need into your own database. While this is a fix, it is not the fix we need to turn the future into an easy way to develop new high-throughput processes. What high-throughput systems ideally need is a single software program to control all of them. Again, this may never happen, unless it is built and implemented locally and run on individual servers at each company or research

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group, or in a truly hypothetical world, there is horizontal integration of all highthroughput robotics, and companies work together to build a system compatible across different software (unlikely).

5 Hypothetical Future and Conclusion Throughout this chapter, a number of problems have been discussed in regard to the current state of high-throughput engineering in filamentous fungi, but assuming it all fits together in the end and works, what does the future hold for this field? The short answer is a lot. The long answer is that looking into a hypothetical not-too-distant future, we can draw parallels across the current production value of high-throughput engineering in other organisms such as E. coli and S. cerevisiae as well as examining the industrial uses of filamentous fungi. We can already see automated setups at various companies such as Zymergen, Ginkgo, and Amyris that fully integrate robotics and software into a functioning high-throughput system for various bacteria and yeasts. This means that in the future, fungal bioengineering should be as simple as mapping some data on the computer and using an integrated software system to run a complex set of experiments to assemble and isolate DNA constructs, transform them into fungi, screen the fungi, and finally test the fungi for production value. Academia could use these systems to greatly increase the amount of basic science and proof-of-concept work being produced for the same or less money. But although industry already harnesses the powers of fungi to efficiently produce a wide variety compounds and products, it could be better. By integrating a fully functional highthroughput platform into industrial processes, the yield improvements of production could increase exponentially by not having to limit to what genetic mutations to test (or as some company’s do, wait for random mutations to arise). This leads to a faster product development and product yield increases. Developing an integrated highthroughput platform for filamentous fungi is a winning situation that will open endless possibilities for academia, industry, and society in general.

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Chapter 7

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories José Arnau, Debbie Yaver, and Carsten M. Hjort

1 The Industrial Enzyme Market The global industrial enzyme market was 6 billion USD in 2017. The market is expected to grow up to 6% in emerging markets in the next years (Global Industrial Enzymes Markets, seventh Edition, Marketresearch.com). The largest segment of industrial enzyme application is food and beverage, amounting to approx. 2 billion USD in 2016, depending on the source (Patel et al. 2016; Chapman et al. 2018). Higher personal spending in developing countries will lead to growth in sales of higher-value products containing or manufactured with enzymes. Market trends also indicate growth in the use of technical enzymes (textile, paper, biodiesel, biofuel) where waste generation using chemicals will be penalized by environmental agencies (Chapman et al. 2018). Enzymes are used today to make more than 700 commercial products. Enzymebased products and solutions are used in over 40 industry sectors, from household care (e.g., detergents) and bioenergy to agriculture, animal health, and food. Food application of enzymes include baking, brewing, beverage, juice, wine, dairy, and oil/fats (Patel et al. 2016). Using the current list of commercial enzymes for food, feed, and technical applications from the European industry association of manufacturers and formulators of enzyme products (AMFEP, list update of 2015), 243 enzymes manufactured by fermentation of microorganisms are commercially available. Of these, 114 (47%) are produced using recombinant host strains. Most of the commercial enzymes are used in food applications (225 or 93%). Currently, over 300 food enzyme dossiers are under evaluation by the European Food Safety Authority (EFSA) under the EU J. Arnau (*) · C. M. Hjort Production Strain Technology, Novozymes A/S, Bagsvaerd, Denmark D. Yaver Production Strain Technology, Novozymes Inc., Davis, CA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_7

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FIAP (Food Improvement Agents Package) regulatory frame. These applications include both the existing commercial enzymes and new enzymes. Approximately 50% of all submitted dossiers include enzymes manufactured from genetically modified microorganisms (GMM). Interestingly, most of the commercial enzyme classes used in food are manufactured using fungal hosts (31 of 38 or 82%; see Table 7.1).

2 Enzymes Used in Food Products: New Solutions to Old and New Problems Fungi, which consist of both yeasts (unicellular eukaryotes) and filamentous fungi (multicellular), are among the most widely used microorganisms in traditional and advanced food production. The yeast Saccharomyces cerevisiae has been extensively used traditionally for food production. In all cases (brewing, baking, wine making), the ability to ferment sugars into ethanol and carbon dioxide is the key feature. Filamentous fungi have also been used for food. Traditional miso processes using Aspergillus oryzae and A. sake (the “koji” mold) for fermentation of soybean, barley, or rice have been used in Asia for more than 10,000 years in spite of the fact that for a very long time, it was not known that the “mold” was present in the inoculate used (Machida et al. 2008). In a few cases, the organism is a component of the final food product, e.g., Penicillium roqueforti strains used in the manufacture of blue cheese or “Quorn”, a product that has been marketed for decades by Marlow Foods in the UK and is made using Fusarium venenatum (Trinci 1992). The original koji isolates were recognized as natural producers of enzyme “cocktails” useful for the food industry more than a century ago. In 1894, manufacturing of an enzyme complex from A. oryzae was established (Takadiastase, Allen 1896; Stone and Wright 1898). A. oryzae was developed to produce an array of different enzyme products in submerged fermentation, still with emphasis on proteases and amylases. Other Aspergillus species like the black aspergilli, A. niger, A. awamori, A. foetidus, A. aculeatus, and A. japonicus were also used mainly for production of glucoamylase. Development of commercial sources of glucoamylase was the basis for the enzyme revolution in the starch industry (Cairns et al. 2018). Traditionally, starch was hydrolyzed to glucose in an acid process using hydrochloric acid followed by neutralization. In this process, several by-products were formed, and heavy salt formation resulted from the neutralization step. By introducing enzymatic hydrolysis, by-product formation was avoided, and a product of a better quality could be manufactured at a lower price. There is an increased attention on healthy food products where the use of new enzymes has shown a clear benefit. Among those, the use of enzymes for reduction of lactose content in food products derived from milk using a bacterial β-galactosidases is a great example (Ugidos-Rodriguez et al. 2018). Additionally,

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Table 7.1 List of approved enzyme classes for use as a processing agent in food in Europe. Only one example of species of the production organism per enzyme is shown. For each product, the fungal or bacterial origin of the host organism is shown Enzyme activity Aminopeptidase Aminopeptidase (leucyl) AMP deaminase Arabinofuranosidase Catalase Cellulase Cellulase Cellulase Dextranase Glucanase (endo-beta) Glucanase (exo-beta) Glucanase (exo-beta) Glucanase (beta) Glucanase (beta) Glucoamylase Glucose oxidase Isoamylase Lactase or galactosidase (beta) Lipase triacylglycerol Lipase triacylglycerol Lipase triacylglycerol Lysophospholipase Alpha amylase Pectinase Phospholipase A Protease (exopeptidase) Protease Protease (mucorpepsin) Protein glutaminase Ribonuclease Tannase Transglutaminase Xylanase Glucanase (beta) Xylanase Pullulanase Pullulanase Xylanase

Production microorganism Aspergillus niger Aspergillus oryzae Aspergillus melleus Aspergillus niger Aspergillus niger Aspergillus niger Penicillium funiculosum Trichoderma reesei Chaetomium erraticum Aspergillus niger Aspergillus niger Trichoderma reesei Aspergillus niger Talaromyces emersonii Aspergillus niger Aspergillus niger Pseudomonas amyloderamosa Aspergillus niger Candida rugosa Rhizopus niveus Rhizopus oryzae Aspergillus niger Microbacterium imperial Aspergillus niger Streptomyces violaceoruber Aspergillus melleus Aspergillus oryzae Rhizomucor miehei Chryseobacterium proteolyticum Penicillium citrinum Aspergillus oryzae Streptoverticillium mobaraense Aspergillus niger Humicola insolens Humicola insolens Pullulanibacillus spp Klebsiella planticola Trichoderma reesei

Host organism Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Bacterial Fungal Fungal Fungal Fungal Fungal Bacterial Fungal Bacterial Fungal Fungal Fungal Bacterial Fungal Fungal Bacterial Fungal Fungal Fungal Bacterial Bacterial Fungal

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using enzymes to reduce the amount of substances with a demonstrated health risk or increasing properties involved in health like higher protein content or a lower salt concentration are worth mentioning. Below are two examples of enzymes manufactured using a fungal host for healthier food products.

2.1

Asparaginase for Acrylamide Reduction in Processed Food

Acrylamide is formed naturally in foods as a by-product during frying or baking at temperatures above 120
C and at low moisture. These conditions are responsible for the flavor and color in toasted, fried, or baked starchy products. Acrylamide is known as a neurotoxin in humans, and it is classified as a probable human carcinogen by the International Agency for Research on Cancer, and its carcinogenic effect has been recently shown (Zhivagi et al. 2019). Furthermore, it is classified as mutagen by the European Union and added to the list of substances of very high concern by the European Chemical Agency (Palermo et al. 2016). It is formed mainly from free asparagine and reducing sugars during high-temperature processing of common foods. The main sources of dietary exposure are fried potato, bakery products, breakfast cereals, and coffee (Pedreschi et al. 2014). A very effective way to substantially reduce acrylamide formation is to convert asparagine to aspartic acid using asparaginase. The use of asparaginase has been suggested as the best approach to reduce acrylamide in food products (Palermo et al. 2016). Different commercial sources of asparaginase are approved for use in food processing by, e.g., the US FDA (https://www.fda.gov/downloads/food/ucm374534. pdf). Importantly, this approach effectively reduces the acrylamide contents (Fig. 7.1a) while maintaining the organoleptic properties of the different food products (Fig. 7.1b) using a recombinant asparaginase manufactured using A. oryzae (Acrylaway®).

2.2

Lipase for the Removal of Unsaturated Fatty Acids

Lipases are used as additives or biocatalysts to manufacture many food ingredients, including hydrolysis of milk fat, acceleration of cheese ripening, improvement of flavor, and extended freshness of bread among other applications (Ferreira-Dias et al. 2013). Baking fats and margarines have either 1) hardened fats with trans-fatty acids or 2) fatty acids removed through a chemical interesterification process. This results in oil of poor quality while also being problematic in terms of both safety and the environment. Current regulations prohibit manufacturers to use partially hydrogenated oils (PHOs) due to the trans-fat content. The World Health Organization launched a program to reduce PHOs and trans fats globally, as their intake is associated to risk for coronary heart disease (Shin et al. 2009). Trans fats are formed when manufacturers use a partial hydrogenation process to give margarines the

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Fig. 7.1 Use of commercial recombinant asparaginase (Acrylaway®) to reduce acrylamide content in potato chips. (a) Dose response test adding different amounts of the enzyme (0 to 20x). (b) Using the 10x dose (“Enzyme”), the potato chips display the same visual (and organoleptic) properties as the “Control.” More information available at https://www.novozymes.com/en/news/news-archive/2013/ 11/new-novozymes-solution-enables-acrylamide-mitigation-in-even-more-product-categories

Fig. 7.2 Color change and by-product formation using chemical esterification (“Chemical,” right flask) compared to the use of a lipase (Lipozyme TL®, a lipase from T. lanuginosus manufactured in A. oryzae; “Enzymatic,” middle flask) using plant oil (“Oil start,” left flask)

correct melting properties and shelf stability. The same quality and avoidance of trans fats formation can be obtained using a different process, interesterification. Using enzymatic interesterification gives a more natural, better tasting product that contains healthier oils (Fig. 7.2; see below).

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In interesterification, the fatty acids of the triglycerides are shifted around randomly—they exchange positions on the glycerol backbone molecule. The most common chemical catalyst is still either sodium methylate (methoxide) or sodium ethylate (ethoxylate). These chemicals are both highly toxic and explosive. The chemical reaction causes by-product formation (including color, Fig. 7.2). Therefore, the reaction is followed by washing, bleaching and deodorization. All these are unit operations, which result in yield loss during the process. By-products of chemical interesterification have been identified as dialkyl ketones (Santoro et al. 2018). Differences between the result of interesterification of a fat blend by chemical and enzymatic interesterification are also partly due to the higher level of diglycerides produced in the chemical process and partly because the enzymatic process does not produce full randomization (Holm et al. 2018). The enzymatic process eliminates the need for chemicals, washing or postbleaching, and it produces no wastewater. This process involves fewer steps and reduces energy costs. Several commercial enzymes are available for this application, e.g., Evonik Accurel MD® (non-recombinant Candida parapsilosis lipase) and Novozymes Lipozyme®. Lipozyme® is a series of lipase products (e.g., C. antarctica lipase B manufactured in A. niger or Thermomyces lanuginosus or Rhizomucor miehei lipase manufactured in A. oryzae) that are used as immobilized enzymes to reduce trans fats in, e.g., margarine. Enzymatic interesterification provides a simple, efficient, and environmentally friendly way to produce margarines without the use of chemicals. The use of enzymes improves product quality and production yields (Ferreira-Dias et al. 2013).

3 A Survey of Innovation in the Field of Biotechnological Production Since cost reduction is a major drive in enzyme manufacturing, we decided to look at the evolution of patenting in the field. The assumption was that since the market for industrial enzymes is growing, an underlying and growing technology development must occur to sustain this. The data presented here are not the result of a comprehensive survey and just serve the purpose of describing the trends in the field. Looking at the number of patent families within the fields of biochemical production, recombinant DNA technology, expression systems, fermentation, production yield enhancement, and enzyme recovery and screening, 2873 patent applications were filed in 2012. These numbers decreased consistently every year (2832 applications in 2013, 2574 in 2014, and 2494 in 2015). In 2016, the total number of patent applications was just 2153. Considering areas closely related to cell factories, numbers for recombinant DNA technology and expression systems have also consistently decreased in the period investigated (664 and 214 in 2012 to 358 and 160 in 2016 for recombinant DNA technology and expression system patents, respectively; Fig. 7.3). In contrast, gene

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Fig. 7.3 Number of patent families filed in the period 2012–2016 in Europe, USA, and World Intellectual Property Organization (WIPO). The numbers refer to the priority counts based on the earliest priority filing dates of the patent families in the dataset and separated by subject (“recombinant DNA technology,” “expression system,” and “gene editing”). The numbers depict the total number of patent families that originate from the identified priority filing dates. The analysis was performed using broad search terms and patent classification codes. The accuracy of this survey is only 60–70% as it was performed using semiautomated techniques and only limited manual review. Data for 2017 cannot be shown as there is an 18-month delay from application to publication date when searches apply

editing patent numbers increased from 13 to 56 in the period 2013–2015 with a small decrease in 2016 (Fig. 7.3). This recent decrease may be due to the uncertainty on the outcome of the court disputes between Broad Institute and the University of Berkeley among others on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats; see Sect. 4.4) rights. In a recent review, the total number of granted patents of CRISPR was described as over 90 (Ferreira et al. 2018). This number correlates with the aggregate numbers described here. However, the estimated number of filed applications under examination is 1300 ranging from CRISPR components to delivery systems and applications in medicine, agriculture, and biotechnology. Except for CRISPR, the overall picture is that the pace at which patents are being filed on technology has slowed down although the number of strategies that can be used for optimization has significantly increased (see Sect. 4.3). It is possible to speculate that many of the existing systems for enzyme production include a mature technology and therefore the number of improvements might have become limiting. Alternative explanations for the steady decrease in the number of patents may include (1) consolidation of the industry, e.g., Danisco being acquired by DuPont in 2011 or BASF expanding its enzyme business in 2013, and (2) a US Supreme Court decision against Myriad Genetics in 2013 that “merely isolating genes that are found in nature does not make them patentable,” i.e., the fact that a DNA sequence alone can no longer be patented.

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4 Fungal Expression Systems and Optimization for Enzyme Production Since the development of the first recombinant commercial enzyme, research in fungal biotechnology has focused on increasing the yield of enzyme(s) produced in a given host species. The reduction in production costs is a main driver for the use of enzymes for, e.g., replacement of cheap chemicals in food applications. Excellent reviews on expression technology for production that describe the tools and the main approaches to achieve high yields of enzyme are available (Fleissner and Dersch 2010; Nevalainen and Peterson 2014; Davy et al. 2017; Xiao et al. 2014). An industrially relevant, “basic” expression system consists of: (a) A host strain with a history of safe use that displays robust and efficient growth and protein production in a low-cost media at industrial (large) scale. (b) A selection system to ensure introduction of the enzyme gene into the host. Several nutritional marker genes (e.g., pyrG, amdS, and niaD) and antibiotic resistance markers (e.g., hygB) can be used. For industrial food enzyme production, the use of antibiotic resistance markers is not recommended due to regulatory requirements in the final strain. (c) A transformation procedure to introduce one or more copies of the enzyme expression cassette into the genome of the strain preferably at specific locations. (d) A strong promoter that enables expression of the enzyme gene under fermentation conditions. An overview of common fungal promoters and their use has been described (Fleissner and Dersch 2010). See also Sect. 4.3.1. Once a strain producing the enzyme of interest is available, several approaches can be followed to increase yields to make it commercially feasible (see Sect. 4.3). Practically, using one of the generic two-way selection systems available in fungi (i.e., selection can be made for either the presence or the lack of the marker gene), it is possible to rationally improve the host strain in sequential steps of gene inactivation (Fig. 7.4a) or gene overexpression. A disruption in the endogenous pyrG gene encoding an orotidine-50 -phosphate decarboxylase results in the requirement of uridine for growth (Mattern et al. 1989). Lack of a functional pyrG can be selected directly by growth in a medium containing 5-fluoro-orotic acid (FOA). FOA is converted into a toxic intermediate only in a strain containing a wild-type pyrG gene. Therefore, selection of pyrG transformants or spontaneous pyrG strains in medium containing FOA allows the direct identification of strains bearing the intended modification. Using a DNA fragment containing the wild-type pyrG gene as a marker flanked by DNA sequences of the target locus (to enable homologous recombination), disruption of the target gene can be achieved. Thus, using the wild-type pyrG and flanking regions of the target gene to be inactivated (e.g., an endogenous secreted protease), it is possible to introduce a deletion by selection in medium without uridine (requires a functional pyrG gene). Including a repeated DNA sequence at both ends of the marker, it is straightforward to loop out the marker by growing in medium containing FOA, thereby resulting in a pyrG strain that can be used for the deletion of other target genes using the same marker (Fig. 7.4a) or to introduce the expression cassette for the enzyme gene.

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Fig. 7.4 Traditional (a) and modern (genome editing, b) methods for construction of improved host strains. (a) Traditional methods can be based on the use of homologous recombination using upstream and downstream sequences (“HR”) of the target gene to be deleted (“Target 1”, “Target 2”) to flank a selective marker (“marker”) as indicated. For each target gene, two consecutive steps (1 and 2) are required for integration of the target site (1) and loop out of the marker (2) before a new target gene can be modified using the same selection (3 and 4). (b) Using CRISPR (illustrated with use of the most used Cas9 nuclease, green box), it is feasible to target modification of two or more genes in a single step using gRNAs directed to each target and a plasmid containing suitable expression cassettes for the Cas9 gene and the gRNAs. If the Cas9 expression cassette is introduced on a plasmid containing, e.g., pyrG as the selective marker, subsequent growth of the resulting strains in medium with FOA results in plasmid (Cas9) loss and enables identification of the desired strains. In the depicted example, two target genes are modified in a single step

With the advent of genome editing, multiple genetic modifications can be performed in a single step (see Sect. 4.3.3, Fig. 7.4b).

4.1

How to Choose the Best Fungal Host Species for Industrial Enzyme Production

One of the challenging tasks to produce a recombinant enzyme is the choice of fungal host. Although S. cerevisiae has been the subject of extensive research and it

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is currently one of the hosts of choice for production of biologicals, the yields typically obtained preclude its use for production of bulk enzymes (Bao et al. 2017). Prediction of the most suitable host is still not possible, and rather a screening for multiple hosts is the standard approach whenever possible. As a rule of thumb, the host should be as taxonomically closely related to the donor organism (i.e., where the enzyme gene originates from) as possible. In this way, requirements for the cellular machinery (secretion, protein folding, etc.) should be somewhat similar in the recombinant host. Yields of 20–100 g/L have been published, but they are normally obtained for homologous or near-homologous enzymes (Meyer et al. 2015). The longer taxonomic distance between the donor organism of the enzyme gene and the host, the lower the likelihood of expression at a feasible level (Singh et al. 2015). Not surprisingly though, there are exceptions to this rule as there are enzymes of bacterial origin that have been successfully produced in fungi, e.g., a Citrobacter braakii phytase used for feed applications that is manufactured in A. oryzae. A bacterial xylanase is produced at high yield in T. reesei (Paloheimo et al. 2003). This illustrates the lack of predictability of a suitable host. Alternatively, a single host can be used in a high-throughput screening approach combining multiple expression tools, strain backgrounds, and growth conditions. Although the hope is to enable production of a wide variety of enzymes, the reality still suggests that only a limited share of the “protein universe” can be produced by a single host. As an example, the Dyadic International Inc. proprietary thermophilic fungal host C1 (Myceliophthora thermophila) discovered in 1992 as a natural neutral cellulase producer was developed extensively aiming at developing a universal system for recombinant protein production. It has shown great potential for cellulase production for biofuel and for the manufacture of pharmaceutical proteins and vaccines, which are proteins that require a much lower yield to be commercially feasible compared to bulk enzymes (Visser et al. 2011). For other enzymes, yields are typically lower than those from more conventionally used species (Berka et al. 2011). In principle, there are additional reasons to choose a host among the most broadly used fungal species for enzyme production including A. niger, A. oryzae, and Trichoderma reesei. All three species have been used for decades and have an extensive history of safe use for enzyme production (Frisvad et al. 2018). Another point of consideration when selecting a host species is the level of endogenous proteases as it can affect yield and stability of the product. Clearly, enzyme yields might not be feasible if the host strain secretes numerous proteases to the medium during fermentation. And protease activity varies depending on the strain, the medium, and the culture conditions. Choosing a host species with a low level of protease like A. vadensis has been proposed (Lubertozzi and Keasling 2009). Alternatively, deletion or reduction of expression of endogenous protease coding genes may result in higher enzyme yields (see Sect. 4.3.3). Additionally, unwanted side activities (i.e., other secreted enzymes that are not suitable for the application) need to be removed. Examples of this include background glucoamylase and alpha-amylase in A. niger and A. oryzae and different cellulases and endoglucanases in T. reesei.

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Finally, the selected host species should not produce any compound of concern during fermentation. As mentioned above, a safety history of use for the most frequently used species is an advantage since the use, e.g., as food additives of many different enzymes in different strain backgrounds, has been documented. As described below (Sect. 6), the major safety concern of a commercial enzyme is not the enzyme but the presence of known mycotoxins in the product. For A. niger, A. oryzae, and T. reesei, safety has been described in a recent review covering this aspect (Frisvad et al. 2018). Scalability is also important. Growth of the host strains and production of the enzyme should be representative from small scale (preferably from a highthroughput screening format) to industrial production scale. This would enable the testing of several thousands of strains to select the most suitable candidates(s) for fermentation upscaling.

4.2

A Generic Fermentation Process for Enzyme Production in Fungal Hosts

During manufacturing at industrial scale, the enzyme should be efficiently secreted into the fermentation broth to ease recovery of the enzyme. A simple and robust process for submerged fermentation is typically used (Fig. 7.5). Shortly, a cell bank vial (typically a working cell bank, WCB) is used to inoculate a solid medium to generate sufficient spores to inoculate a medium-size seed tank (seed fermenter). This step enables the generation of suitable fungal biomass optimally without induction of enzyme production. The biomass is used as inoculum for the main tank (5000 L or larger) where induction of enzyme production occurs. Growth of filamentous fungi in a fermenter presents a number of challenges in terms of the morphology, pellet vs. dispersed growth, high viscosity, and transfer of oxygen to the cells compared to, e.g., unicellular, round yeast cells or bacteria. High viscosity typically limits oxygen transfer and result in loss of productivity (enzyme yield). Mycelial morphology during fermentation has an important effect on aeration and productivity (Amanullah et al. 2002). In submerged cultures, the observed macroscopic morphology of filamentous fungi varies from freely dispersed mycelium to dense pellets consisting of a more or less condensed and intertwined network of hyphae. Morphology and productivity are influenced by the environment and can be controlled by inoculum concentration and viability, pH value, temperature, dissolved oxygen concentration, medium composition, mechanical stress, or process mode as well as through the addition of inorganic salts (Walisko et al. 2015). Depending on the promoter used to control expression of the enzyme gene, the feed rate can be adjusted to enable high productivity and low carbon catabolite repression by, e.g., glucose using the most common fungal-inducible promoters (see Sect. 4.3.1).

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Fig. 7.5 Overview of a generic fermentation process for enzyme production in a fungal host (see main text for explanation). Typically, a defined medium is used and sterilized. A vial of the production strain is used for cultivation in an inoculation flask. Spores or cells are harvested after 5–10 days depending on the host and used to inoculate a seed fermenter. After 1–4 days, the culture is used to inoculate a larger tank where induction of enzyme production occurs by, e.g., feeding with an inducer. Both medium pH and temperature are controlled, the latter by, e.g., cooling water

Production processes involving fungi are often highly aerobic in nature, which implies these cultures are routinely subject to oxidative stress (Li et al. 2011). Most of the enzymes produced in fungal hosts and used commercially are secreted into the medium during fermentation. Once the fermentation is completed, the enzyme is recovered from the fermentation broth. Recovery consists of a step for removal of the cellular debris and a step to concentrate the cell-free fraction, e.g., ultrafiltration, followed by stabilization, sterile filtration, and formulation (Fig. 7.6). Thus, contrary to production of biopharmaceuticals, the enzyme product is a concentrate of the culture supernatant with no physical/biochemical separation of molecules as in a chromatography column. This is a point of concern for the safety evaluation of food and feed enzymes by the authorities. Impurities and compounds of concern like mycotoxins should not be present in the enzyme product (see Sect. 6). Significant work is devoted to optimizing the fermentation and recovery processes from media optimization, growth conditions, scale up from lab fermenter, etc. (Fig. 7.7).

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Fig. 7.6 Overview of a generic recovery process for enzyme production. The fermented broth undergoes different steps to remove the cellular debris (flocculation, filtration, or centrifugation). Subsequently the product can be concentrated by ultrafiltration. Product stabilizers are added prior to filter sterilization (bacterial filtration). The enzyme preparation is used for formulation of a liquid or a solid (granulate) enzyme product

4.3

Strategies for the Optimization of Enzyme Yields

Product development in industrial biotechnology includes a continuous challenge to increase enzyme titers at large scale to reduce costs. Two major approaches have been used for this purpose in the last decades to attempt yield increase. The first one is based on classical mutagenesis and screening. Here, the specific genetic modification is not pre-defined. The main requirement is a screening assay that has the sensitivity and range of detection to allow the identification of mutants producing higher enzyme titers. Classical mutagenesis and screening strategies have been used for decades to improve small molecule (Adrio and Demain 2006) and enzyme production. The original Penicillium notatum strain isolated by Fleming contained a single copy of the penicillin biosynthetic genes, and the published yield using optimized strains that contain multiple copies is 70 g/L (Adrio and Demain 2006). Over several decades, a pedigree of A. niger strains with increased production of glucoamylase (GlaA) was developed by classical mutagenesis. Analysis of this pedigree identified both deletions and amplifications including repeated amplification of a 216 kb region that includes the glaA gene. The highest producing strain contained ten copies of glaA compared to two copies in the original parent strain (Cherry et al. 2010). High-throughput screening enables large numbers of mutants to be screened in search for the desired phenotype, i.e., higher enzyme yields. The increase in copy number typically leads to an increase in product formation, but it also reaches a maximum when transcription is not limiting and other cellular processes like secretion become a bottleneck (Gressler et al. 2015).

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Fig. 7.7 Overview of generic approaches used for enzyme yield increase in fungal cell factories. A simplified model of a fungal tip is depicted showing the apical cell, the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, the secretory vesicles containing proteins to be secreted (green circles) that are transported to the hyphal tip through tubulin filaments (green lines), the chitosomes (red circles) containing enzymes involved in the synthesis of cell wall that are directed through actin filaments (red lines), and the Spitzenkörper, where secretory vesicles accumulate before exocytosis into the extracellular environment. Secretory vesicles can also be directed to vacuoles (v) for recycling. Genome editing can be used both as a rational and high-throughput method for functional genomics analysis

The second approach based on rational design includes numerous strategies including the use of stronger promoters and multicopy strains to ensure high expression of the gene of interest; codon-optimized gene sequences to improve translation; deletion of genes encoding endogenous proteases; gene fusions for enhanced secretion; and overexpression of accessory cell machinery to alleviate the burden of producing high amounts of protein (chaperones, protein fusions, etc.). Excellent reviews have been published on strategies for cell factory engineering (Fleissner and Dersch 2010; Nevalainen and Peterson 2014; Davy et al. 2017; Xiao et al. 2014). Rational strategies also include the removal of unwanted side activities that could lead to a negative impact in the purity or the application (i.e., endogenous lipase activity in enzymes used for cheese manufacturing). Other approaches are directed to reduce endogenous protease levels in the host strain for yield and stability of the produced enzyme (Laustsen and Nielsson 1995). Altering the amino acid sequence of the enzyme for increased secretion and/or yield may also be a valid approach (Katakura et al. 1999; Held et al. 2018). In industrial production strains, high-level production of a protein may trigger several bottlenecks in the cellular machinery for secretion of the enzyme of interest

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into the medium. Assuming high levels of transcription (result of strong promoters, high gene copy numbers, and sufficient mRNA stability) and translation (result of optimized codon usage, etc.), the next obvious bottlenecks might be encountered in the secretory pathway (translocation into the ER, protein folding, glycosylation, protein degradation, etc.; Fig. 7.7). More targeted strategies to engineer fungi for high-level enzyme production can be hypothesized using omics tools like transcriptomics, proteomics, and metabolomics to identify cellular bottlenecks and suitable targets for yield optimization. However, multiple candidate target genes are normally identified as being upregulated or downregulated at specific growth conditions or by comparing strains overproducing different enzymes (see Sect. 4.3.2; Zubieta et al. 2018). Gene editing can be used to functionally test several to hundreds of targets. Most of this work is performed in model strains and at laboratory scale. Therefore, careful experimental design and correlation to large-scale production need to be taken into consideration.

4.3.1

Maximizing Transcription of the Enzyme Gene of Interest

A strong and regulated promoter is an important prerequisite for high yield. The stronger the promoter, the lower the requirement for copy number of the expression cassette to be inserted into the production strain to reach optimal enzyme titers. The advantages of using a regulated promoter include the possibility to design a fermentation process composed of a biomass production phase followed by induction of enzyme production. Strong and regulated promoters can be identified, e.g., looking at relatively abundant enzymes or mRNAs. The great majority of highly expressed genes include those encoding secreted enzymes that are an essential part of the life strategy of fungi, i.e., for degradation of polymers such as cellulose, starch, etc. (Wang et al. 2018; Yoder and Lehmbeck 2004). Examples of promoters that are used in industrial enzyme production include the Trichoderma reesei cbhI promoter (PcbhI), the A. oryzae TAKA amylase (PTAKA; Christensen et al. 1988) and the A. niger neutral amylase, and glucoamylase promoters (PamyB and PglaA). All the above promoters are strong and regulated. Induction is obtained in medium containing cellulose-like substrates like sophorose (PcbhI) or starch, maltose, and maltodextrin (PTAKA, PglaA, or PamyB). They are all subjected to carbon catabolite repression (CCR), and their use may negatively affect productivity as glucose is produced during growth. Many industrial fungal hosts have therefore been mutated in functions regulating CCR. One example is the mutation identified in the cre1 gene in T. reesei strains that produce cellulase during growth in glucose. Cre1 is the major CCR transcription factor that binds to a specific motif present in promoter of genes involved in glucose repression. Introducing the mutation in a production strain increases the enzyme yield without affecting growth (Nakari-Setälä et al. 2009). Various non-native promoters have been engineered to control gene expression in fungi. Synthetic promoter systems (SES) have been developed that are useful for several yeast and fungal species and that show yields comparable to PcbhI for cellulase production. SES consists of a promoter with a core sequence and of a

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heterologous transcription factor which binds to repeated activating sequences placed in the promoter (Rantasalo et al. 2018). Other promoters like the Aspergillus tet-on/tet-off system are useful for research purposes rather than for industrial production (Wanka et al. 2016; Kluge et al. 2018). As mentioned above, a source of novel promoters may be obtained investigating mRNA levels and choosing the promoter of the highest expressed genes under relevant growth conditions. One challenge of this approach is the fact that unforeseen bottlenecks (titration of accessory functions like transcription factors) may appear when using these promoters at high copy number in a production strain background. Another strategy to increase transcription from strong native promoters is artificial transcription factors that can be engineered to improve production of secreted heterologous enzymes. A recent paper highlighted improving cellulase expression in Trichoderma reesei by introducing artificial transcription activators (ATAs). The ATAs were constructed by linking the C-terminus of XYR1, ACE1, and ACE2 with an activation domain of herpes simplex virus protein VP16. Strains engineered with one of the three artificial transcription activators displayed different phenotypes of improved cellulase production (Zhang et al. 2018). Yet another approach is the use of polycistronic RNAs under the control of a regulated promoter for secondary metabolite production in A. niger (Geib and Brock 2017). Long noncoding RNAs that are involved in gene regulation have been identified in yeast and filamentous fungi (Till et al. 2018a). Identification and manipulation of long noncoding RNAs represent another means to engineer filamentous fungi for improved heterologous protein production. Recently a long noncoding RNA, Hax1, that enhances cellulase expression was discovered in T. reesei (Till et al. 2018b). With low-cost sequencing, it is possible to sequence RNA to identify possible long noncoding RNAs present under relevant fermentation conditions for production followed by functional testing to determine if they have an impact on protein production. The choice of promoter influences the fermentation process that can be developed, e.g., a process based on glucose as carbon source may not be suited if expression of the enzyme gene is under control of a promoter that is repressed totally or partially by glucose.

4.3.2

Optimization of Protein Secretion: Still a Black Box

Secretion is a complex cellular process that involves many steps, proteins and organelles in the cell. Shortly, secretion consists of the transfer of a nascent protein to the endoplasmic reticulum (ER), its appropriate folding, posttranslational modification and maturation (Golgi), sorting of vesicles to either early endosomes or secretory vesicles, directional transport of secretory vesicles to the growing hyphal tip, and fusion of the vesicles with the plasma membrane to release the enzyme to the extracellular environment (Fig. 7.7). Early endosomes might also be involved in the delivery of secretory proteins to the vacuole for degradation and recycling of nutrients (Hernandez-Gonzalez et al. 2018).

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In 1994 a review was published in which protein secretion in filamentous fungi was called a “highly productive black box” because despite the use of these organisms to produce high yields of commercial enzymes applied in many industrial applications, very little was understood about the cellular mechanism of secretion (Peberdy 1994). Since this publication, research has broadened the understanding of secretion in filamentous fungi; however, many reports on attempts to improve “secretion” of a protein have been published, and no broadly successful strategy to manipulate the secretion pathway exists. Therefore, secretion optimization is probably one of the most challenging tasks to increase enzyme yields. Fungi like A. niger and A. oryzae adapt the protein load in the secretory pathway, probably as an evolutionary advantage of their lifestyle in nature. In addition, we continue to learn unique aspects of secretion in fungi as exemplified by recent insights into protein trafficking in A. nidulans. A genetic dissection of the exocytic pathway in A. nidulans has been described. It reveals numerous and yet unexplored cellular functions like Rab1and Sed5 (early Golgi), Sec7 and Trs120 (trans Golgi network), and Rab5 (early endosome) among others (Hernandez-Gonzalez et al. 2018). To efficiently direct a protein for secretion via the ER, it is normally necessary to use of an optimal signal peptide (SP). A comprehensive review on the structure, features, and application of SPs has been published (Owji et al. 2018). In principle, SPs derived from efficiently secreted enzymes of the host should be suitable. Screening of different known signal peptides to find the most suitable sequence can be performed as described for the yeast Pichia pastoris. Although some signal peptides (e.g., the sequence derived from the yeast mating factor) result in an overall higher yield in yeast, no secretion was obtained using this sequence for other proteins (Obst et al. 2017). Suitable SPs may be obtained by identifying the secreted proteins of a fungal host and selecting the SP of the most abundant ones (Wang et al. 2018), although in vivo screening is needed as theoretical values generated by SignalP (a prediction software for SPs) are not associated with high levels of secretion in yeast (Mori et al. 2015). Overexpression of secretory enzyme genes causes an overload of the secretory route. Excellent reviews have been published on the subject (Aviram and Schuldiner 2017; Meyer et al. 2015). To restore homeostasis, both ER-associated protein degradation and the unfolded protein response (UPR) aid in removing or folding the accumulated protein. The UPR is activated when misfolded proteins accumulate in the ER. It leads to the alternative splicing of the HAC1 gene product and the activation of UPR-regulated genes. In yeast, the secretion of α-amylase was significantly enhanced through the overexpression of HAC1 (Valkonen et al. 2003). In addition, many genes become transcriptionally downregulated, maybe because they are less important for growth and survival under the specific growth condition. This phenomenon is called repression under secretion stress (RESS) and was first observed in cellulase production strains of T. reesei (Pakula et al. 2003). To illustrate the complexity of enzyme secretion, it was shown that glucoamylase overproduction in A. niger is associated with a change in the expression of 1500 genes, including upregulation of genes that are involved in ER translocation, protein folding and glycosylation, and vesicle transport (Kwon et al. 2012).

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If folding is a bottleneck in enzyme overproducing strains, an obvious target for optimizing enzyme yield is overexpression of ER-resident chaperones (Lubertozzi and Keasling 2009; Davy et al. 2017). Significant upregulation of bipA, clxA, and pdiA among others was reported for glucoamylase production in A. niger (Fleissner and Dersch 2010; Kwon et al. 2012). The challenge is the choice of chaperone and the appropriate level of overexpression that may be necessary to boost yields since different enzymes or different sequence variants of the same enzyme may require different cellular functions. In S. cerevisiae, moderate expression of SEC16 has been shown to increase secretion of two heterologous enzymes (Bao et al. 2017) adding extra complexity to the process of optimization of enzyme secretion. Another approach to improve secretion of a desired protein is a fusion of the protein to a well-expressed, secreted protein from the host. The rationale for this approach is that the homologous protein may facilitate passage through the secretory pathway, although the precise mechanism is not understood (Yoder and Lehmbeck 2004). For production of calf chymosin in A. awamori, a protein fusion to the endogenous glucoamylase was used to increase yields (Ward et al. 1990). Given that there are no clear strategies to engineer filamentous fungi for increased secretion of a protein of interest, the ability to build and identify recombinant strains with improved secretion of a given enzyme requires methods to be able to construct and test many different combinations of transcriptional control elements, signal peptides, and genome modifications. Automation and high-throughput screening that correlate with production scale are required.

4.3.3

Improving Protein Quality and Stability

The involvement of endogenous proteases on the yield is suspected whenever degradation of the enzyme of interest is observed during fermentation or during storage. Production and secretion of endogenous proteases and peptidases is rather widespread among fungal species. Filamentous fungi have the potential to produce numerous endogenous protein-degrading enzymes. To identify the proteases responsible for product degradation, investigation of the proteins secreted into the medium can be performed. This may also be a substantial task since the number of secreted proteins in fungal cultivations may vary from 300 to 700 or more, depending on the growth conditions and the species (Jun et al. 2013; Schmoll et al. 2016; Terfrüchte et al. 2018). In A. oryzae, more than 130 exoand endopeptidase genes have been identified (Kobayashi et al. 2007). In Ustilago maydis, the use of proteomics identified 477 proteins in submerged cultivation of which 21 were annotated as proteases or peptidases. Surprisingly, engineered strains in which deletion of major protease genes has been introduced and not displaying detectable protease activity in casein-indicator plates still contained a significant protease activity (Terfrüchte et al. 2018). As in many other fungi, the arsenal of proteases present in wild-type strains ensures that available protein sources can be degraded under different growth conditions even if loss of function mutations in major protease genes occur. In T. reesei, strains engineered for production of

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enzymes for biomass degradation, protease levels (e.g., PepM and Asp1) vary depending on growth conditions (Jun et al. 2013; Stappler et al. 2017). Additionally, a strain containing deletions in the seven genes encoding the more predominant proteases was developed, using a combination of inhibitor studies and protease purification, to enable production of biopharmaceuticals in T. reesei (Landowski et al. 2015). Identifying major secreted proteases has resulted in the development of improved industrial strains carrying deletions in one or more protease genes (Laustsen and Nielsson 1995; Wang et al. 2018). An alternative approach is to introduce a deletion of the gene encoding a transcription factor, PrtT, that controls the expression of multiple protease genes (Hjort et al. 2001; Punt et al. 2008). A protease regulator, Pea1, has been identified in T. reesei and Fusarium sp. that is required for expression of several proteases. Interestingly, Pea1 does not share similarity to other known regulatory proteins (Paloheimo et al. 2018).

4.3.4

Modification of Other Important Features of the Host

Filamentous fungi have the potential to produce a large number of secondary metabolites (SM). Some SM display antimicrobial or antioxidant activity and are used as a defense mechanism in nature. These compounds are normally not produced during growth in the laboratory or in large-scale growth in a fermenter. Mycotoxins are a small group of SM representing a health risk for animal and humans. Mycotoxins are acutely or chronically toxic and pose health hazards or death in humans and other vertebrates when acquired in small amounts via a natural route, i.e., orally, by inhalation, or via the skin (Frisvad et al. 2018). Genes responsible for mycotoxin biosynthesis are normally grouped in gene clusters. Although mycotoxin production is not normally associated to submerged growth of pure cultures, the potential for production of these compounds can be eliminated by deletion of the responsible gene cluster resulting in strains with a reduced toxigenic potential. To do this, the genes responsible for production of the mycotoxin need to be identified. In A. oryzae, two neighboring gene clusters involved in the synthesis of cyclopiazonic acid and aflatoxin, respectively, have been removed from the genome in production strains (Christensen et al. 1999; Olempska-Beer et al. 2006). Similarly, genes responsible for production of most relevant fungal mycotoxins like fumonisin and ochratoxin (Khaldi and Wolfe 2011; Gill-Serna et al. 2018) are known, and strains lacking the potential to produce them can be engineered. An alternative to this approach, e.g., in cases where the genes responsible for production of a mycotoxin are not known, is to monitor the presence of relevant mycotoxin in production and in the enzyme product. As mentioned earlier, to keep COGs low, production processes do not include purification steps. Purity of the product is solely controlled by the level of production of the enzyme product and the concentration of other secreted proteins in the recovered and formulated product. Many of the industrial fungal host species produce one or more secreted enzymes at reasonable titers, i.e., amylase, cellulases, and glucoamylase by A. niger, A. oryzae, and T. reesei. This ability to produce high

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levels of a secreted enzyme(s) has been exploited both for production of these native enzymes as well as for heterologous enzymes. In strains producing a heterologous enzyme, genes coding for abundant native secreted enzymes are deleted or classical mutants that are unable to produce the native enzymes have been isolated. T. reesei produces a complex set of cellulases and hemicellulases that provides the capability to degrade lignocellulose. Deletion of the most abundant enzyme cellobiohydrolase I (cbh1) gene leads to an increase in production of cellobiohydrolase II (cbh2), whereas deletion of endoglucanase I or endoglucanase II increased levels of both cbh1 and cbh2 (Seiboth et al. 1997).

4.4

Genome Editing as a Revolutionizing Tool for Production Strain Development

Genome editing has become a true game changer in research, medicine, and biotechnology. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym whose impact has reached even outside the scientific community through headlines in everyday news as a tool to combat diseases and provide food for a growing population. CRISPR is a natural mechanism that allows bacterial cells to detect and destroy the viruses that attack them. Since the publication describing how CRISPR and the Cas9 nuclease can be used to edit DNA sequences (Jinek et al. 2012), the possibilities for application of genome editing have extended to medicine and agriculture. In industrial biotechnology, the use of CRISPR has enabled a faster method for strain construction. A recent review was published highlighting the recent applications of CRISPR/Cas9 in industrial biotechnology as well as the patent landscape (Ferreira et al. 2018). A review on CRISPR systems and tools for genome editing in filamentous fungi has recently been published (Deng et al. 2017). RNA-guided Cas9 endonuclease can be used to cleave any DNA sequence in a fungal host with high specificity, and repair of the double-strand breaks can be mediated by error-prone, nonhomologous end joining (NHEJ) or by the introduction of specific changes via homologous recombination using donor DNA. In strains deficient in nonhomologous end joining (NHEJ), highly efficient marker-free gene targeting can be performed. Even singlestranded oligonucleotides work efficiently as repair templates for Cas9-induced DNA double-strand breaks in different Aspergillus species (Nødvig et al. 2018). This indicates that this type of repair may be widespread in filamentous fungi. Using oligonucleotides for CRISPR-Cas9-mediated gene editing, it is now possible to introduce specific point mutations as well as gene deletions at efficiencies approaching 100%. In A. nidulans, up to three editing events can be achieved in one transformation (Nødvig et al. 2018). For research purposes in, e.g., functional genomics, CRISPR has already shown a tremendous impact. CRISPR-Cas9 and the nuclease inactivated variant of Cas9

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(Cas9dead or Cas9d) can be used for studies of gene regulation (activation and repression), to reconstruct and modify metabolic pathways among numerous other approaches (Deng et al. 2017). The ability to perform multiple modifications in a single step using CRISPR (Fig. 7.4b) will enable faster construction of yieldoptimized strains and will fuel its use for rational design in strain construction as well as for more genome-wide searches for gene modifications that improve production of an enzyme. Genome editing in the thermophilic fungus Myceliophthora was used to increase cellulase production by 13-fold by editing up to four genes at one time (Liu et al. 2017). Having the possibility to modify a target sequence regardless of the number of copies present in the genome using CRISPR, yet intractable but important microorganisms should now be amenable for strain improvement (e.g., diploid fungi).

4.5

Automated Strain Construction, Systems Biology, and Synthetic Biology Approaches

The emergence of inexpensive, base-perfect genome editing along with low-cost sequencing is revolutionizing biology. Modern industrial biotechnology exploits the advances in genome editing in combination with plug and play strain construction systems, automation, analytics, and data integration to build high-throughput automated strain engineering pipelines (Marcellin and Nielsen 2018). In this way, strain construction can be accelerated and expression results from a large number of unique strains can be used to begin to develop machine learning models to predict optimal expression cassette elements and genome modifications. Systems biology provides rational approaches and analytical tools to understand complex biological processes like manufacture of industrial enzymes using fungal cell factories and to enable production of novel molecules (Martins-Santana et al. 2018; Hammer and Avalos 2017). Mathematical modeling is central in systems biology to integrate information and quantitatively analyze phenotypes to enable predictions about the behavior of the organism or elucidating mechanisms underlying experimental observations. For this purpose, a genome model of the host species provides the “scaffold” for the integration of the large datasets that are generated and that enable computational predictions (Campbell et al. 2017). These approaches address a key issue in the construction of efficient cell factories, i.e., the ability to control where and when chemical reactions take place in the cell (Jakobson et al. 2018). An overview of systems biology research conducted in Aspergillus has recently been published (Brandl and Andersen 2018). A review on the impact of systems biology on cell factories including those producing heterologous proteins in different bacterial and fungal hosts like A. niger highlights possible applications of this approach for improving protein production (Campbell et al. 2017). Synthetic biology approaches can also be used to engineer strains for improved yield and productivity; it provides an opportunity to engineer biology in a more

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standardized and rational fashion, and several examples using a synthetic yeast genome have been described (Liu et al. 2018). One of these approaches is the rewiring of metabolic flux by deleting genes that negatively affect enzyme production. Using protein localization tags, beneficial modifications (by engineering the cellular location of the reaction) of the metabolic flux can be obtained (MartinsSantana et al. 2018). In yeast, several organelles (mitochondria, peroxisomes, and even the cell wall) have been redesigned to improve processes such as bioethanol production (Hammer and Avalos 2017).

4.6

The Evolution of Methods for Construction of Production Strains to Adapt to Regulatory Requirements

In the early years of modern biotechnology, production strains were constructed using free replicating plasmids and antibiotic resistance as a dominant selective marker on the plasmid containing the expression cassette for the gene of interest. Although not a strict requirement for technical enzymes, the presence of genes encoding antibiotic resistance imposes additional requirements from regulatory authorities on food and feed enzymes (see Sect. 6). Thus, auxotrophic markers or markers that enable growth in specific nitrogen or carbon sources have been introduced, e.g., the amdS gene (Christensen et al. 1988). Genetic stability issues have also resulted in the scarcity of strains constructed using free replicating plasmid based on, e.g., the Aspergillus AMA1 fragment (Aleksensko and Clutterbuck 1997). Even though transformation frequencies are much lower than for free replicating plasmids, current production strains are therefore constructed using chromosomal integration. Because NHEJ is the dominant mechanism for DNA integration in industrial fungal strains, initially, production strains were the result of ectopic (nonhomologous) integration. The regulatory requirement—determination of the site of integration of the expression cassette—has driven the construction of strains through homologous recombination at one of more defined chromosomal locations. A suitable host strain should harbor multiple and reusable integration loci (i.e., a “plug and play” system) in such a way that newly identified yield improvements can be readily incorporated to produce different enzymes (Udagawa 2017).

5 Environmental Aspects of the Yield Improvement in Industrial Recombinant Enzyme Production Enzymes enable the increase in quality, speed, and yield of numerous processes and reduce both energy consumption and the use of chemicals. Life cycle assessment of industrial enzyme production has been performed. The fermentation step accounts for half of the environmental impact of enzyme manufacturing (energy

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consumption), while recovery and waste management represent 1/8 of the total contribution in the commercial setting at Novozymes A/S in Denmark (Nielsen et al. 2007). The environmental impact resulting from the optimization of enzyme yields from using recombinant technologies has been compared to non-recombinant enzyme production. Considerable and consistent improvements (e.g., energy consumption, global warming, nutrient enrichment, ozone formation) have been clearly demonstrated for the use of optimized recombinant strains (Nielsen et al. 2007). The environmental impact of enzymes in some applications is quite striking. The textile industry has a reputation as one of the most polluting industries in the world. The problem is the traditional, extensive use of chemicals, energy, and water to meet the needs of the fashion industry. For instance, it takes about 150,000 L of clean water to make one ton of knitwear. The estimated savings if the world’s textile industry implements the whole range of available enzymatic solutions in the production of cotton textiles include 28% of water consumption which is 1250 billion liters of water. Additionally, the use of textile enzymes will save 80% of chemical consumption—that is, 10 mn tons of chemicals—and up to 25% of the energy consumption. Numbers are based on a world production of 25 million tons of cotton textiles per year which is a very conservative estimate and LCA studies made by Novozymes.

6 Approval of Enzymes for Use as Food Processing Aids and in Feed Applications In principle, the same safety considerations apply to enzymes derived from native or recombinant microorganisms. The main aspect of the safety of food enzymes is the production strain and the potential risk of producing compounds with a toxicogenic or pathogenic effect (e.g., mycotoxins) during manufacturing, i.e., mainly during fermentation (Pariza and Johnson 2001). Contrary to biopharmaceuticals, industrial enzymes are typically not purified products but rather derive from a concentrated fermentation broth where the production organism has been removed. This principle of “contained use” where the enzyme product does not contain material from the production organism is important for the classification of the product and requirements from regulatory authorities. Furthermore, the use of antibiotic resistance genes as selective markers for strain construction should be avoided due to concerns of its transferability and effect on the spread of resistance to antibiotics normally used to treat infections in humans. In this section, a short overview of the requirements for the description and characterization of the production organism used to manufacture enzymes for use as food processing aids and in feed applications is presented. A comprehensive review on the regulations, definitions, and approval processes in different regions of the world is available (Magnuson et al. 2013). Significantly lower requirements for the strain description applies for technical and detergent enzymes. However, the

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general trend is that the requirements for the characterization for the production strain used for food and feed applications increase steadily.

6.1

Regulatory Considerations for the Approval of Recombinant Food Aids and Feed Enzymes in the European Union

The European legislation for the use of additives, flavorings, and enzymes in foods is set out in the Food Improvement Agents Package (FIAP). A regulatory authority (European Commission, Directorate General for Health and Consumers) and an advisory scientific body responsible for the risk assessment [EFSA (European Food Safety Authority)] participate in the approval process to ensure, e.g., the safety of GM food products for animal and human consumption (Aguilera et al. 2013). These regulations were introduced in 2009 and reached a key milestone on March 11, 2015, the first deadline for food enzyme manufacturers to submit dossiers on all food enzymes currently sold or used in Europe under the new regulation. More than 300 enzyme dossiers were submitted. These submissions are required to ensure compliance of the enzyme industry and its customers when FIAP gets fully implemented in 2021. An update of the requirements for the characterization of the production strain has recently been implemented (EFSA 2018). For enzymes manufactured using a recombinant strain, a comprehensive description of the genetic elements and methods used to construct the strain from a publicly available parental strain is required. One major aspect is the requirement to demonstrate the absence of the production organism and DNA in the final enzyme product to enable the approval as a Category 2 product (“Complex products in which both GMMs and newly introduced genes are no longer present”) as opposed to the use of, e.g., a recombinant organism directly in the food. To issue an opinion on the safety of the enzyme, EFSA requires—among many other aspects relating to, e.g., the technical need or calculation of the margin of safety for intake—a description of each modification step from the parental to the actual production strain, in addition to data on the taxonomical identification, safety, pathogenicity, toxicological studies, history of safe use, genetic stability and the absence of vector or any other sequence used during construction steps that is not intended for insertion into the genome, etc. (Aguilera et al. 2013). In the latest update, and although not yet a requirement for fungal hosts, the genome sequence (i.e., a comparison between the parental and production strain) can be used as the basis for the description of the changes introduced in the production strain. Additionally, further constrains have been adopted for the PCR method used for demonstration of the absence of DNA in the product (EFSA 2018). In the current stage, where EFSA has a backlog of submitted dossiers in 2015 and the expected publication of a positive enzyme list in 2021, it is difficult to predict the time that will be required to obtain approval of a new food or feed enzyme in Europe.

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Regulatory Considerations for the Approval of Recombinant Food Aids and Feed Enzymes in the US

In the US, FDA and the GRAS (generally regarded as safe) notification system provides a relatively short standard review and approval time. GRAS food ingredients are generally recognized, among qualified experts, to be safe under the conditions for its intended use (GRAS final rule 21CFR170.250(b)). In the past, FDA reviewed GRAS affirmation petitions for enzymes. This process has been replaced by a voluntary notification program under the FDA’s proposed regulation. A successful notification process leads to an FDA letter to the manufacturer stating the agency has no questions on the manufacturer’s conclusion that the use of the enzyme for the described application is safe (Olempska-Beer et al. 2006). There is no specific regulation governing enzymes. Depending on the intended use and the method, enzymes are regulated as direct (i.e., added to the food product) or—most common—secondary additives (i.e., processing aids in food manufacturing to fulfil a technological purpose) or GRAS substances (Magnuson et al. 2013). In 2017, the accumulated number of submitted GRAS notifications in a recent pilot program (1997–2015) was 678, including enzymes, fats and oils, probiotics, sweeteners, and carotenoids. With a total of 70 enzyme notifications, the average review time by the FDA was 168 days. In 68 of the notifications, the application did not trigger questions whereas two applications were withdrawn (Hanlon et al. 2017). The science-based process used by FDA for risk assessment is more of a productcentric approach where safety is assessed on the product and not on the methodology used to bring about the product (i.e., GM or not, CRISPR or not, etc.). Requirements for strain description and characterization are not as comprehensive as in other geographies. The data included in the enzyme dossier can be accessed by third parties following the freedom of information act (FOIA, a federal law that allows for the full or partial disclosure of previously unreleased information and documents controlled by agencies like FDA). This has direct consequences on the extent of the information that enzyme manufactures can disclose on, e.g., strain construction and gene expression technology in a GRAS notification.

6.3

Evolution of Regulatory Requirements in the Rest of the World

In the rest of the world, the trend is also a steady increase in the requirements for the characterization of the production strain fuelled by the development of new technologies and methods. Currently, data on the absence of DNA in the enzyme product that has been a requirement in Europe and Japan for several years, are also requested in China. Although FIAP has created a comprehensive, high-level standard for the characterization of production strains, countries like China have additional requirements compared to, e.g., FIAP that include a comprehensive genetic and expression

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stability study that spans to five consecutive cultivations of the production strain. Similarly, a requirement to identify the chromosomal location of the inserted DNA exists in Japan. Although technically feasible with novel sequencing technology, this can be rather challenging if the production strain is constructed using ectopic integration in multiple copies at single or multiple locations, considering the relatively high level of repeated DNA sequences in a fungal genome. Finally, alignment of requirements between regulatory authorities would enable the characterization of the production strain and the submission of dossiers according to one standard. As an example, the modification of the host strain can be described with less detail in a GRAS notification than in a FIAP dossier where each step needs to be described in detail and supported with data confirming that the modification has happened as intended. Alignment of requirements for strain characterization in enzyme dossiers would represent a tremendous advantage for industrial biotechnology companies.

7 The Future of Industrial Enzyme Production in Fungal Hosts Although many commercial enzymes have been successfully produced in filamentous fungi, it is still not possible to predict the necessary combination of engineered cellular functions and expression cassette elements required for optimal production of a novel enzyme in a fungal host. Increasing numbers of fungal genome sequences are available, but gene annotation is still a limitation. A large number of putative genes still have an unknown function. Assigning functions to these genes using the appropriate method (transcriptomics, proteomics, etc.) will improve the knowledge about the biology and physiology of the host strains and contribute to our understanding of protein production and secretion in filamentous fungi. With increasing accessibility to CRISPR and “omics” technologies, the expected growth of genomic and experimental datasets is of paramount importance to accelerate knowledge about complex cellular processes related to enzyme production and the development of new and improved fungal hosts. Being able to construct many strains using targeted integration should enable genome-wide screening in fungi as it has been performed in yeast to identify optimization candidate genes (e.g., via knockout libraries). Novel technologies allowing for even larger number of strains to be tested (e.g., ultra-HTS) will contribute to this. Combining these approaches with emerging genome models and available “omics” will accelerate the capability of fungal cell factories to produce more diverse classes of enzymes at higher yields. This will enable production of many novel enzyme classes for existing and novel applications. The development of novel fungal species as hosts may also facilitate production of difficult to express enzymes using traditional systems.

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Increasing knowledge of SM pathways and genes involved will enable the construction of safer strains containing deletions in one or more SM gene clusters, especially for novel host species. The continuous improvement of technologies like genomics to characterize the production strains will impact the requirements from regulatory authorities for the characterization of the strains used for food and feed enzyme production. The promise of industrial biotechnology becomes more relevant as we face the need of new biological solutions to replace chemicals and to support sustainability in a growing world. Acknowledgments We thank Amitabh Gupta (Novozymes) for the support in the patent survey.

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Chapter 8

Meeting a Challenge: A View on Studying Transcriptional Control of Genes Involved in Plant Biomass Degradation in Aspergillus niger Jing Niu, Arthur F. J. Ram, and Peter J. Punt

1 Introduction In any organism, it is the regulated pattern of gene expression that determines the phenotype. Gene regulation is the means by which cells orchestrate gene expression to ensure that the right genes are expressed at the right time. The proper control of gene expression is important for cells to adapt to changing conditions such as nutrient availability and environmental stresses. Industrially relevant filamentous fungi such as Aspergillus niger and Trichoderma reesei are well-known producers of enzymes. They produce and secrete these enzymes such as cellulases, xylanases, and amylase in the medium in large quantities reaching 30–100 g of enzymes per liter. The expression of these enzymes and the metabolic network required for the metabolism of the monosugars liberated from the polymeric substrates is tightly regulated to ensure that the genes are highly expressed only when the substrate is present. Our knowledge on how these enzyme networks are controlled has expanded dramatically in the last few years due to recent developments in genome sequencing, and development of gene editing and gene knockout methods, which in combination allow new and efficient transcriptome analysis and forward genomics approaches to study gene regulation. With the coming of age of genome and transcriptome sequencing and genome editing technologies for filamentous fungi, not only the amount of available genetic data from these organisms has enormously expanded, but also the way molecular genetic research is performed has completely changed. J. Niu · A. F. J. Ram Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands P. J. Punt (*) Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands Dutch DNA Biotech, Hugo R Kruytgebouw, Utrecht, The Netherlands e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_8

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This is, in particular, relevant for the study of regulatory gene networks since the use of single-gene-based molecular genetic approaches have shown to provide limitations in elucidating these seemingly simple but, as we now know, actually rather complicated networks. As we will describe in this chapter, we now have the opportunity to switch back from molecular genetic approaches targeted on single (or a few) genes to biological approaches more resembling those in the time of classical genetics, but now with the benefit of using of systems biology approaches. The ease of using genome-wide technologies allows the straightforward analysis of individual mutant strains obtained from the classical screening and selection approaches. In this review, we will describe functional genomics and transcriptomics approaches to study fungal gene function and gene regulation in relation to controlling expression of enzymes involved in plant cell biomass degradation. As an example for this approach, we review the recent breakthrough results obtained for understanding the regulatory network involved in the breakdown and utilization of pectin in A. niger.

2 Functional Genomics Approaches 2.1

Genome Sequencing Technologies

Sequencing DNA molecules contributes greatly to research progress in biology and medicine. During the last 10 years, considerable progress has been made in DNA sequencing technologies, allowing individual researchers to sequence fungal genomes within a few weeks and allow transcriptome analysis to study gene regulation without the need to generate microarrays. DNA sequencing techniques have been going through three generations. Sanger sequencing is the most important first-generation sequencing technique. It has been the most widely used sequencing technique before being replaced by the next-generation sequencing, such as Roche 454, Illumina, and ABI/SOLiD which allows sequencing DNA samples in high throughput. Most recently, the third-generation sequencing techniques (PacBio and MinION) were developed for single molecule sequencing. Table 8.1 shows the characteristics of different DNA sequencing methods. Starting from the second-generation approaches, these different sequencing technologies provide a powerful tool for research on functional genomics of fungi. One important application of these technologies is whole genome sequencing. There has been substantial investment in sequencing of filamentous fungi genomes, with a clear focus on sequence analysis of a very important class of fungi, the Aspergilli. The first sequenced filamentous fungus was Neurospora crassa, a well-established filamentous fungus for basic fundamental research (Galagan et al. 2003; Mannhaupt et al. 2003). Analysis of the sequencing data has provided unprecedented overview of various aspects of Neurospora biology that helped to identify genes involved in secondary metabolism. The first sequenced Aspergillus genomes include the model organism Aspergillus nidulans, as well as Aspergillus fumigatus and Aspergillus oryzae in 2005

a

2005

2006

2nd

2nd

3rd

3rd

PacBio

Oxford Nanopore MinION

Approach Synthesis with dye terminators Sequencing by synthesis Sequencing by synthesis Sequencing by synthesis Direct sequencing 4,000,000

400,000 10,000

1300

25–50

100

>200,000 30,000,000

Average read length (bp) 800

Reads per run ~100

60–70

80–90

>95

>95

% accuracy >99

Website: https://flxlexblog.wordpress.com/2016/07/08/developments-in-high-throughput-sequencing-july-2016-edition/

2015

2011

Year 2002

Generation 1st

Technology Sanger ABI 3730xI 454/Roche FLX Illumina/Solexa

Table 8.1 Characteristics of different DNA sequencing methods used for whole genome sequencing

Mikheyev and Tin (2014), Laver et al. (2015) Websitea

References Rhoads and Au (2015), Kerstens (2010) Kerstens (2010) Websitea Kerstens (2010) Websitea Websitea

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(Galagan et al. 2005; Machida et al. 2005; Margulies et al. 2005). Comparison of the genomes of these species has revealed specifically enriched sequences for genes involved in metabolism in A. oryzae (Machida et al. 2005) and provided insights into the evolution of the eukaryotic genome (Galagan et al. 2005). Subsequently, genomes of Aspergillus flavus, Aspergillus fischeri, Aspergillus clavatus, and Aspergillus terreus were also sequenced (Payne and Loomis 2006; Fedorova et al. 2008; Arnaud et al. 2012). Two Aspergillus niger strains CBS513.88 and ATCC1015 were sequenced in 2007 and 2011, respectively (Pel et al. 2007; Andersen et al. 2011). In a more recent study, ten more Aspergillus strains were sequenced and annotated (de Vries et al. 2017). Most recently, the genomes of 26 Aspergillus species of section Nigri were completely sequenced and annotated (Vesth et al. 2018) which in combination with the other analyses allowed for inter- and intraspecies comparison of 32 isolates in the Nigri section. Comparative genome analysis using the sequence data obtained in these studies has facilitated to establish a correlation between genotype and phenotype and provided insights into fungal evolution. These genome sequence data apparently have provided a resource-rich platform for evolutionary and functional genomics studies and provided reference genomes for transcriptomic studies via RNA sequencing. On the other hand, however, identification of responsible mutations for specific characteristics in random mutants just by sequencing is challenging and inefficient. Other methods are therefore needed to be combined with genome sequencing to more efficiently identify target mutations. In the following sections, the use of these platforms will be described in relation to the genetic characterization of mutants and to study gene functions.

2.2

Methods to Genetically Identify Mutant Genes from Forward Genetic Screens

During the last decades, forward genetic screens have identified many new genes in various species and contributed greatly to our understanding of gene functions. The essence of a forward genetic screen is to make random mutations to create mutants with specific phenotypes and to identify the genetic basis of the mutations responsible for these phenotypes. In forward genetic screens, chemicals (e.g., ethyl methanesulfonate, EMS) or radiation-based mutagens (e.g., UV) are commonly used to generate random mutants, which are then screened for interesting mutant phenotypes. For the genetic identification of mutations from a forward screen, different approaches can be used. In the pre-genomic era, genetic linkage analyses in combination with chromosome walking and complementation approaches with cosmid libraries were generally used.

2.2.1

Genetic Linkage Analysis-Based Methods

Genetic linkage analysis utilizes genetic markers to map the mutation of interest. Depending on the genetic background of the species, a variety of crossing schemes

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can be used to map the mutation that causes the phenotype of interest to a specific region of the genome. During the crossing process, markers which are closely linked to the causal mutation will be co-segregated with the causal mutation due to infrequent recombination between them. Therefore, there is a distinct allele distribution of the mutation and the closely linked markers in the progeny from a cross. Once the mutation region is mapped, a targeted search, e.g., via chromosome walking, a technique that has been developed for identifying genomic sequences contiguous to a known piece of DNA, by generating genomic DNA libraries and identifying those clones in these libraries containing these contiguous genomic DNA sequences, can be conducted to find the actual causal mutation within that region by sequence analysis (see for review Schneeberger 2014). Genetic mapping by this method is largely dependent on the density of the polymorphic markers genotyped. Moreover, as this method can only locate the genomic region that contains the causal mutation, further sequencing within this region is required (Schneeberger 2014).

2.2.2

Complementation Analysis-Based Methods

Confirmation of the mutated candidate gene responsible for the phenotype can also be achieved by complementation analysis. In this approach, a gene library is constructed by ligating genomic DNA fragments into, e.g., a cosmid vector. Introduction of the cosmid library into the mutant strain allows selection of transformants functionally complementing the causal mutation. If the cosmid clone contains a wild-type allele of the mutated gene, it can rescue the phenotype by complementing the endogenous disrupted allele. Further analysis of complementing cosmid clones will reveal the gene contained in the complementing sequences (Damveld et al. 2008; Punt et al. 2008; Meyer et al. 2009). This method has been successfully used in A. niger, for example, for the identification of PrtT, a unique regulator of extracellular protease encoding genes (Punt et al. 2008). However, the complementation method is time- and labor-intensive and has some limitations, such as that the gene might be lacking in the library, that it is limited to recessive mutations, and that certain mutant phenotypes are difficult to screen for complementation among thousands of transformants.

2.2.3

Next-Generation Sequencing-Based Methods

With the advent of next-generation sequencing (NGS) techniques, it is possible to directly sequence individual mutant genomes to identify causal mutations (Srivatsan et al. 2008). However, multiple mutations might be found in mutants and requiring a lot of research to identify the mutation responsible for the phenotype. Therefore, several approaches have been developed to facilitate identification of the mutation related to the phenotype of the mutant. Recently, the combination of the classical bulked segregant analysis (BSA) (Michelmore et al. 1991) with NGS has proven to greatly accelerate this process, leading to the development of an approach named

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mapping-by-sequencing (Schneeberger et al. 2009; Niu et al. 2016; Downes et al. 2014; Tan et al. 2014). BSA is traditionally used to identify makers linked to gene(s) of interest (Michelmore et al. 1991). It involves comparing the pooled DNA sample of mutant segregants with that of wild-type segregants. Both segregants result from a single cross of the parental strains. The individuals in each pool have the same version of the target gene (either wild type or mutated) but are arbitrary in all the other genes. By genome sequencing, single-nucleotide polymorphisms (SNPs) are analyzed between the two parental strains and serve as markers. Markers that are homogeneously polymorphic between the two segregant pools are within physical proximity of the mutation and thus genetically linked to the locus of the target mutation (Michelmore et al. 1991; Lister et al. 2009). This approach, combining BSA with NGS, allows simultaneous mapping and identification of the target mutation. In our lab, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation gene laeA, which is responsible for the non-acidifying phenotype in A. niger (Niu et al. 2015). In case of a very specific mutant selection approach, spontaneous mutants with the same mutant phenotype can be directly used to identify the causal mutations without bulk segregant analysis as there are less nontargeted mutations and the selection scheme only results in one phenotype in multiple mutants. In our lab, directly sequencing of several individual A. niger mutants revealed a transcriptional repressor which controls expression of genes for D-galacturonic acid utilization (Niu et al. 2017).

2.3

Genomics-Based Functional Analysis of Regulatory Genes

The availability of high-quality genome sequence of A. niger in combination with improved annotations of the genome has resulted in the identification of 11,800 potential genes (Niu et al. 2015), of which most still await further functional analysis. There are two common ways to study the function of a gene in vivo: deletion analysis or overexpression analysis. In this review, we will focus on two more recent and highly efficient technologies for making gene deletion mutants: the split marker approach and the CRISPR-Cas9 system studying the function of regulatory genes in particular.

2.3.1

NHEJ Mutants Combined with the Split Marker Approach

Targeted deletion of a gene of interest (GOI) requires homologous recombination (HR). However, random integration of DNA via the nonhomologous end joining (NHEJ) pathway is the dominant or preferred way of DNA integration in filamentous fungi, resulting in less frequent occurrence of HR and therefore low efficiencies in

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obtaining gene deletion mutants (Haber 2000). Therefore, approaches were required to make this more efficient. An important breakthrough in fungal genetics was the discovery (Ninomiya et al. 2004) of genes encoding proteins responsible for NHEJ in fungi. Making NHEJ-deficient mutants was first performed in N. crassa via deletion of the ku70 gene, and deletion of the ku70 gene was shown to lead high frequencies of HR and consequently high efficiencies of obtaining targeted mutants (Ninomiya et al. 2004). In addition to ku70, deleting other components of the NHEJ machinery, such as ku80 and lig4, resulted in fungal NHEJ-deficient recipient strain for gene-targeted deletion (for review, see Hruby et al. (2010) and references therein). Gene-targeted deletion is usually performed by constructing a linear DNA fragment that contains the 50 and 30 flanks of the gene of interest (GOI) and a selection marker between them. The easiest way to generate these fragments is by fusion PCR in which the three fragments (50 flank, selection marker, and 30 flank) are fused together by primer overlap extension. Although these methods in general work well, the full-length PCR fragments are quite large in size (4–5 kb, depending on the size of the flanking sequence and selection marker used) with sometimes leads to PCR problems and low yields. To circumvent amplification of these large fragments, the split marker approach was developed. In split marker approach, the gene deletion cassette consists of two fragments. The first fragment contains the 50 flank of the GOI fused with a 30 truncated version of the selection marker. The second fragment contains a 50 truncated version of the selection marker that still overlaps with the first one and is fused with 30 flank of the GOI (Fairhead et al. 1996; Nielsen et al. 2006; Goswami 2012). Using this approach, PCR fragments are smaller in size (3 kb) thereby increasing success rate and yield of the PCR reaction. Both fragments are transformed simultaneously to the strain of choice. Strategies using the split marker approach lead to more efficient gene deletion in strains with an intact NHEJ machinery (Nielsen et al. 2006). We have used the spilt marker approach in combination with NHEJ mutants for generating in an even more efficient way gene deletion mutants (Niu et al. 2016; Arentshorst et al. 2015). Although these two methods can be used separately, the NHEJ mutants help to significantly increase the frequency of homologous recombination when using the split marker approach.

2.3.2

CRISPR-Cas9 Approaches

Genome editing technologies that allow deletion, insertion, and modification of DNA sequences have greatly accelerated our understanding of the functional organization of the genome. Currently, the most rapidly developing genome editing technique is the CRISPR-Cas9 system, a RNA-guided DNA editing technique that originates from type II CRISPR-Cas systems. In bacteria, CRISPRs (clustered regularly interspaced short palindromic repeats) provide acquired immunity against viruses and plasmids (Horvath and Barrangou 2010; Wiedenheft et al. 2012). Typical CRISPR loci consist of a CRISPR array of repeated sequences separated by variable sequences called spacers, which match the sequences within the invading

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foreign DNA (protospacer) and are often adjacent to CRISPR-associated (Cas) genes that encode RNA-guided DNA nucleases (Hsu et al. 2014). During adaptive immunity, certain Cas enzymes incorporate segments of the invading DNA into the CRISPR array as spacers. In type II CRISPR-Cas systems, the CRISPR array is firstly transcribed into pre-CRISPR RNA (pre-crRNA). A trans-activating crRNA (tracrRNA) then hybridizes with pre-crRNA to form a RNA duplex, which can be cleaved and processed by RNAse III to produce mature tracrRNA:crRNA hybrids. In the hybrid, the small crRNA contains a repeat portion that hybridizes with tracrRNA and a spacer portion that can recognize the target DNA sequence by base pairing. The tracrRNA:crRNA duplex then pairs with the target DNA sequence and directs the Cas protein to introduce a site-specific double-strand break (DSB) in the DNA (Hsu et al. 2014; Doudna and Charpentier 2014; Sander and Joung 2014). In the to date most commonly used CRISPR-Cas9 genome editing system, the tracrRNA:crRNA duplex is engineered as a single guide RNA (sgRNA). By redesigning crRNA, the CRISPR-Cas9 system can target any region of interest in the genome as long as it is adjacent to a protospacer adjacent motif (PAM). Due to ease of use and efficiency of this technique, it holds great promise to help us understand gene function. The CRISPR-Cas9 system has been tested in several Aspergillus species and has been shown to be effective in targeting genes. For example, CRISPR-Cas9 can efficiently introduce directed mutations into the yA gene in A. nidulans, the albA and pyrG gene in A. aculeatus, and albA homologs in five Aspergilli (A. brasiliensis, A. carbonarius, A. luchuensis, A. niger, and A. tubingensis) (Nodvig et al. 2015). Moreover, it has been reported that a strain generated by CRISPR-Cas9 and containing a pyrG marker is capable for iterative gene targeting (Nodvig et al. 2015). Combining CRISPR-Cas9 gene targeting with transformation with “repair DNA” allows not only disrupting a gene but also specific gene editing. Currently, multiple approaches for in vivo expression of Cas9 and of the sgRNA have been described for filamentous fungi, in particular Aspergillus species (e.g., Sarkari et al. 2017; Song et al. 2018; Zheng et al. 2017, 2018, and references therein). Furthermore, also in vitro synthesized and assembled Cas9ribonucleoprotein complexes have successfully been used in filamentous fungi (Al Abdallah et al. 2017, 2018). Also, approaches allowing the use of oligonucleotide-mediated marker-free gene editing have been developed for filamentous fungi (Nodvig et al. 2018). In fact, as shown in Penicillium chrysogenum, both marker-based and marker-free CRISPR/Cas9 tools are available in filamentous fungi to make genome modifications (Pohl et al. 2016). On the other hand, the CRISPR/Cas9 system also has its limitations, the major one of which is the off-target effect. Currently, the off-target activity of the technique is still quite high (Zhang et al. 2015), which may compromise the genotype-phenotype correlation identified. Further developments are therefore needed to improve the specificity of the current CRISPR gene editing system by using alternative guide RNA and/or nuclease variants. Recently, a new genome editing system based on the RNA-guided endonuclease Cpf1, with clearly different characteristics from Cas9, has been developed in Saccharomyces cerevisiae which could also be an excellent addition to the current genome editing toolbox for filamentous fungi (Verwaal et al. 2018). Nonetheless,

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CRISPR-Cas9-based gene editing is still a very promising technique to employ genetic engineering in these fungi and holds great potential in helping us understand their biology.

2.3.3

Overexpression Analysis

Yet another way to study gene function is by overexpressing the GOI and study the phenotypic effects of overexpression. The most common strategy of overexpressing a gene is to put the gene under control of a strong constitutive promoter or using an inducible promoter system such as the Tet-on system (Vogt et al. 2005; Meyer et al. 2011). The Tet system is involving the repressor protein TetR from Escherichia coli, which binds to the operator sequence (tetO) of the Tn10 in the absence of tetracyclines and prevents the transcription of the operon. In the presence of tetracycline, TetR dissociates from tetO, initiating the transcription of the operon (Beck et al. 1982). This system was modified to generate a hybrid transactivator tTA by combining the TetR with the minimal transcriptional activation domain derived from the herpes simplex virus protein 16 (VP16) for application in eukaryotic systems. In this system (Tet-off system), tTA stimulates gene expression in the absence of tetracycline. Alternatively, a Tet-on system has been developed. In the Tet-on system, the reverse hybrid transactivator rtTA was generated by introduction of mutations to TetR, which lead to induction of gene expression in the presence of tetracycline instead of repression. The Tet-on system can be used for maximum expression levels by placing several copies of the tetO sequence upstream of a minimal promoter. Both the Tet-ON system and the Tet-OFF system have been adapted to be functional in A. niger (Meyer et al. 2011; Wanka et al. 2016). Overexpression using these strategies often gives rise to an exaggerated phenotype due to overexpression of the targeted genes of the regulatory network, which directly imply the function of the gene.

3 Transcriptomics and Related Technologies to Study Regulatory Networks As described above, to dissect the role of regulatory proteins such as transcriptional activators in the regulatory networks, overexpression and deletion strategies are frequently used to study their effects on the expression of their target genes. The set of genes that are regulated as a unit or controlled by the same regulatory gene comprise a regulon. The regulon includes genes whose expression is collectively controlled and likely to be involved in a specific functional program. The general approaches described below allow us to study gene regulation on a large scale to identify these regulons. In Sect. 4, we review a study on a specific Aspergillus niger regulatory circuit on pectin utilization in which several of these technologies have been used.

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First-Generation Genome-Wide Transcriptome Analysis: Microarrays

Traditional approaches of detecting gene expression include northern blot analysis, in situ hybridization, and quantitative reverse transcriptase-PCR (Q-PCR). While they are useful for studying single or a few genes, it is not possible to systematically survey genome-wide gene expression using these traditional methods. The invention of DNA microarrays has greatly transformed the traditional way of studying gene expression and allowed to detect and quantify tens of thousands of genes simultaneously (Kurella et al. 2001). Since its conception in 1995, DNA microarrays have developed into a powerful tool for surveying gene expression efficiently and comprehensively on a genomic scale. Despite their widespread use, DNA microarrays continue to have some limitations, which include accuracy of genome annotation, inflexible probe design, and strain variations which may influence hybridization signals of genes containing multiple DNA polymorphisms. Moreover, DNA microarrays can only be developed for species whose genome sequence has been determined. With the advent of even cheaper high-throughput DNA sequencing technology, DNA microarray is rapidly replaced by RNA sequencing (RNA-Seq).

3.2

RNA-Seq

RNA-Seq is a fast emerging technology that uses next-generation sequencing to map and quantify transcriptomes. It provides a powerful tool to reveal many different properties of the transcriptome and to accurately measure all transcripts of an organism, including messenger RNAs, microRNAs, small interfering RNAs, and long noncoding RNAs (Wang et al. 2009). The typical protocol for RNA-Seq is to extract RNA, convert it into a library of cDNA fragments and attach them to sequencing adaptors, and sequence the cDNA library using high-throughput sequencing technology. After sequencing, the resulting reads, including exonic reads, junction reads, and poly (A) end reads, can be mapped to a reference genome or de novo assembled if the genome is unknown. This generates a base resolution expression profile for each gene in the genome. Comparing with microarrays, RNA-Seq has several key advantages. First, it can be used for species whose genomic sequences have not yet been determined and does not require an optimal genome annotation to predict open reading frames. Second, it can actually reveal transcript structure to a single-base resolution. Many properties of transcript structure (e.g., the precise location of transcription boundaries, the connectivity of exons, etc.) can therefore be accurately determined, making it useful for studying complex transcriptomes. Third, it has much lower background signals for sequence mapping and a higher dynamic range for measurement of transcriptional levels compared to microarrays. Fourth, RNA-Seq is highly accurate for measurement of expression levels, and the results have high levels of reproducibility. Finally, RNA-Seq needs less RNA sample because no cloning steps are required.

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ChIP-Seq Analysis

Essential components of any gene regulatory network are DNA-binding proteins, such as transcription factors. Transcription factors can be activators, whereby activation of the transcription factor stimulates gene expression, but transcription factors can also act as repressors. DNA-binding transcription factors (either acting as an activator or repressor) often bind to specific transcription factor binding sites in the promoter of target genes, thereby controlling their expression. Therefore, DNA– protein interactions play a fundamental role in the regulation of gene expression. Historically, DNA–protein interactions can be identified by EMSA, footprinting, and chromatin immunoprecipitation (ChIP) experiments. In ChIP studies, DNA-binding proteins are immunoprecipitated with a protein-specific antibody, the precipitated protein–DNA complexes are then purified, and the bound DNA is characterized. Further development of ChIP combines this technique with genomewide microarrays, leading to the invention of ChIP-chip method which allows hybridizing fluorescently labelled bound DNA to an appropriate microarray at a relatively high throughput (Ren et al. 2000). With recent advances in the nextgeneration sequencing, ChIP sequencing (ChIP-seq) was developed to sequence the released bound DNA with short reads at a higher throughput. The short reads delivered in ChIP-seq allow identification of interaction sites with more precision. ChIP-seq has first been applied to identify the binding sites of STAT1 and NRSF at the genome-wide scale (Johnson et al. 2007; Robertson et al. 2007) and has been used in several studies to define direct binding sites. CHIP-seq has also revealed that the transcription factors CLR-1 and CLR-2 in N. crassa form a homocomplex rather than a heterocomplex to regulate genes involved in plant cell wall deconstruction (Craig et al. 2015). This finding provides important insights into the interconnection between transcriptional circuits when the fungus responds to environmental signals. The ChIP-seq technology represents a powerful tool to verify direct binding of a transcription factor to a promoter element.

4 Regulation of Gene Expression for Pectin Utilization in A. niger With the rapid development of DNA and RNA sequencing technologies, more and more genomic sequences and transcriptional data of fungi are available. Bioinformaticians assemble whole genomic sequence for each species and create websites to store the sequences information and related protein information and other information as soon as they are available. Several websites are accessible such as JGI (http://genome.jgi.doe.gov), fungal special database FungiDB (http://fungidb. org/fungidb/), or Aspergillus genome database AspGD (http://www.aspgd.org/) to view or download information. The study of gene regulation is clearly focused on studying the role of pathway-specific and wide domain regulatory proteins, which, in

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the majority of cases, are DNA-binding proteins governing transcription. These so-called transcription factors consist of two or more domains. One is a DNA-binding domain (DBD), which attaches to a specific DNA sequence that is present upstream to the translational start site of a regulated gene. The second is a transactivation domain (TAD) to which other proteins (co-regulatory proteins) bind. DBD domains are commonly classified into different types including zinc finger, helix-turn-helix, leucine zipper, and helix-loop-helix based on the secondary structure. Zinc fingers are categorized into three main classes Cys2His2 (C2H2), Cys4 (C4), and Cys6 (C6) (MacPherson et al. 2006; Shelest 2017). Proteins with a Zn(II) 2Cys6 domain are found exclusively in fungi and yeasts. Chang et al. conducted genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus resulting in 199 genes encoding proteins with a Cys6 domain (Chang and Ehrlich 2013). Detailed genome mining in A. niger revealed the presence of 694 putative DNA-binding transcription factor of which 453 belong to the Zn(II)2Cys6 zinc cluster family (Ram, unpublished). Furthermore, a recent study, by performing genome-scale surveys of transcription factors, compared the distribution of regulator genes across different fungal species and showed that the Zn2Cys6 and fungal-specific domain transcription factors are most abundant in ascomycetes (Todd et al. 2014). A. niger is an industrially important enzyme producer; it can produce a wide range of enzymes involved in modification and degradation of plant polysaccharides, such as starch, inulin, cellulose, hemicellulose (mainly xylan and arabinan), galactomannan, and pectin (de Vries and Visser 2001). Polysaccharides are polymeric carbohydrates, composed of ten to up to several thousand monosaccharides linked together by glycosidic linkages. Plant cell wall polysaccharides can be classified into storage components (starch and inulin) and structure components such as cellulose, hemicellulose, and pectin. The most common monosaccharides that appear as parts of polysaccharides are glucose, fructose, xylose, arabinose, galactose, rhamnose, and mannose. In addition, galacturonic acid (GA) is the most important sugar acid in plant cell wall and present as the main component of pectin.

4.1

Pectin

Pectin is the main constituent of the middle lamella as found in the outermost layer of plant cell wall. Galacturonic acid (GA) is the most abundant component of pectin. Pectin is a collective name for GA-rich structures, and four substructures have been defined which include (1) homogalacturonan (HGA) or polygalacturonic acids (PGA), (2) xylogalacturonan (XGA), (3) rhamnogalacturonan I (RG-I), and (4) rhamnogalacturonan II (RG-II) (reviewed in Mohnen 2008). PGA is a linear polymer, consisting of α-1,4-linked D-galacturonic acid residues. The backbones of XGA and RG-II are made up of α-1,4-linked D-galacturonic acid residues. In XGA, β-D-xylose residues are β-1,3-linked to GA residues of the PGA backbone. The backbone of RG-I is made up of alternating GA and L-rhamnose residues (reviewed

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in Mohnen 2008; Leijdekkers et al. 2015). The side chains of RG-I are mainly arabinan and arabinogalactan comprising of L-arabinose and D-galactose residues (Mohnen 2008). RG-II is the most complex structure, and side chains of RG-II are composed of up to 12 different types of monosaccharides in more than 20 different linkages (Mohnen 2008). The abundance of each substructure varies with plant species, but typically homogalacturonan is the most abundant polysaccharide in pectin (65%) followed by rhamnogalacturonan I (25–30%). Xylogalacturonan and rhamnogalacturonan II comprise less than 10% of the total pectin (Mohnen 2008).

4.2

Degradation of Pectin by A. niger

A. niger is a typical saprophytic fungus feeding on plant litter. Saprophytic fungi convert plant polysaccharides into mainly monosaccharides before uptake of the monosaccharides into the cell for further catabolism. They degrade plant litter by secreting substrates-specific enzymes (mainly hydrolytic enzymes). The expression and consequent secretion of these enzymes is tightly controlled and dependent on which carbon source is available. Like many other filamentous fungi, A. niger has a rich arsenal of different enzymes able to plant polysaccharides including pectin. Pectin-degrading enzymes are mainly produced in nature by saprophytes and by bacterial and fungal plant pathogenic species that degrade the plant cell wall of its host. Commercial pectinase preparations are primarily derived from A. niger (Voragen and Pilnik 1989). Genome mining has revealed a large array of extracellular pectinolytic enzymes in A. niger (Coutinho et al. 2009; Martens-Uzunova and Schaap 2009). Pectin-degrading enzymes can be grouped in two major classes, “pectinases” and “accessory enzymes,” according to the complex structure of pectin. The “pectinases” attack the backbone of pectin, and “accessory enzymes” degrade the side chains of pectin. Homogalacturonan (HGA) is most abundant component in pectin (Harholt et al. 2010). During HGA degradation, pectin methylesterases hydrolyze methoxy groups in pectin to yield pectate and methanol. Endopolygalacturonases and exo-polygalacturonases are hydrolytic enzymes that hydrolyze pectate, producing oligogalacturonic acid and GA, respectively. Pectate lyases are endo-acting enzymes that catalyze pectate to unsaturated oligogalacturonides with an eliminative cleavage mechanism. Pectin lyases are endo-acting enzymes with an eliminative cleavage mechanism on naturally methylated pectin (Hsiao et al. 2008). The backbone XGA can be degraded by endo-xylogalacturonan and exo-polygalacturonan hydrolases, whereas RGI requires the additional activity of rhamnogalacturonan hydrolases and rhamnogalacturonan lyases. To utilize GA as a carbon source, GA have to be taken up into the cell by specific sugar transporters (Sloothaak et al. 2014). GA can be metabolized both by bacteria and in eukaryotes using different enzymatic pathways. In bacteria, GA is metabolized in a five-step pathway via D-tagaturonate, D-altronate, 2-keto-3-deoxy-gluconate, and 2-keto-3-deoxy-6-phospho-gluconate resulting in formation of pyruvate and glyceraldehyde-3-phosphate (Ashwell et al. 1960; Huisjes et al. 2012). In

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eukaryotes, the metabolism of GA takes a different metabolic route. Metabolism of GA in fungi is well studied and involves four enzymatic reactions to convert GA into glycerol and pyruvate. The genes encoding these enzymes (gaaA, gaaB, gaaC, and gaaD in A. niger) have been identified, and the biochemical properties of the enzymes have been determined (Kuorelahti et al. 2005, 2006; Liepins et al. 2006; Hilditch et al. 2007; Mojzita et al. 2010; Wiebe et al. 2010; Zhang et al. 2011; Kuivanen et al. 2012). Specific sugar transporters that are able to transport GA over the plasma membrane have recently been identified and characterized in N. crassa (Benz et al. 2014) as well as in A. niger (Sloothaak et al. 2014) and Botrytis cinerea (Zhang et al. 2014).

4.3

State of the Art of Understanding the Regulation of Pectinolytic Genes

As described above, A. niger can secrete a wide range of enzymes to synergistically degrade pectin. The expression of these enzymes is tightly regulated in filamentous fungi including A. niger. In many cases, the expression is under the control of substratespecific transcriptional activators, which belong to the fungal specific transcription factors with a Zn(II)2Cys6 DNA-binding motif (Todd and Andrianopoulos 1997). Their expression of the genes encoding the extracellular enzymes, sugar transporters, intracellular metabolic enzymes, and in some cases also the transcriptional activator is also controlled by wide-domain regulators, such as carbon catabolite repressor CreA and the ambient pH regulator PacC which are both members of the C2H2 family of transcription factors. Here, we will focus on the recently identified transcriptional module consisting of a transcriptional activator GaaR and a transcriptional repressor (GaaX) that control the expression of enzymes related to pectin utilization. Based on previous research, it was well established that the expression of polygalacturonic acid or pectin-degrading enzymes was tightly regulated. Early studies in A. nidulans performed by Dean and Timberlake showed that the pelA mRNA (encoding a pectate lyase) was detectable on polygalacturonic acid as carbon source and undetectable on glucose or acetate as carbon source (Dean and Timberlake 1989). Similarly, it was shown in A. niger that several pectinases are specifically induced on GA or pectin (de Vries et al. 2002). In addition, it was found that the expression of polygalacturonase and pectate lyase in A. nidulans is completely repressed by glucose due to carbon catabolite repression (Dean and Timberlake 1989; de Vries et al. 2002). With the possibility of performing genome-wide expression studies using Affymetrix gene arrays, it was shown that at least 11 genes were specifically induced on GA (Martens-Uzunova and Schaap 2008). Analysis of the promoter region of these 11 genes identified a conserved promoter element 50 -YCCNCCAAT-30 (Martens-Uzunova and Schaap 2008) which was suggested to play an important role in the regulation and co-expression of these genes. These results indicate that the expression of genes encoding enzymes

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involved in pectin degradation is specifically induced by polygalacturonic acid or galacturonic acid. Taken together, genes encoding enzymes involved in pectin/ polygalacturonic acid degradation are specifically induced on polygalacturonic acid or galacturonic acid and are under carbon catabolite repression control. The co-regulation of pectin-degrading enzymes and the conserved promoter element in the co-regulated genes (Martens-Uzunova and Schaap 2008) strongly suggested the existence of a transcriptional activator coordinating the activation of gene expression of these GA-induced genes in response to GA or pectin. Whereas over the last few years several new transcription factors involved in plant cell wall degradation have been identified (Kowalczyk et al. 2014), a possible transcription factor involved in the regulation of pectinases was not identified at the start of our research. To identify TFs involved in the regulation of pectin degradation enzymes, we have used several of the functional genomics approaches as described in the first part of this chapter including both targeted and nontargeted screening approaches. As a targeted approach, we screened our collection of 240 TF mutants in A. niger (unpublished data) for reduced growth on pectin or GA, which did not identify a single pectin or GA non-utilizing mutant. Therefore, we also resided to nontargeted functional genomic approaches. In one such approach, we designed a forward genetic screen to isolate mutants with constitutive expression of enzymes related to PGA degradation. Since pectin is a complex polysaccharide composed of various different monosaccharides, we hypothesized that several transcription factors with partially overlapping functions could be involved in pectin degradation and that single deletion of one TF was not sufficient to reduce growth sufficiently to be detected by plate growth assays. In our approach, we designed an amdS reporter driven by the promoter of a GA-induced gene, pgaX, containing a putative conserved galacturonic acid-responsive element (GARE; 50 -YCCNCCAAT-30 ) in its promoter region. As this promoter also was under the control of carbon catabolite repression, the promoter-reporter in creA null background was used to perform forward genetic screens for inducer-independent mutants, which may contain a mutation responsible for constitutive activation in a GA-specific transcription factor. In our studies, we identified gaaR as relevant regulatory factor (Alazi et al. 2016, 2018). In parallel, this same gene was also identified by its homology to Botrytis cinerea GaaR (Zhang et al. 2016). A. niger GaaR is about 50% identical to B. cinerea GaaR on the amino acid level. Deletion of gaaR in A. niger showed strongly reduced growth on GA and PGA and a little reduced growth on sugar beet pectin (SBP) compared to parental strain. The growth phenotype indicates that GaaR is required for the utilization and release of GA from pectin. Further transcriptomic analysis of the gaaR deletion strain by RNA sequencing showed genes encoding 25 pectinolytic enzymes are not induced on GA in the ΔgaaR mutant, indicating that GaaR is required for the induction of these genes on GA. Other genes involved in the degradation of pectin side chains and involved in catabolism of L-rhamnose, Larabinose, and D-xylose were still expressed in ΔgaaR, indicating that the degradation and metabolism of pectin sugars other than GA support the growth of ΔgaaR on SBP (Alazi et al. 2016).

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GaaR is a member of the family of Zn(II)2Cys6 transcription factor. Both in A. niger and B. cinerea, it controls expression of genes involved in GA utilization. The GaaR-encoding gene in A. niger gaaR is 2476 bp long and contains five introns resulting in a 740-amino acid long protein. GaaR contains a fungal-specific DNA-binding domain Zn(II)2Cys6 with the pattern of CX2CX6CX6CX2CX6C at residues 26–56 and a fungal transcription factor regulatory middle homology region (fungal_TF_MHR) at residues 139–518. Orthologs of GaaR were found across all 20 Aspergillus species except Aspergillus glaucus based on the information from the Aspergillus genome database (http://www.aspgd.org). A. glaucus is not able to grow on medium with GA as single carbon source (http://www.fung-growth.org/) corresponding with the absence of gaaR in A. glaucus, in agreement with the fact that gaaR is responsible for the utilization of GA. The PpgaX-amdS reporter strain used to screen for inducer-independent mutants with constitutive expression of pectinases also resulted in the identification of a second gene different from gaaR (Niu et al. 2017). This gene was discovered by whole genome sequencing of five constitutive non-gaaR mutants. The sequencing results revealed allelic mutations in one particular gene encoding a previously uncharacterized protein which we named gaaX. Subsequently, we performed targeted deletion and transcriptomic analysis of the mutant strain to study the function of gaaX. The ΔgaaX mutant grows normally on a variety of C-sources including GA, PGA, and pectin. Deletion of gaaX was also shown to result in constitutive pectinolytic activity in plates assay. RNAseq analysis revealed that 37 genes were upregulated in ΔgaaX mutant (FDR < 0.001, FC > 4.0). Gene ontology (GO) enrichment analysis using FetGOat (Nitsche et al. 2011) and manual inspection of the genes upregulated in the ΔgaaX mutant indicated that genes involved in pectin catabolism were highly enriched. Of the 37 genes, 16 are predicted to encode extracellular enzymes acting on the GA backbone of pectin or acting on pectin side chains (Niu et al. 2017). Nine genes of the 37 upregulated genes are predicted to encode intracellular proteins. Four of these nine genes (gaaA–gaaD) are required for the conversion of GA into pyruvate and glycerol (Martens-Uzunova and Schaap 2008). The exact role of the other five genes and their possible role in pectin catabolism are currently unknown. Genomic localization of gaaX (NRRL3_08194) showed the gaaX gene is next to the GA-specific transcriptional factor gaaR (NRRL3_08195). Among 19 Aspergillus species, gaaR and gaaX orthologs showed a strongly conserved genomic clustering pattern: either next to each other or separated by only one or a few genes (Niu et al. 2017). The A. niger GaaX protein is predicted to be 697 aa long. Sequence alignment and BLASTP searches displayed similarity of GaaX to the last three domains in the C-terminal half of the AROM protein, which is a large (1586 aa in A. niger) pentafunctional protein composed of five domains involved in different enzymatic steps of the prechorismate shikimate pathway. Like GaaX, the previously identified quinate repressor protein QutR shows similarity to the last three C-terminal domains of AROM (Lamb et al. 1996a). The genomic clustering of gaaX and gaaR is analogous to that of the quinic acid utilization transcriptional activator (QutA/Qa-1F) and repressor (QutR/ Qa-1S) in

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A. nidulans and N. crassa, respectively (Geever et al. 1989; Levesley et al. 1996). The amino acid homology to the quinate repressor, the similarity in chromosomal organization of gaaR/gaaX compared to the known activator/repressor genes qutA/ qutR, and the constitutive phenotype of the isolated gaaX mutants point to the possibility that gaaX encodes a repressor protein that controls the activity of GaaR and keeps GaaR inactive under non-inducing conditions, similar as what was predicted for QutA/QutR (Lamb et al. 1996b; Levett et al. 2000; Watts et al. 2002). Recent evidence from our group has shown that the GA-metabolic pathway intermediate 2-keto-3-deoxy-L-galactonate (2-KDG) is likely to act as the inducer (Alazi et al. 2017). We propose a model (Fig. 8.1) in which it is postulated that under inducing conditions, the inducer, 2-KDG, will bind to repressor protein GaaX. We speculate that the interaction of the inducer with the repressor results in the release of active GaaR from GaaX. Whether this occurs in the cytosol or nucleus remains to be determined, but preliminary experiments suggest that GaaR is present in the nucleus under inducing and non-inducing conditions (Alazi et al. 2018) suggesting that the interaction takes place in the nucleus. Active GaaR not only induces the expression of GA-responsive genes involved in GA release, uptake, and metabolism but also induces the expression of gaaX (Niu et al. 2017; Martens-Uzunova and Schaap 2008). Under inducing conditions, a sufficient amount of the inducer is likely to keep GaaX dissociated from GaaR, and consequently, the GaaR transcription factor will be active. When the concentration of inducer decreases (non-inducing conditions), the amount of inducer is insufficient to bind to the repressor protein thereby allowing interaction between GaaR and the repressor GaaX to inactivate the GaaR transcriptional activator. An interaction of GaaR and GaaX to prevent GaaR from being active is also supported by the observation that overexpression of GaaR results in inducer-independent expression of pectinases (Alazi et al. 2018) The exact mechanism by which GaaX controls GaaR activity is unknown. However, such a mechanism allows an elegant way which enables the cell to activate GA-induced gene expression when the inducer is present in the cell and to stop expression of GA-responsible genes when inducer levels decrease. It also ensures the rapid response to the presence of GA as no de novo synthesis of GaaR is required. The proposed model on how GaaR/GaaX regulate gene expression not only is similar to what is shown for QutR/Qa-1S) but also shows similarities to the wellknown galactose regulatory system from S. cerevisiae. In this system, three proteins, Gal3p/Gal4p/Gal80p, are involved in the regulation of galactose utilization. Gal4p works as a transcriptional activator, Gal80p works as a transcriptional repressor, and Gal3p possibly works as a galactose sensor. Gal80p can bind to both of Gal4p and Gal3p. In the absence of galactose, Gal80p binds to Gal4p preventing GAL gene expression. In the presence of galactose, galactose triggers an association between Gal3p and Gal80p, by binding of galactose to Gal3p relieving Gal4p from Gal80p (Diep et al. 2008; Jiang et al. 2009; Platt et al. 2000; Timson et al. 2002). The differences between the GA utilization system and Gal system are that in the GA case, there are only two genes found to be involved in the GA utilization system so far. At this point, we cannot exclude that there is a third gene involved in the GA utilization system. However, GaaX does not show homology to Gal80p or Gal3p,

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B. Non-inducing condition

A. Inducing condition

PGA

GaaX GaaRactive GA gaaX

GaaA

?

L-galactonate Other transcription factors unknown

GaaB

2-keto-3-deoxyL-galactonate

GaaRinactive 25 pectinolytic enzymes

GaaC

pyruvate

GaaX

L-glyceraldehyde GaaD

glycerol

GA specific transporters

metabolic genes gaaA, gaaB, gaaC, gaaD

Fig. 8.1 (a) Model describing GaaR/GaaX-controlled gene expression in response to galacturonic acid (GA). Under inducing conditions (presence of (poly)galacturonic acid), the inducer 2-keto-3deoxy-L-galactonate (red dot) is predicted to bind to GaaX. The binding of the inducer to GaaX is expected to cause dissociation of the GaaX/GaaR complex resulting in non-GaaX-bound GaaR which can drive the expression of GA-responsive genes. These genes include gaaX (MartensUzunova and Schaap 2008), other transcription factors with unknown function (Supplementary Table 3 in Niu et al. 2017), 25 pectinolytic genes which involved in GA release (Niu et al. 2017; Alazi et al. 2016), GA-specific transporters which take up GA into the cell, and GA intracellular metabolism genes gaaA, gaaB, gaaC, and gaaD (Niu et al. 2017; Alazi et al. 2016). Under inducing condition, gaaX expression is induced, but the presence of the inducer is thought to keep GaaX inactive. (b) Under non-inducing conditions (absence of pectin or (poly)galacturonic acid), no inducer is present to bind to the repressor protein thereby allowing interaction between the repressor GaaX and the transcription activator GaaR to inactivate GaaR, preventing the expression of GA-responsible genes

making it unlikely that the two systems are evolutionarily related. To further elucidate the regulation mechanism of the GA utilization system, future research should be aimed at understanding the biophysical and biochemical interactions between GaaR, GaaX, and the inducer. From our research, it is clear that with the elucidation of the GaaR/GaaX activator/repressor system, we have identified yet another mechanism of gene regulation involving a Zn(II)2Cys6 transcription factor. Interestingly, this mechanism is not conserved for pectin catabolism in N. crassa where features of A. niger

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GaaR and RhaR (Diep et al. 2008) are combined in a single transcription factor PDR-1 (Jiang et al. 2009).

5 Outlook As described above the transcriptional network regulating a major part of the pectinolytic pathway is governed by an activator/repressor system, including a canonical Zn(II)2Cys6 transcription factor (GaaR). However, it is clear from previous work on Zn(II)2Cys6 transcription factor-mediated transcriptional networks related to other carbohydrate catabolic pathways like cellulose, hemicellulose, xylan, starch, and inulin that a wide range of variations on the activator/repressor theme exist on how these transcription factors perform their regulatory role. A very comprehensive overview of these different modes of regulation is recently published (Benocci et al. 2017). These variations include (1) induction-dependent nuclear import or export, (2) posttranscriptional modifications, (3) binding site competition with other regulatory factors including those involved in carbon catabolite repression, and (4) (hetero-)dimerization. Regarding the origin of these different modes of regulation, it is even more interesting to note that these different modes of regulation are not conserved among the same pathways in different species, not even in orthologous transcription factors. How the diversity of regulatory circuits in different filamentous fungi has evolved from an ancestral regulator protein is the subject of our continuing research interests in this field. Funding JN was supported by a grant from the Chinese Scholarship Council.

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Part III

Towards Bioeconomy: Potential of Fungal Biotechnology

Chapter 9

The Economic Potential of Arbuscular Mycorrhizal Fungi in Agriculture Maya Benami, Yochai Isack, Dan Grotsky, Danny Levy, and Yossi Kofman

1 Introduction 1.1

Sustainable and Regenerative Agriculture

With an increasing worldwide population, the demand for food is driving the need for crop enhancement products, including biological inoculants. World population will increase to over 8 billion in 2025 according to UN Department of Economic and Social Affairs. Thus, there is a global need for increased agricultural production which may be partially met by addition of agricultural inoculants in order to promote crop yield growth and nutrition. In order to satisfy the imminent requirements of a growing population and to attain food security in the context of climate change, an upsurge in food production will need to happen, preferably in a manner which minimizes harmful environmental impacts (Foley et al. 2011). Sustainable intensification of agriculture (Garnett et al. 2013), also referred to as ecological intensification, encompasses important aspects of conservation agriculture (Hobbs et al. 2008; Giller et al. 2015). The Food and Agriculture Organization (FAO) of the United Nations states that the three fundamental principles of conservation agriculture include minimal mechanical soil disturbance practices (e.g., no till), continuous soil organic cover (e.g., coverage with cover crops or residues), and species diversification practices (i.e., varying crop sequencing, multi-cropping, and crop rotations) (FAO 2015). The addition of biofertilizers or bio-inoculants can be key components to achieving sustainable or even regenerative agriculture.

M. Benami · Y. Isack · D. Grotsky · D. Levy · Y. Kofman (*) Groundwork BioAg Ltd., Moshav Mazor, Israel e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_9

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Biofertilizers

Bio-inoculants for agricultural purposes are also known as biofertilizers, agricultural inoculants, soil inoculants, or microbial inoculants. They can be broadly defined as preparations of active or latent strains of microorganisms (generally bacteria or fungi alone, or in combination) which, directly or indirectly, stimulate edaphic microbial activity in the rhizosphere of plants. This activity thereby increases mobilization of nutrients from the soil into the plant and potentially enhances crop yields. Specialized biological inoculants induce beneficial processes in the plant that help to deliver nutrients and suppress diseases, thus promoting plant growth. Biofertilizers consist of tailored formulations of microorganisms and/or their functional attributes which are customized to assist a range of soil systems and cropping patterns in order to attain agricultural sustainability. The need for various agricultural inoculants in the global marketplace stems from various microbial characteristics, such as their ability to form symbiotic relationships for mutual benefits and provide required nutrients and minerals to plants. In comparison with chemical-based, conventional, or synthetic pesticides and fertilizers, microbial inoculants have several advantages as they (Berg 2009): 1. 2. 3. 4. 5. 6. 7. 8.

Cause less environmental damage Generally demonstrate lower risk to human health as they are safer to handle Can exhibit more targeted activity Can be effective in small quantities Replicate themselves Decompose more quickly than conventional chemical pesticides Show reduced resistance (in host plants or target pathogens) Can also be utilized in conventional or integrated pest management systems

There are several model organisms for plant growth promotion or disease suppression which are well-studied in their mode of action and regulation. These comprise members of the bacterial genera Azospirillum (Cassán and Diaz-Zorita 2016; Okon and Labandera-Gonzalez 1994), Bacillus (Jacobsen et al. 2004), Pseudomonas (Haas and Défago 2005; Loper et al. 2007), Rhizobium (Long 2001), Serratia (de Vleeschauwer and Höfte 2003), Stenotrophomonas (Ryan et al. 2009), and Streptomyces (Schrey and Tarkka 2008) and the fungal genera Ampelomyces, Coniothyrium, Glomus (Ozgonen and Erkilic 2007), and Trichoderma (Harman et al. 2004). The practical use of biological fertilizers is well below its full potential, mainly due to nonavailability of suitable inoculants, which are often difficult to produce cost-effectively. Therefore, further studies on bio-inoculant formulations and their exploration will certainly aid in our understanding of the complexity and dynamism of microbial functioning and interactions in soils (Suyal et al. 2016). While many types of biofertilizers based on bacterial formulations have shown beneficial effects in agriculture, this chapter will focus specifically on how biofertilizers based on fungi are used in sustainable and regenerative agriculture.

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241

Applications of Fungi in Sustainable and Regenerative Agriculture

Efficient nutrient cycling indicates healthy and productive soils. There is a growing appreciation for how microbes contribute to agricultural sustainability, including providing plant-symbiotic associations and enhancing soil biogeochemical cycling (Bender et al. 2016; Mader 2002; Wagg et al. 2014). An important microbial constituent which enhances plant nutrient uptake is the common natural symbiosis between arbuscular mycorrhizal fungi (AMF) and plant roots. These soil fungi penetrate the root tissue of the plant and form mycorrhizae (in Greek: myco ¼ fungus, rhiza ¼ root). Mycorrhizae create a secure relationship of carbon extraction from the host roots in exchange for providing important, plant-usable nutrients to the host plant. The fungal hyphae of mycorrhizal species grow inside and outside of the plant root and effectively increase the surface area of the root, thus improving processes of water, nutrient, and solute uptake (see Fig. 9.1). In addition, mycorrhizal fungi contribute to changing the plant and soil hormonal balance, and improving soil structure and stability by producing glomalin; and some evidence shows that mycorrhizae provide enhanced resistance to root-damaging pathogens (such as Fusarium, Pythium, Phytophthora, and nematodes). Much research has been dedicated to investigating and managing AMF symbiosis in agriculture in order to enhance agricultural sustainability. The prominent reasons for these research efforts are as follows: (1) AMF symbiosis is carried out in 90% of plant species; (2) AMF symbiosis plays a multifunctional role in enhancing plant nutrition, pathogen protection, stress tolerance, and soil structure; and (3) many agricultural practices (e.g., tillage, fertilization, nonhost crops) can deplete AMF abundance and diversity (Rillig et al. 2016).

Fig. 9.1 Root hairs: Endomycorrhiza colonizing and extending an eggplant root, 2018 (Photo credit: Groundwork BioAg, Ltd.)

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Arbuscular mycorrhizal fungi (AMF) are constituents of the phylum Mucoromycota subphylum Glomeromycota of fungi which are very common and important members of soil microbiology (Spatafora et al. 2016). The over 200 species included in Glomeromycota have been widely researched and have been estimated to comprise 4–10% of total soil microbial biomass (Fitter et al. 2011). As obligate symbionts, Glomeromycota form arbuscular mycorrhizae. Arbuscular mycorrhizae are intimate symbiotic relationships with the thalli of bryophytes (nonvascular land plants) or the roots of vascular terrestrial plant species. The most well-known and well-studied mycorrhizal symbioses pertain to arbuscular mycorrhizae, due to their abundance. Ectomycorrhizae exclusively associate with woody plants (mostly tree and shrub species). However, several other types of mycorrhizal relationships also exist, e.g., ericoid, arbutoid, monotropoid, orchidaceous, and ectendomycorrhizae (Aggarwal et al. 2011). In highly dependent, susceptible plants, there are many documented cases of mycorrhizae enhancing plant growth and yields. Plant responses to mycorrhiza can vary due to a variety of factors. However, in most cases positive plant responses to mycorrhizal fungi stem from an increase in usable root area for water and nutrient extrication, since the mycorrhizal hyphal network functions as a natural extension of the plant root system (FAO 2015). Although the plants benefit from the AMF in the form of better overall plant nutrition and uptake, soil structure, and other ecosystem services, their high-affinity phosphorus uptake mechanism makes them ideal partners in the role to increase host plant phosphorus (Smith and Smith 2012). Plant uptake of other minerals and nutrient forms such as nitrogen has also been recorded to be helped by effective AMF host plant colonization (George et al. 1995). The scientific community has yet to reach consensus regarding the benefits of AMF inoculants. Hart et al. (2018) reviewed several parameters that could affect AMF efficacy in the field, such as soil fertility, inoculation timing, site disturbance level, and perhaps partner coadaptation. Ryan and Graham (2018) suggested that the AMF community may be more resilient to many agricultural practices than often assumed. In contrast, Rillig et al. (2019) discussed the contribution of AMF to agricultural system performance and sustainability and proposed several high-priority research questions for better understanding of the AMF inoculation application. A meta-analysis by Zhang et al. (2018) has shown an overall effect of 16% increase in grain yield, and indicates the importance of pH and cultivar on the mycorrhizal effect. This important debate reflects the challenges that remain in the production of commercial AMF inocula and the need for further research to improve mycorrhizal inoculants.

1.4

AMF-Induced Yield Enhancements

Growth, nutrient, and quality enhancements of vegetable crops after mycorrhizal inoculation have been described in a variety of diverse species and cultivars (Baum et al. 2015). Research performed by Affokpon et al. (2011) documented a range of yield and growth enhancements after field applications of a mixture of 20 indigenous West-African AMF strains. These strains increased tomato (Solanum lycopersicum) yields up to 26%, and remarkably, carrot (Daucus carota) yields up to 300% (Affokpon et al. 2011).

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Crop Cultivar and Variety-Specific Responsiveness to AMF

It is important to note that differing varieties and cultivars of the same species of plants may respond differently to mycorrhizal colonization. The degree of plant growth change associated with arbuscular mycorrhizal colonization is expressed as mycorrhizal dependency (MD). Tawaraya (2003) extensively reviewed the mean values of MD for dozens of crops and compared them among similar varieties and cultivars. The MD results found overall in this research were 44% for field crops (37 species), 56% for forage crops (46 species), 70% for wild grasses and other herbaceous flowering plants (140 species), 79% for trees (26 species), and 56% for all plants (250 species). Importantly, Tawaraya indicated that tested cultivated plant species showed a lower MD than wild plant species. This research suggested that AMF inoculation in low-input systems should be executed while bearing in mind differences in MD (Tawaraya 2003). Individual investigations into MD across a variety of plant species have found a range of plant responses depending on the mycorrhizal fungi type and plant cultivar. Khalil et al. (1994) performed greenhouse studies with AMF (Gigaspora margarita or Glomus intraradices) in low P soil originating from Iowa. They evaluated the mycorrhizal dependency in terms of growth response, nutrient uptake (N, P, K, Ca, Mg, and Zn), and root phosphatase activity on three improved and three unimproved corn (Zea mays L.) and soybean [Glycine soja Siebold & Zucc. and Glycine max (L.) Merr.] cultivars. They found that the addition of AMF to the roots increased colonization from 62 to 87% for soybean and 49 to 68% for corn. Overall these soybean cultivars demonstrated higher mycorrhizal colonization than the corn cultivars, but considerable variation occurred within soybean cultivars. Relative positive growth of the two unimproved soybean cultivars was significantly greater with AMF colonization than without (Soja, >1900%; Mandarin, >400%). In the improved cultivars, there existed less relative growth with colonization (BSR 201, Richland, and Swift cultivars averaged ~200% greater growth with AMF than without AMF colonization). An unimproved corn cultivar, Reid Yellow Dent, was unresponsive to mycorrhizal colonization, whereas another unimproved cultivar, Argentine Pop, increased growth 400% after AMF colonization. In addition, total uptakes of N, P, K, Ca, Mg, and Zn were significantly greater ( p < 0.001) in the mycorrhizal plants. These results suggest that substantial variability exists in mycorrhizal colonization for corn and soybean cultivars when grown in P-deficient soils and that this variability can exist in both improved and unimproved cultivars (Khalil et al. 1994). Mohammad et al. (1998) researched the growth and yield potential of winter wheat (Triticum aestivum) inoculated with Glomus intraradices at two P levels, 30 kg/ha and zero additional P, under field conditions in Lind, Washington (Mohammad et al. 1998). Results showed that regardless of P status, inoculation with Glomus intraradices increased grain yields by more than 25% when P was not added to the soil compared to the non-inoculated plants. When the same research group created a similar experiment with Glomus intraradices applied to the Swift

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wheat cultivar and added 5, 10, or 20 kg/ha of P, they found that the wheat plants produced significantly more grain per spike at P0 than at other P levels (Mohammad et al. 2004). Their overall results suggested that sheared-root inoculum of Glomus intraradices may contribute to reducing P fertilizer applications. In contrast, nonresponsiveness to the colonization of AMF was described in host crops like cucumber (Cucumis sativa) and pea (Pisum sativum) (Smith and Smith 2011). Some vegetable crops like tomato (Solanum lycopersicum) exhibited growth promotion (see Table 9.1) after AMF inoculation, whereas the same host species but differing cultivar or crop variety demonstrated to be less, nonresponsive, or even inhibited in their growth after AMF inoculation (Smith and Smith 2011). Tawaraya (2003) found that MD between differing plant types and cultivars within the plant type was negatively correlated with root morphological characteristics such as root length, root dry weight, root hair length, density of root hairs, the ability of roots to obtain phosphate from soil, and the P utilization efficiency of the host plant (Tawaraya 2003). However, these “rules” differ among crop types. For example, MD in wheat was shown to be affected more by root and root/shoot ratio dry weights, but neither MD nor mycorrhizal infection levels were directly affected by nitrogen, phosphorus, potassium, calcium, or magnesium concentrations in plant tissues (Azcon and Ocampo 1981). In a meta-analysis performed by Hoeksema et al. (2010), the researchers found that in most subsets of the data, host plant functional group and nitrogen fertilization were more significant factors in estimating plant responses to mycorrhizal inoculation than other parameters. They found that nonnitrogen-fixing forbs, woody plants, and C4 grasses responded more positively to mycorrhizal inoculation than plants with nitrogen-fixing bacterial symbionts and C3 grasses. In their laboratory studies of the arbuscular mycorrhizal symbiosis, plant responses were more positive when the soil community was more complex. Their univariate analyses upheld their supposition that plants received the most positive benefits when they were P-limited rather than nitrogen-limited. Their results emphasize that mycorrhizal function depends on a complex mixture of both abiotic and biotic contexts (Hoeksema et al. 2010). Overall, AMF’s influence on crop and cultivar-specific growth promotion can be highly dependent upon (1) the type(s) of mycorrhizal fungi introduced to the crop, (2) the crop response and mycorrhizal dependency to the specific mycorrhiza type, (3) soil fertility, (4) soil biocomplexity, and (5) specific environmental conditions (e.g., pH and salinity levels) (Hoeksema et al. 2010). The major factors contributing to crop mycorrhizal dependency and why certain varieties and cultivars within the same crop species respond better to mycorrhizal inoculation will certainly continue to be a topic of important future research. In order to facilitate tests on ecological and evolutionary contexts in which the addition of mycorrhizal fungi is considered beneficial or parasitic to host plants, a large database of mycorrhizal inoculation experiments linked with plant and fungal phylogenies was recently created. MycoDB is a database and an over 10-year meta-analysis that contains data on plant productivity responses to mycorrhizal fungi from over 4000 studies (Chaudhary et al. 2016). Encouraging participation in projects like MycoDB will greatly benefit both the mycorrhizal industry’s and the scientific community’s understanding of mycorrhizal symbioses and plant responses.

+P, +K, +Zn, +Cu, +Fe; Na +P, +K +P, Zn

+Shoot, root biomass, early flowering +Shoot, root and fruit biomass

Shoot and root biomass

Glomus mosseae, G. etunicatum

Soil indigenous AMF population

+Shoot and root dry matter

+P, +Zn, +nutrient uptake

+Shoot biomass

Glomus coronatum, G. intraradices, G. claroideum, G. mosseae Glomus mosseae, G. clarum, G. etunicatum, G. intraradices, G. caledonium Glomus mosseae

Glomus mosseae

N, P

+Shoot biomass

+P, +Zn

+N

+Tolerance of salt stress +Tolerance of salt stress Water use efficiency n.t.

+Tolerance of drought stress +Tolerance of drought stress n.t.

n.t. n.t.

+P, +Zn Cd, +P

Glomus mosseae, G. fasciculatum

n.t.

n.t.

Stress tolerance n.t.

+P, +Zn

+P, +Zn

Shoot element concentrations +P

Glomus fasciculatum, G. albidum, G. macrocarpum Glomus mosseae, G. etunicatum Glomus caledonium, G. versiforme

Growth response of the host plant +Shoot biomass +Shoot biomass, numbers of flowers, fruit yield +Plant high, numbers of flowers, fruit yield +Shoot and root dry matter +Shoot biomass

Glomus fasciculatum

Fungal inoculum species Glomus intraradices

Abdel Latef and Chaoxing (2011) Watts-Williams and Cavagnaro (2012) Ortas (2012)

Al-Karaki (2006)

Marulanda et al. (2003) Ortas et al. (2013)

Tobar et al. (1994)

Reference Deressa and Schenk (2008) Bagyaraj and Sreeramulu (1982) Sreeramulu and Bagyaraj (1986) Ortas (2012) Hu et al. (2013)

n.t.—not tested,  decreased, + increased,  not affected Note: Reprinted from “Increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: A review,” 187, Baum, El-Tohamy Gruda, 133, Copyright 2015, with permission from Elsevier

Corn (Zea mays)

Tomato (Solanum lycopersicum)

Water spinach (Ipomoea aquatica) Lettuce (Lactuca sativa)

Host plant species Onion (Allium cepa) Pepper (Capsicum annuum)

Table 9.1 Effects of inoculation of vegetable crops with arbuscular mycorrhizal fungi (AMF) on growth and stress tolerance of host plants

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Product Type, Application Rates, and Methods Influencing Crop Yield Responses to AMF

Application methods and minimum quantities of mycorrhizal inoculants have been shown to be important for effective plant infectivity. In Tarbell and Koske (2007), eight commercial inocula of arbuscular mycorrhizal fungi (AMF) were tested for their ability to colonize plant roots in the sand/peat medium specified by the US Golf Association for use in putting greens (Tarbell and Koske 2007). The failure of five of the eight commercial inocula to colonize roots when applied at the recommended rate suggests that many mycorrhizal inoculants are ineffective at their recommended rates. The authors stated that for those AMF products that declared to have high propagule concentrations but had low or no ability to colonize, one of the likely causes may have been far fewer spores or propagules present than claimed.

1.5

Beneficial Fungi and Abiotic Stress

Recent studies indicate that fitness benefits conferred by mutualistic fungi contribute to or are responsible for plant adaptation to stress (Rodriguez et al. 2004). Collectively, mutualistic fungi may confer tolerance to drought, metals, disease, heat, and herbivory and/or promote growth and nutrient acquisition. It has become apparent that at least some plants are unable to tolerate habitat-imposed abiotic and biotic stresses in the absence of fungal endophytes (Redman et al. 2002). Abiotic stresses, such as drought, salinity, extreme temperatures (heat and cold), heavy metal toxicity, and oxidative stress, are serious threats to agriculture and result in the deterioration of the environment (Wang et al. 2003b). Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Oerke 2006). Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth and productivity (Wang et al. 2001). Drought, salinity, extreme temperatures, and oxidative stress are often interconnected and may induce similar cellular damage (Wang et al. 2003b). For example, oxidative stress, which frequently accompanies high temperatures, salinity, or drought stress, may cause denaturation of functional and structural proteins in plants (Wang et al. 2003a). AMF have been widely researched as crop growth enhancers while also improving plants’ abilities to tolerate stress by increasing water uptake and transforming nutrients from the soil which are normally out of reach to the plant. This enhanced nutrient uptake also allows AMF to fortify the crops against less-than-ideal growing conditions. Evidence from in vitro studies on the Glomus genus of mycorrhiza has shown to be one of the more popularly studied and effective mycorrhizal fungi genera to produce positive yield enhancements in plants under edaphic stress conditions (Baum et al. 2015). Figure 9.2 shows a larger healthier corn plant treated with a mycorrhizal inoculant versus an untreated plant. Specific studies have shown

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Fig. 9.2 Corn plants and ears: On the left, plants and ears of corn plant treated with mycorrhizal inoculant, on the right untreated corn plant. Ohio, USA, 2019 (Photo credit: Groundwork BioAg)

that certain powerful species of the Glomus genus, specifically Glomus intraradices (also known as Rhizophagus irregularis or Rhizophagus intraradices), may help crops better withstand petroleum, heavy metal, salinity, nutrient-deficient, and drought-induced edaphic stresses (Table 9.1; Krüger et al. 2012; Lenoir et al. 2016). It is important to note that most former studies and references only refer to Glomus intraradices as Rhizophagus irregularis, but only in recent years has Rhizophagus irregularis been recategorized and recognized more accurately as Rhizophagus intraradices (Krüger et al. 2012).

1.6

Beneficial Fungi and Biotic Stress

Biocontrol fungi are beneficial, nonpathogenic microorganisms that can reduce the negative effects of fungal and insect-based plant pathogens while also promoting positive responses in the plant (Shoresh et al. 2010). Beneficial fungal biocontrol mechanisms include upregulation of antibiotic production, establishing parasitic and

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nutrient competitive interactions with the target organisms in the rhizosphere, mycoparasitism, and inducing plant genetic expression changes which cause systemic resistance inside the plant (Howell 2003; Pozo et al. 2002; Shoresh et al. 2010). Some fungal species have been extensively used and researched as biocontrol agents. At least 40 fungal species have been reported or tested to have potential biocontrol capacity against powdery mildews (Kiss 2003). Several nonpathogenic and saprotrophic species of Rhizoctonia, Fusarium, and Trichoderma have been utilized to reduce damage (root rots, wilts, damping off, and bare patches) caused by genetically and phenotypically similar pathogenic fungi from the same genera and other pathogenic fungi (e.g., Pythium, Sclerotium, Verticillium). Several fungal species within the Trichoderma and Glomus genera are known biocontrol agents for the control of soil-borne and plant diseases (Harman et al. 2004; Pozo et al. 2002). Several studies reported increased plant growth when associated with strains of Trichoderma, Glomus intraradices, Glomus mosseae, and other plant growthpromoting microorganisms. However many of these positive growth effects were found to be more pronounced when the plants were under suboptimal conditions in the arenas of biotic, abiotic, or physiological stresses (Pozo et al. 2002). Several reports have demonstrated that beneficial fungi may induce systemic resistance to plant pathogens by upregulating specific genes in the plant related to pathogen resistance (Shoresh et al. 2010). Plants may often be concurrently confronted by pathogens and insects which trigger a range of responses that may be beneficial or detrimental to the plant (Bostock et al. 2001). Induced resistance is a plant response mechanism which is activated when confronted with certain abiotic or biotic factors to create an active resistance state. This process involves reapportioning carbon and nitrogen resources from plant growth and reproduction, and instead, creating long-lasting and systemic resistance of the plant to a broad array of pathogens and detrimental insects (Walling 2001). Localized and systemic induced resistance has been demonstrated in most plants in response to attack by harmful organisms, physical damage due to insects or other factors, chemical treatments, and the presence of nonpathogenic root-dwelling microorganisms (Harman et al. 2004). Significant research efforts have been dedicated to the protective, induced, and internal responses of plants to herbivory. Some of this research has focused on the plants’ resistance mechanisms to avoid further herbivore attack, and some of these mechanisms include the plants’ abilities to create changes in itself which reduce the preference for, or effect of, herbivores on the damaged plant (Bostock et al. 2001; Maschinski and Whitham 1989; Schuman and Baldwin 2016). Several plant physiological pathways can be activated in response to different threats; however, it appears that some crosstalk or competition may exist between pathways (Ward 1991). Localized and induced systemic resistance reactions have been demonstrated to be involved in pathogen control in mycorrhizal and nonmycorrhizal plants. For example, there are several reports of Glomeromycota division of arbuscular mycorrhizal fungi (Glomus mosseae, Glomus intraradices, Glomus etunicatum, Glomus fasciculatum, and Gigaspora margarita) conferring bioprotection in tomato roots and pepper plants

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and reducing disease symptoms from Phytophthora parasitica and Phytophthora capsici by using a combination of local and systemic mechanisms (Ozgonen and Erkilic 2007; Pozo et al. 2002). Via the accumulation of phenolics, fungitoxic properties, and other plant cell defense responses, these AMF species have decreased pathogen development and associated damage they create in mycorrhizal and nonmycorrhizal parts of plant root systems (Liu et al. 2007; Ozgonen and Erkilic 2007). Other well-known examples of fungi which have been used as biocontrol agents include Trichoderma spp. and the recently described Sebacinales spp. Species within both fungal genera have demonstrated numerous capabilities to control many foliar, root, and fruit pathogens, along with invertebrates such as nematodes. Glomus, Arthrobotrys, Nematophthora, Dactylella, and Verticillium fungi have been investigated as potent potential nematode-controlling agents as they were able to capture, kill, digest, or reduce the damaging effects of nematodes on a variety of host plants (Table 9.2) (Davies and Spiegel 2011). Although some fungal species have demonstrated exemplary potential for wider use in an array of biocontrol applications, they are not utilized to their fullest extent. This is mainly due to stringent and sometimes unclear rules regarding their use, application, safety, and the technical challenges associated with applying and maintaining a specific, singular strain of fungi in the soil. To overcome some of these challenges, one would need to address the following: (1) be aware of factors affecting their viability rates in soils; (2) understand which fungal strains were best for specific crops and environmental conditions; (3) be knowledgeable on the most appropriate formulation, farm management, and application practices; (4) educate the growers on the best use of this technology; and (5) overcome stringent and relatively expensive regulatory barriers in target markets in order to claim products as biopesticides (FAO 2018).

1.7 1.7.1

The Role of Mycorrhizae in Sustainable and Regenerative Agriculture Evolution and History

The symbiosis between plants and AMF originated approximately 450 million years ago and is thought to have played an important role in the shift of plants surviving outside of water to becoming terrestrial (Redecker 2000). Some research focusing on the fossil record suggest that glomalean fungi were present before the first vascular plants arose (Redecker 2000). The symbiosis between AMF and a majority of species in the plant kingdom is believed to be a principal driver of the biodiversity, ecosystem variability, and productivity of plant communities (Chagnon et al. 2013; van der Heijden et al. 1998). A recent study of global biogeography of AM fungi examined DNA from 1014 plant root samples collected worldwide. Although AM

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Table 9.2 Effects of inoculation of vegetable crops with arbuscular mycorrhizal fungi on the resistance of the host plants to nematode attacks Fungal inoculum Glomus spp.

Nematode species Meloidogyne incognita

Glomus intraradices, G. mosseae, G. versiforme

Meloidogyne incognita

Carrot (Daucus carota)

20 indigenous AMF

Meloidogyne spp.

Tomato (Solanum lycopersicum cv. Marmande) Tomato (Solanum lycopersicum)

Glomus mosseae

Meloidogyne incognita

20 indigenous AMF

Meloidogyne spp.

Host plant Watermelon (Citrullus lanatus) Cucumber (Cucumis sativus)

Impact on nematodes and host plant  Plant attacks, + Early plant establishment + Early fruit yield  Root gall index  Number of eggs per root system  Number of female nematodes + Field application of AMF increased carrot yields by over 300% compared with non-AMF control treatments  Significant suppression of nematode multiplication over two crop cycles  Significant decrease of root galling damage over two crop cycles  Penetration of roots  Nematodes mobility

+ Field application of AMF increased tomato yields by 26% compared with non-AMF control treatments  Significant suppression of nematode multiplication over two crop cycles  Significant decrease of root galling damage over two crop cycles

Reference Westphal et al. (2008) Zhang et al. (2008)

Affokpon et al. (2011)

Vos et al. (2013)

Affokpon et al. (2011)

 decreased, + increased,  not affected Note: Reprinted from “Increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: A review,” 187, Baum, El-Tohamy Gruda, 137, Copyright 2015, with permission from Elsevier

fungal communities reflected local environmental conditions and the spatial distance between sites, 93% of AMF taxa were found on multiple continents and 34% on all six continents surveyed. This suggests that AMF effectively and efficiently dispersed throughout the world, probably via both abiotic and biotic vectors, including human intervention (Davison et al. 2015). Therefore, much research has been dedicated to understanding the mechanisms through which AMF influence a wide range of plant responses in different environmental contexts.

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Compatibility with Cultivation Methods and Products

It is now widely accepted that the methodologies used and soil conditions prevalent in sustainable agriculture are likely to be more favorable to AMF than those under conventional agriculture (Harrier and Watson 2004). Any agricultural operation that disturbs the natural ecosystem will have repercussions on the mycorrhizal system (Mosse 1986). The conventional agricultural disturbances which may disrupt the mycorrhizal fungi’s ability to colonize or survive include systemic pesticide application (Trappe et al. 1984), tilling (McGonigle and Miller 1993), fallowing (Allen et al. 2001), residual phosphorus (Vivekanandan and Fixen 1991), and inorganic fertilizer inputs (Breuillin et al. 2010). However, the effects of all of these conventional agricultural practices may be transitory and depend on (1) the AMF type (its robustness); (2) at which plant growth stage the chemicals were applied; (3) types of pesticides, the application method, and the respective concentrations which were applied; and (4) the host plant species (Abd-Alla et al. 2000; Azcón-Aguilar and Barea 1997). In addition, while many systemic and soil drench pesticides are deleterious to AMF, pesticides which are intended for foliar application generally do not have significant impacts on AMF (Trappe et al. 1984). In addition, it has been demonstrated both in small plot trials and in commercial-scale agriculture that preceding crops affect the growth and yield of subsequent crops and this cannot be explained entirely by nutritional effects (Altieri and Farrell 2018; Arihara and Karasawa 2000). The inclusion of nonmycorrhizal crops within rotations has been shown to decrease both AMF colonization and yield of following crops. Proper integrated pest and farm management practices which take into account AMF sensitivities are recommended for effective host crop AMF inoculation. Figure 9.3 illustrates the effects of application of mycorrhizal fungi in soybean. Common soil and weather stresses such as high salinity, acidity, alkalinity, insufficient or overabundance of water, heavy metal stresses, and nutrient deficiencies are known phenomena to which AMF have adapted: and much research has been dedicated to how AMF assist in plant survival under these common crop stress conditions (Table 9.1; Lenoir et al. 2016). Many examples have been provided to show how AMF have contributed to positive impacts in landscape regeneration, horticulture, alleviation of desertification, and the bioremediation of contaminated soils (Jeffries et al. 2003). The next sections summarize 30 years of research on mycorrhizal technologies, products, and enterprises. It highlights major challenges, identifies gaps in the research, and proposes strategies to enhance mycorrhizal inoculation, return on investment opportunities, and successful marketing and benefits of mycorrhizal technologies. We compiled data from recent mycorrhiza research, case studies from both developed and developing countries, as well as practical insights from the Israeli mycorrhizal inoculant producer, Groundwork BioAg Ltd. The major topics related to the use of mycorrhizal inoculants that are discussed in this review are (1) regional and global markets; (2) techno-economic analyses; (3) business strategies of successful companies; (4) survey of global mycorrhizal regulation; and (5) challenges for new mycorrhizal ventures.

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Fig. 9.3 Field: Soybean field in Mato Grosso, Brazil, 2019. Right side treated with mycorrhizal inoculant; left side untreated. (Photo credit: NovaTero BioAg)

2 The Mycorrhizal Marketplace 2.1

Defining Mycorrhizal Technology

Overall, we define mycorrhizal technology generally to involve one or more of the following activities: the research, production, distribution, marketing, and utilization of mycorrhizal inoculum. The focus in applied mycorrhizal research for sustainable agriculture entails developing mycorrhizal technology in order to more efficiently produce, apply minimum effective propagule combinations onto crops, identify species, and analyze mycorrhizal viability. Out of these segments, the production and application of mycorrhizal fungal inocula have been major foci in the mycorrhizal market as these tasks have proven challenging. The production of costeffective mycorrhizal inoculants has been an elusive goal over the history of this market. Nonetheless, mycorrhizal inoculation into agricultural fields have exhibited substantial yield benefits across a variety of crop types, as documented in numerous field trials (Hijri 2016; Pellegrino et al. 2015). Nevertheless, it can be debated that production and inoculation concerns should not be the only development foci for next-generation mycorrhizal technology

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(Rillig et al. 2016). Recent research compiled by Rillig et al. (2016) describes the components of mycorrhizal technology, their current status, and specific research needs in more depth. Rillig et al. propose some key elements of future needs in mycorrhizal technology including novel monitoring, agricultural management, database tools, plant breeding, and ecological engineering of communities of mycorrhizal fungi (also known as “myco-engineering”) and their associated microbiota. Gianinazzi and Vosátka (2004) also elaborate on the needs within future mycorrhizal technology including (1) the creation of genetic or sensor technologies for tracking mycorrhizal inocula in field settings, (2) enhancing data acquisition on the ecophysiology of mycorrhizae in stressed ecosystems, (3) increasing knowledge on the interactions of mycorrhizae with other rhizosphere microorganisms, and (4) selection of appropriate or novel plant varieties with enhanced mycorrhizal traits and/or enhance mycorrhizae with new symbiotic traits. They argue that one of the principal and more challenging tasks for both mycorrhizal producers and researchers is to raise public awareness with regard to the benefits mycorrhizal technology can provide for sustainable crop production and soil conservation efforts (Gianinazzi and Vosátka 2004).

2.2

Global Biofertilizer Market: A Snapshot

In the past decade, there has been a great increase in AMF inocula production, marketing, and related services for the retail and wholesale segments (Singh et al. 2016). While specific numbers regarding sales have yet to be compiled, one could infer the tremendous growth potential based on the global biofertilizer market. The global biofertilizer market size was estimated at USD $787.8 million in 2016 and is estimated to reach USD $1.65–2.31 billion by 2022 (Biofertilizers Market Size, Share, and Trends Analysis Report 2018; Biofertilizers Market by Type 2018). Global market demand is projected to increase at a strong compound annual growth rate of 12.9% during a forecasted period of 2017 to 2025 (Transparency Market Research 2018). This tremendous increase in demand is due to a number of factors such as growing public recognition regarding the health and environmental benefits of biofertilizers, transition from chemical-based farming techniques to organic practices, increased use of biofertilizers in soil fertility management activities, growth of the organic food industry, and rising monetary and environmental costs associated with chemical fertilizers, pesticides, and related agrochemicals (e.g., nutrient inhibitors). Despite these trends, lack of awareness in potential emerging markets, insufficient infrastructure, funding, and know-how deficiencies on important mycorrhizal parameters hamper mycorrhizal market expansion. The factors which hinder prosperity of the biofertilizer and mycorrhizal markets include unknown storage stability or shelf life, production constraints, and the time and labor requirements needed to produce adequate propagule quantities, along with technological challenges.

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Mycorrhizal Technology Potential per Territory

To date, there are only a handful of basic mycorrhizal inoculant producers (companies producing inoculum for direct retail) worldwide—primarily located in the USA, Europe, and Asia. The most developed and diversified retail market for mycorrhizal products is in North America. Brazil is a world leader in export of agricultural products, but mycorrhizal inoculants were not commercially available in Brazil until 2018, when the Israeli mycorrhizal inoculant producer Groundwork BioAg became the first company to obtain product registration in that territory, in conjunction with its Brazilian counterpart, NovaTero. In Asia during the past few years and specifically in India, a boom in mycorrhizal inoculant production and distribution enterprises have also been noticed. Substantial commercial operations in the mycorrhizal inoculant market have attracted venture capital funding and acquisitions by larger companies. For example, Roots Inc. was acquired by Danish Novozymes in 2001, and A/S and Valent BioSciences bought Mycorrhizal Applications in 2015 (Gianinazzi and Vosátka 2004; Valent BioSciences Corporation Acquires Mycorrhizal Applications, Inc. 2015). A small number of mycorrhizal inoculant production companies in the USA and Canada have demonstrated proactive marketing activity and distribution on an international scale. Most other mycorrhizal production operations in the rest of the world remain “immature,” with many producers sticking to developing distribution in their local and regional markets. Competition remains limited due to legislative barriers restricting export and import of biological products along with minor penetration by other suppliers into domestic markets.

2.4

Mycorrhizal Technology Market Segments

Vosátka et al. (2008) also detail mycorrhizal market opportunities for each of the following sections below. Here we attempt to summarize some key takeaways and relay new insights.

2.4.1

Hobby Garden Sector

Many mycorrhizal producers have focused on the hobby garden market with hundreds of products formulated and packaged for both on-line markets and on-site retail garden centers. Most formulations for this market are mainly comprised of singular AMF species, ectomycorrhizal fungi, or the combination of both with other rhizosphere microorganisms and are marketed to help plant growth. Vosátka et al. (2008) described the one of the principal marketing messages in this center as a “more professional” planting practice for those who wish to enhance general garden plants. Vosátka et al. explain that part of the difficulty of selling in this sector is often

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in the placement of the products in the “organic” or “fertilizer” sections of the garden center. As products are not always certified organic nor considered a replacement for fertilizer, this gives mixed messages to the gardener. The true role of a mycorrhizal product is to add a secondary root system which is able to support the host plant’s acquisition of nutrients for its entire lifetime, not to offer a finite nutrient resource like a fertilizer.

2.4.2

Horticulture and Commercial Nurseries

One definition of horticulture can be described as “the art and science of plant production for beauty, value, and utility.” Horticulture is a practice in growing fruits, vegetables, flowers, or ornamental plants, but a broader definition also incorporates plant conservation, landscape restoration, soil management, landscape and garden design, construction, maintenance, and arboriculture (Solaiman and Mickan 2014). The horticulture definition generally excludes large-scale crop production operations. The horticulture sector remains a significant and very attractive market for mycorrhizal fungi products, as many high-value ornamental and edible horticultural crops form arbuscular mycorrhiza (Azcón-Aguilar and Barea 1997; Vosátka et al. 2008). The interest of horticulturists in mycorrhizal technology is due to: the ability of AMF to increase the uptake of phosphorus and other nutrients; enhance survival rate and development of micropropagated plantlets and earlier flowering and fruiting; increase crop uniformity; improve rooting of cuttings; augment fruit production; and increase resistance to biotic and abiotic stresses (Azcón-Aguilar and Barea 1997). One of the primary avenues from which the mycorrhizal industry can profit from in this sector includes selling to large-scale operations with repeated seasonal inoculum needs. Trials have shown yield enhancements of up to 50% in field-grown bulb flowers and over 15% annual yield increases in flower production, thereby demonstrating how the application of mycorrhiza in flower production can be economically advantageous (Vosátka et al. 2008). Plant propagation in horticultural systems generally starts from seedlings, cuttings, or graftings produced or developed in soil or mixed media substrates which have been chemically or temperature-treated to reduce pathogen risks (AzcónAguilar and Barea 1997). This part of the horticultural arena, micropropagation, is a promising area for the introduction of AMF inoculants as the sterilization process also kills the beneficial rhizosphere microbes in in vitro cultivation. After transplantation, these plants can significantly benefit from the introduction of mycorrhiza to reduce transplantation shock (Vestberg et al. 2002). A unique area that the mycorrhizal market can exploit in horticulture is the essential oil marketplace. Essential oils from a variety of plants are widely used in the cosmetic, pharmaceutical, food, and flavoring industries (Copetta et al. 2006). Mycorrhizal fungi have demonstrated tangible benefits in this arena. Two AMF species—Glomus macrocarpum and Glomus fasciculatum—were tested on fennel

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(Foeniculum vulgare Mill) essential oil production, and Glomus fasciculatum was found to significantly improve essential oil concentrations up to 78%, especially of anethol (Kapoor et al. 2004). Glomus fasciculatum was also demonstrated to enhance root colonization, growth, essential oil yield, and nitrogen, phosphorus, and potassium acquisition of three cultivars of menthol mint (Mentha arvensis). Gigaspora margarita BEG 34 was shown to increase biomass, root branching and length, and total amount of essential oil yield of basil (Ocimum basilicum L. var. Genovese) (Copetta et al. 2006). Specialty and nonspecialty commodity crops grown exclusively in nurseries (e.g., Cannabis) could especially benefit from mycorrhizal inoculants as often the media used to grow many of the nursery plants in this sector are sterilized. Challenges in this arena include understanding the proper mycorrhizal strain which would provide the greatest benefit to the host crop, proper application rate, and application methods. There are a variety of ways mycorrhizal inocula can be applied to nursery media. However it is important to understand which mycorrhizal strain will provide the greatest benefit to the host crop; proper application rate; and proper application methods. For example, one must understand if the strain inoculum is best applied by mixing the propagules with the media from the start, inserting inoculum in-furrow, or adding inoculum into the irrigation system. Application methods, rates, and associated challenges will be elaborated further in Sect. 3.

2.4.3

Forestry and Landscaping

Many tree species are naturally associated with AMF. Indeed, seedlings of tree species raised in nursery settings may need to be associated with AMF in order to survive transplantation shock (Urgiles et al. 2009). Results from field trials in Ecuador showed that maintenance of tree seedlings was significantly improved by inoculation with mycorrhizal fungi of either the individual tree species or a mixture of mycorrhizal inocula from four trap species (Urgiles et al. 2009). While it can be difficult to assess which mycorrhizal species are most appropriate for the host plant, studies suggest that introducing diverse AMF to seedlings may be advantageous after planting in the field. Particularly in tropical settings, the natural environment, edaphic, and climate conditions will select for the best-adapted species (Allen et al. 2005; Urgiles et al. 2009; Zahawi et al. 2015). Targeting AMF inoculants as well as ectomycorrhizal fungi for use in operations involving large-scale, fast-growing trees (e.g., willow, poplar, and alder) for biomass or timber plantations are also potential and emerging market opportunities (Vosátka et al. 2008). This market opportunity is due to the rise of rapidly growing trees being planted for the timber market in tropical regions in Latin America and Africa on clear-cut sites left after rainforest cropping (Vosátka et al. 2008). For the landscaping sector, mycorrhizal inoculant application could be advantageous as many general landscaping practices occur in places where soils can be fertility-deficient, compacted, or even polluted after mining activities or extensive

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agrochemical application. As mycorrhizae can help with both soil fertility and compaction concerns, mycorrhizal inoculant application in this sector is particularly beneficial on sites where aftercare is difficult, for example when planting occurs on steep slopes or highway verges, making fertilization and irrigations measures difficult to implement. In many landscaping environments, soils are disturbed, absent of native mycorrhizal fungi or containing very low levels. In these types of situations, mycorrhizae may take years to reestablish naturally. Inoculation with mycorrhizae at planting would assist in establishing stable, functional fungal root systems, and may aid in reducing transplantation shock. Increasingly, landscaping endeavors are being financed by customers from private corporations, businesses, private homes, regional governments, or municipal agencies (e.g., highway and road maintenance) which seek to promote low-cost, natural, more ecologically diverse, and aesthetically pleasing solutions for their landscaping needs. Introducing these sectors to mycorrhizal inoculants fits to address these stated goals and can be marketed accordingly in order to encourage native planting and best reclamation practices to establish natural or seminatural ecosystems (Vosátka et al. 2008). Specifically, sports fields, road medians, golf courses, public and private parks, and gardens could benefit from the vast array of positive mycorrhizal services.

2.4.4

Revegetation and Remediation

Reclamation sites may have problems similar to those found in the landscaping sector: soils may be subfertile, compacted, not compacted enough, or polluted from extensive agrochemical application. An additional issue at reclamation sites may be pollution from mining, including heavy metal contamination depending on prior land use. In reclamation sites where native fungi are absent, reduced, or reduced in viability, the introduction of mycorrhizal fungi into the soil is a reasonable measure. Much research has been dedicated to the positive effects that mycorrhizae can provide to soil containing volcanic ash, mine spoils, waste deposits, other anthropogenically polluted sites, or sites where inappropriate soil and vegetation management has been employed (Gosling et al. 2006; Harrier and Watson 2003; Plenchette et al. 2005). Mycorrhizae can ameliorate many of these problems, especially in reducing heavy metal pollutants and improving insufficient soil compaction (Khan et al. 2000). In addition, if the reclamation sites are to be used for crop production, many studies have described the capability of some AMF, depending on the host plant, to reduce translocation of some heavy metals such as lead (Wu et al. 2016), cadmium, and arsenic (Chaturvedi et al. 2018) and possibly other pollutants into the edible parts of food crops, e.g., vegetables and tobacco (Hall 2002; Janoušková et al. 2005). Challenges in this sector include the fact that different host plants selectively uptake different heavy metals. Not all compounds are immobilized when entering the host plant. In response to mycorrhizal inoculation, many deleterious compounds

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may accumulate inside the host plant. These accumulations may result in vegetation becoming unsuitable for human consumption, for animal grazing, and for other direct contact uses. In comparison to common agricultural practices, mycorrhizal fungi have repeatedly proven to provide a more natural and sustainable approach to plant establishment, fitness, vigor, yield, and nutrition enhancements when introduced into stressed and disturbed environments (see Sect. 1.5). Much of this sector remains an untapped marketing opportunity for mycorrhizal inoculants. Bioremediation and phytoremediation stand as the next frontier for research and sales in this arena.

2.4.5

Agricultural Market Opportunities in Developed and Developing Regions

Development of distribution efforts into the agricultural sector is perhaps the largest challenge for new and existing mycorrhizal inoculum producers both in terms of potential revenues and in global benefits. One of the more profitable niches mycorrhizal technologies in both developed and developing regions could enter into is the “organic” market. At least in developed regions, it is probable that the highest value and profit margins could exist in the organic agricultural markets as mycorrhizal inocula could accompany or potentially replace traditional conventional and chemically based fertilizers (Vosátka et al. 2008). This avenue remains largely untapped due to the financial, labor, and equipment capital and profit margins needed to sustainably produce large amounts of inoculum in both developed and developing regions. Due to the waste that would result, current and future mycorrhizal inoculum application is unlikely to be accomplished via broadcast methods. Inoculum is best positioned near to, or onto, the seed or root zone. Precision application alternatives have already been demonstrated in commercial agricultural systems. These include seed drilling, or treating seeds initially with finely-ground, high-density inoculum. Other possibilities that are actively employed and encouraged by several mycorrhizal inoculant producers worldwide include various forms of in-furrow and planting hole applications. Many of these newer options for appropriate, large-scale inoculum placement require specific, sophisticated, and potentially expensive equipment. Also required is knowledge of the mycorrhizal fungi type to be applied; education on the mycorrhizal sensitivity of the host crop; information on which stage of the growth process is most effective for application of mycorrhizal inoculum; minimum application rates and methods; and which agrochemicals and concentrations may hinder or even kill mycorrhizal development. These challenges limit mycorrhizal usage in the developed agricultural sector, and limit usage in developing regions even more profoundly. Additionally, culture of mycorrhizal fungi is conventionally laborintensive and harvesting of the inoculum requires large-scale production of plants either in pots or nursery beds. For developing regions, Mukhongo et al. (2016) argue that the costs associated with the technology of inoculum production are borne by farmers and nursery

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owners. These costs include sterility requirements and establishment of single cultures of AMF species, shipping and handling, and development of the carrier substrate. In addition, the value chains in developing countries are deeper, so prices for growers are much higher (Mango et al. 2018). This makes mycorrhizal technology expensive in developing regions. Commercial inoculum produced using a variety of mycorrhizal production systems is generally available in more welldeveloped regions in North America, Asia, and Europe. While larger profit margins probably exist in marketing mycorrhizal technology to the developed world due to the more efficient and large-scale production of commodity crops, agricultural producers in impoverished regions would probably benefit the most from mycorrhizal application due to the large-scale nutrition and climatic vulnerabilities they constantly face. Agricultural productivity in many areas around the world, but especially in developing areas, is gradually declining and attributed to increasing water stress, climate changes, low soil fertility (especially insufficient nitrogen, phosphorus, and potassium), herbivorous insects, pathogen infestation, and associated diseases. The limited knowledge, lack of financial capital, and inadequate equipment-based resources most developing nations and smallholder farmers possess in order to combat these problems are the largest differences between developed and developing nation approaches to mycorrhizal inoculant technology. In addition, the practice of continuous cropping without soil inputs has exacerbated declining soil fertility. Yet, improvement of soil fertility, crop yields, and nutrition provided by mycorrhizal inoculum application onto corn and other vegetables in developing regions have demonstrated AMF’s incredible capacity to change the pace of food scarcity in these regions if applied correctly. For example, acidic conditions limit crop production in 40% of the world’s soils, mostly because many plants do not absorb nutrients (especially phosphorus) well in acidic (pH < 5.0) or in alkaline (pH > 7.5) soils. In sodic and saline soils (estimated at 5–15% of agricultural land), too much sodium, boron, and chloride frequently reduce crop production. Toxic concentrations of manganese and iron can materialize from waterlogged or flooded soils (Oliver and Gregory 2015). Beyond providing appropriate education and financial capital, effectively developing production, marketing (often in multiple regional languages and dialects), and distribution networks in economically disadvantaged regions also exist as limiting factors to expansion of mycorrhizal inoculant technology. Research performed by Vosátka et al. (2008) expands upon the concept of future collaboration between researchers and commercial producers in the quest for a consensus on efficacy limits that if defined could help producers develop this sector. Vosátka et al. provide examples and argue that, even in developed markets, new AMF inoculant production enhancements are required in order to cost-effectively increase the propagule concentration using current standard production methods, at the same unit cost. Regardless, whether trying to sell in developing or developed markets, mycorrhizal inoculants need to be cost-effective, simple-to-apply, and accessible, and the populace in both types of markets need further education in its wide spectrum of benefits.

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Mycorrhizal Fungi Technology Potential per Crop

Robust research performed by Tawaraya (2003) describes an extensive list of plant species, cultivars, and their relative dependencies on specific mycorrhizal types. This research suggests substantial market potential for cultivated mycorrhizal inoculants. As many major global food crops top the list of highly mycorrhizal-dependent plant species, these crop commodities could benefit from the addition of appropriate AMF inoculants and thereby enhance global food production.

3 Business Strategies of Successful Mycorrhizae Companies 3.1

Adoption of New Agricultural Technology

Successful adoption of new technologies by farmers is key for adapting in an everchanging market along with enhancing their chances of increasing outputs and profits. Klerkx et al. (2012) summarized the main shifts of agricultural innovation over approximately five decades and showed increasing complexity due to enhanced access to more information, changing regulatory processes and incentives, and current regional and/or global mindsets regarding agriculture (e.g., push toward organic and less environmentally harmful crop production). There exists the idea that new, demonstratively effective technology is at the heart of any successful innovation and is one primary reason new agricultural concepts are adopted. However, there are several other factors that can affect the adoption of agricultural innovation such as policy, legislation, infrastructure, funding, and market developments (Klerkx et al. 2012). Social network inputs and firsthand knowledge can influence a farmer’s decision to adopt a certain technology. Even without full, detailed knowledge about the new technology, observation of successful operation and results such as positive results from field experiments can lead a farmer to have a more positive impression on the use of the new technology. Once a farmer successfully utilizes and publicizes the new technology, he creates awareness in his local social network that could lead other farmers to adopt the same technology (Llewellyn et al. 2012; Maertens and Barrett 2013). While observation of local experimentation may be limited to the farmer’s social network, positive results from local network and industry R&D experimentation could also have a large impact on diverse clients in different regions (Evenson 2001). On the other hand, some farming decisions are directed more by constraints or tradition, and may not be as easily influenced by external efforts (Suri 2011). From an Israeli mycorrhizal inoculant producer’s perspective, Groundwork BioAg’s overall experience is that farmers who demonstrate open minds and motivation to look for more efficient, new products and procedures are a good target market for new technology adoption. In addition, as mycorrhizal inoculants can make host crops more profitable, demonstration of these positive results on the respective host crop or local field conditions increases the likelihood and motivation of the farmer to adopt mycorrhizal technology and products.

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Table 9.3 Advantages and disadvantages of common mycorrhizal product formulations Application method Growth Mixing media

Formulation Granules

Root treatment

Coating

Powder

Seed treatment

Dusting

Powder

Coating

Filler and binder

Concentrated product, accurate, can be combined with pesticides, suited for non-tillage practices

Drip irrigation/infurrow

Wettable powder

In-furrow

Fine powder

Application during plant growth; can use existing irrigation system Application during plowing or sowing

Soil drench

a

Advantages Multiple mixing options; effective in commercial nursery; seedlings can be well-coated; highly cost-effective Highly potent inoculum; can be applied to an expansive root system; high potential for high root activity Specialized equipment not necessary; highly cost-effective

Disadvantages Not concentrated; cannot apply after transplantation; particle size can influence effective application Only applied during replanting; timeconsuming; respiratory-sized particles could pose health hazard Inexact application rate; low uniformity; respiratorysized particles could pose health hazard Specialized equipment needed; strong competition by other companies

Application is not directly applied to root Unreliable and nonhomogeneous application rate; respiratory-sized particles could pose health hazard

References Bashan (1998), Mahanty et al. (2017) Picone (2002)

Bashan (1998), Nuyttens et al. (2013)a Oliveira et al. (2016), Rocha et al. (2018) Bashan (1998)

Bashan (1998)

Nuyttens et al. (2013) discuss pesticide formulation advantages/disadvantages

3.2

Understanding Mycorrhizal Inoculant Formulation Advantages/Disadvantages

Mycorrhizal inoculant quality has a great impact on the reputation and success of a company. Mycorrhizal product formulations generally consist of the fungi, a carrier, and additives (e.g., stabilizer, fertilizer, etc.) and can be designed to accommodate differing host plant or client application needs (Table 9.3). Fungal propagules such as colonized root fragments, fragments of fungal mycelium, and spores are generally the main living constituents of a mycorrhizal product. It is these living constituents that colonize plant roots in order to confer the many benefits of mycorrhizae to the plant. Therefore, it is important that commercial mycorrhizal inoculant producers

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include many living, infective mycorrhizal components in product formulations in order to ensure effective colonization onto and into the host roots. There is no official standard or thorough research on the minimum infective dosages of inoculable mycorrhiza for every host plant. Therefore, the amount of active ingredient in product formulations can vary widely between mycorrhizal inoculant producers. For example, Faye et al. (2013) demonstrated the importance of mycorrhizal propagule quantity and quality by assessing 12 commercial mycorrhizal inoculants after inoculating maize in greenhouse conditions. Of the 12 assessed products, nine claimed to contain 13–100 propagules per 1 cc, another product claimed an incredible 200,000 propagules/g; and two products did not mention the number of propagules. Moreover, colonization rates of roots did not correlate with the number of propagules. At six weeks after planting, they found seven of the inoculants to enhance root colonization levels compared with control soil; however, only three inoculants marginally increased the maize shoot biomass (Faye et al. 2013). Tarbell and Koske (2007) also tested eight mycorrhizal commercial products for their ability to colonize Zea mays (corn) roots in containers of sand/peat media when applied at both the manufacturers’ recommended rates and at five to ten times the recommended rates. Only three of the eight tested inocula formed mycorrhizae when used at the recommended rate, and the extent of colonization ranged from 0.4 to 8%. Of the eight products, increasing the inocula rate enhanced colonization levels from 8.6 to 72.5% at the highest rate (10). Strangely, one product did not colonize at both the recommended and 5 rate but did produce 8.6% colonization at 10. Another product did not colonize at any rate but did contain an identified root pathogen (Tarbell and Koske 2007). Collectively, these studies determine that the significant failure rate of commercial inocula to colonize host roots when applied at recommended rates suggests that preliminary trials should be made before commercial AMF inoculants are utilized for important plantings. Another important factor to consider upon application of mycorrhizal inoculants is how and when the inoculants are applied to roots at different plant physiological stages, as ideal root inoculation times may differ among host plant types. Therefore, selection of an appropriate carrier, or the material to which the mycorrhizal particles adhere and maintain viability, is very important. Many agricultural products use common carriers such as perlite, peat, inorganic clay, zeolite, vermiculite, sand, and more. One must understand the plant type and ideal application method for that crop. The application method will be dictated by the carrier type which suits the crop and application method (Bashan et al. 2014; Gianinazzi and Vosátka 2004; Mukhongo et al. 2016; Mahanty et al. 2017) (Table 9.3). The formulation could be prepared from singular or multiple ingredients depending on client demands and host plant needs (Rodrigues and Rodrigues 2017, 2018). An effective mycorrhizal product must satisfy the regulatory agency’s storage stability requirements, must be certifiably free of any pathogens or other contamination types, and must contain and maintain at least the minimum infective mycorrhizal component amounts stated on the label. In nature, AMF propagules can survive and maintain their infectivity for years in soil even without direct host root contact. For example, Guadarrama et al. (2006)

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evaluated the mycorrhizal propagules in tropical dry forest plots which had been previously subjected to slash-and-burn agriculture and posterior abandonment. The plots were in different stages of secondary vegetation, from 0 to 27 years after abandonment. They discovered high field root colonization rates (44–69%), viable spores (23–34 spores/100 g dry soil), and infective propagule rates (4–37 infective propagules/100 g dry soil) that did not correlate with the time of abandonment: The longer time of abandonment did not correlate with lower colonization, viability, or infectivity rates (Guadarrama et al. 2006). McGee et al. (1997) isolated mycorrhizal spores from freshly sampled soils and found 4–200 spores/g and their respective viability ranged from 16–21% viability. The viability of spores dropped to 6–7% after 24 months. Haugen and Smith (1992) found that the mycorrhizal fungus Glomus intraradices can retain its infectivity in well-watered soil, at high temperatures (22–38  C) for up to 6 weeks. They found that after 6 weeks, the inoculum infectivity decreased faster at higher temperatures (43  C). Therefore, formulation features will ideally support spore survival by creating a buffered environment which maintains optimal moisture, nutrient, and temperature conditions needed during the storage life of the mycorrhizal product (de Santana et al. 2014; Mahanty et al. 2017; Rodrigues and Rodrigues 2018). Additionally, the mycorrhizal formulation, packaging, and storage conditions need to be contaminant-free and/or discourage the introduction or growth of potential contaminants. Microbial contamination prevention steps during the mycorrhizal production process could include employing strict aseptic growing practices, clean storage and packaging protocols, hygienic production environment, and sterilization of the equipment and formulation ingredients. These steps can support the creation of mycorrhizal products which are free from non-biological contaminants, pathogens, or other uninvited microorganisms (Gianinazzi and Vosátka 2004). For the creation of an organic mycorrhizal product, the formulation components, growth protocol, and production process need to follow additional, strict regulation standards set forth by the organic regulatory agency. Therefore all ingredients and processes must be accredited and sometimes independently tested for veracity of content or process (Picone 2002; Mahanty et al. 2017).

4 Mycorrhizal Techno-Economic Analyses 4.1

Grower Business Model and Return on Investment

In a grower business model, the amount of return from investment calculations ranks high on the list of considerations when choosing mycorrhizal products above other biofertilizer or conventional fertilizer products. Typically, yield increases are the main factor that is considered. While mycorrhizae have been shown to significantly enhance plant tolerance to abiotic and biotic stressors such as to petroleum, heavy metals, salinity, nutrient deficiencies, and drought-induced conditions (Table 9.1; Lenoir et al. 2016), one would assume that mycorrhizal inoculants can therefore reduce irrigation,

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fertilizer, and possible pesticide inputs. Return on investment is mostly a function of yield increase. However, return on investment values remain largely dependent on local crop prices and related input prices (e.g., fertilizer, irrigation equipment, water prices, labor, etc.). Moreover, depending on the mycorrhizal strain, the crop cultivar type and the combination of that cultivar’s mycorrhizal dependency to a specific mycorrhizal strain, the overall yield enhancements to a crop can vary drastically per season (Tawaraya 2003). The base soil nutrient levels, pesticide residues left in the soil, previously planted crop, weather conditions, soil contaminants, and pathogens can also affect mycorrhizal effectiveness and return on investment calculations. Few producers are capable of developing highly concentrated, high-quality, efficacious mycorrhizal inocula due to complexities in the production process which entails simultaneous production of the fungi and the host plants. Most mycorrhizal fungi do not lend themselves well to production in bioreactors or fermenters. Therefore, inocula must be produced under lengthy and labor-intensive processes. Few companies have succeeded in overcoming the challenges associated with the cost-effective production of mycorrhizal inoculants. Exact financial calculations that mycorrhiza could contribute economically in this regard remain vague and mostly unexplored. The academic world remains undecided on which parameters influence mycorrhizal effectiveness most significantly. Regardless of the academic context, farmers desperately seek cost-effective products in this market in order to increase yields and profits while minimizing losses and costs due to climate change, volatile market prices, and legislative barriers. Rillig et al. (2016) recommend further research into agricultural management approaches in order to further understand what factors may interfere with mycorrhizal-mediated benefits. Advancement in this arena would further define which processes are ultimately more helpful or damaging to potential mycorrhizal crop benefits and therefore provide more realistic expectations for stakeholders and for setting management priorities. In order to maximize potential benefits of mycorrhizae through inoculation in field conditions and to achieve a respectable return on investment, customers should investigate and have a thorough understanding of the following: soil nutrient profile and deficiencies; field soil heterogeneity; published research on if that plant and cultivar has responded well to mycorrhiza and which type of mycorrhiza; previous field-applied pesticide use which may delay successful mycorrhizal colonization; previously planted crop (mycorrhizal-sensitive or not); past and present plant and soil pathogen contamination and severity; appropriate inoculant application and inoculation conditions (e.g., best time and method to inoculate depending on crop’s physiological growth stage); and proper aftercare (e.g., no systemic pesticide use, reduced tilling, etc.). It is recommended that prior to making a significant investment, the grower should treat a small portion of his/her field with the mycorrhizal inoculant and inoculate the proposed plant cultivar in order to understand how the mycorrhizal product will react to the respective plant cultivar and soil conditions. Average yield values of treated crops against a control could provide a respectable baseline in order to calculate if the combination of the mycorrhizal inoculant and that

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crop cultivar would meet a full return on investment after successful inoculation within one crop season. Vosatka and Dodd (2002) also argue that based on current evidence and that local soils are likely to respond better, it may be more advantageous to inoculate using native mycorrhizal formulations. To reduce startup costs, these inocula should be produced locally, as there are also important regulatory and possible environmental concerns in transferring non-autochthonous microbial species to new areas (Schwartz et al. 2006). Therefore, local production of native AMF strains could address both of these concerns. Some manufacturers profess to include several localized strains in their product formulations to address these concerns. The mere presence of mycorrhizae does not indicate benefits are being transferred to a plant. There are questions of whether the native AMF species are as effective as they could be in serving the diversified or specific native, local plant populations. It may be advantageous to test and see if different and more efficient AMF isolates, e.g., faster and more robustly colonizing species, could substitute for less effective, native AMF already present in the soil (Adholeya et al. 2005). An overall recommendation would be to ensure that a mycorrhizal inoculant contains multiple, highly concentrated, infective, localized, and non-native yet proven robust strains for application onto a variety of crop types. Nevertheless, some global case studies have made reasonable attempts to calculate return on investment opportunities in cassava, peppers, and soybeans after inoculation with the AMF and specifically the endomycorrhiza Glomus intraradices (also known as Rhizophagus irregularis or Rhizophagus intraradices). The studies below describe mycorrhizal return on investment opportunities when AMF are combined with differing P fertilizer regimes and other bio-stimulants.

4.1.1

Cassava and Mycorrhiza Case Study

Ceballos et al. (2013) evaluated the effect of in vitro mass-produced Rhizophagus irregularis inoculum on the yield of cassava crops, a very popular and important global food crop, at two locations in Colombia. They applied an economic analysis which fully assessed and compared production costs in the different treatments, profits, cash flow, and profitability (return on investment—ROI%). At both locations they calculated the cost-benefit and ROI% of cassava after the application of six treatments. Treatments included three phosphorus levels: 0 P, 50% of standard application levels and standard P fertilization, with and without AMF inoculation. In addition, they considered agricultural interest rates for calculations involving monetary value, corresponding to an effective rate of 11% annually–the most conservative interest rate they considered for such analyses. They carried out an economic simulation using different inoculum prices to calculate the economic feasibility of using the product and to find the maximum inoculum price at which the farmer would increase profits than by using the traditional crop management techniques. Additionally, they performed an identical simulation when applying varying phosphate fertilizer prices in order to examine the economic feasibility of utilizing Rhizophagus irregularis while

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considering possible future P fertilizer prices. They calculated inoculum prices using European prices of the product as at the time these prices were not currently available or publicized for the Colombian market. Overall, they found a significant effect of Rhizophagus irregularis inoculation on cassava yields at both sites. The first site demonstrated yield increases regardless of the P fertilization level. At the second site, with only half of the normally applied P, Rhizophagus irregularis inoculation produced the highest yields. However, even though Rhizophagus irregularis inoculation resulted in greater food production, their economic analyses revealed that it did not produce a greater return on investment when compared to conventional cultivation methods. However, it is important to note that the amount of applied inoculum was twice the recommended application rate and the return on investment calculations were calculated using European, not Colombian, inoculum prices.

4.1.2

Peppers and Mycorrhiza Case Study

In a field trial performed in the Arava desert in Israel under the auspices of the Agricultural Research Organization (ARO), Volcani Center, peppers of the Celica variety were treated with Glomus intraradices. The peppers were treated at two levels of fertilization and three levels of irrigation (50%, 75%, and 100% of standard irrigation levels) (Cohen et al. 2017). The fertilizer treatments were full fertilization, according to the accepted practice, and low P fertilization. They found the effect of the level of P fertilization as highly significant ( p < 0.05)—full fertilization was better than low P fertilization. In the low P treatment, there was no effect of the irrigation rate, while a significantly ( p < 0.05) higher yield was obtained when inoculated with mycorrhiza. In the full fertilization treatment, there were significant effects ( p < 0.05) of both mycorrhiza and irrigation rate—in both cases a higher yield was obtained in the presence of mycorrhiza and when irrigated at 100% of the recommended level. There was no correlation between the irrigation rate and the presence of mycorrhiza: Therefore, they concluded that one variable cannot be used to compensate for the other. From a financial point of view, results showed that it was possible that, under a full fertilization regime, mycorrhizae could contribute a revenue increase in the order of US$1800/1000 m2 when the price rate of peppers is set at $1.4/kg. When multiplying these results by 45,000 m2 per farm, a substantial addition of approximately $86,000 per farm could be established in return for an investment of approximately $40/1000 m2 or ~$1800 for the price of inoculating an area of 45,000 m2 of peppers with mycorrhizal fungi.

4.1.3

Soybean and Mycorrhiza Case Study

Two years of field experiments in Raipur, India, were executed to find the best soybean [Glycine max (L.) Merr.] yield- and growth-enhancing P source and level with and without the addition of P-solubilizing microbial seed treatments (Sarawgi

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et al. 2012). Soil type tested was vertisol, and its characteristics included neutral to slightly alkaline, medium in organic carbon, low in available nitrogen and P, and high in available potassium. P fractions were studied after crop harvest. Results from this study showed that, in comparison to the standard soybean treatments, the addition of microbial inoculants including bacteria and mycorrhizal fungi produced significantly higher yield and economic benefits. Results showed that application of 30 kg P2O5/ha through rock phosphate (RP) + phosphate-solubilizing bacteria (PSB) + Rhizobium inoculation (RI) + vesicular arbuscular mycorrhizae (VAM) produced significantly higher seed yields, net return, and invested rupee returns in P when compared to application of only 60 kg P2O5/ha through rock phosphate without biofertilizers. They discovered that the P supplied through rock phosphate and inoculated with PSB, RI, and VAM augmented the overall nitrogen and P soil content, while PSB and VAM application improved the availability of different inorganic P fractions in the soybeans.

5 Challenges in Registration and Regulation of Agricultural-Based Fungi Applications 5.1

Mycorrhizal Registration Features

Producers who wish to successfully market, export, and sell mycorrhizal products will find it advantageous to legally register their products both locally and abroad. Two major concerns of regulatory authorities that dominate the mycorrhizal market are proving that the mycorrhizal inoculants are (1) safe and (2) effective. At this time there are no standardized regulations worldwide which dictate how mycorrhizal inoculants are to be considered safe or effective. Generally, many US states as well as other countries consider laboratory tests sufficient which prove inoculum purity (no additional or unannounced microbial or chemical additives/contamination) and inoculum viability within the expiration date (storage stability). Some regulatory bodies may insist that producers provide literature review data and field trial results on how that specific mycorrhizal species, product, and/or combination is effective in increasing plant productivity (yield or nutrient growth) in specific areas (e.g., US states South Dakota and Arkansas). However, it is tricky to make claims and promises that there exist mycorrhizal “superstrains” capable of increasing plant biomass under any environmental and soil conditions (Vosatka and Dodd 2002). Certified organic agencies such as the North American Organic Materials Review Institute (OMRI, https://www.omri.org/) and the European Ecocert (Ecocert, www. ecocert.com) also currently require proof of the mycorrhizal production process. To be considered “suitable for organic farming” according to these agencies, the mycorrhizal production process should not include conventional, nonorganic certified fertilizers; pesticides (herbicides, insecticides, fungicides, etc.); or growth regulators. Independent testing of mycorrhizal products could generally identify

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residues of noncertified organic products. When those residues are identified, the products will not pass stringent standards set by those organic certification agencies.

5.2

Regulation of Mycorrhizal Products

There are efforts to create overarching regulatory bodies which take the necessary measures to ensure that inoculum producers respect defined quality criteria. For instance, the internationally recognized IMS (International Mycorrhizal Society, https://mycorrhizas.org/) is both a scientific society involved in the advancement of education, research, and development in the area of mycorrhizal symbiosis, and it is also geared to help corporations producing inocula to promote their products’ name and quality (Vosátka et al. 2012). In order to promote uniform legislation, encourage effective sampling and fertilizer analytical methods, and develop high standards of fertilizer inspection techniques and enforcement practices, the Association of American Plant Food Control Officials (AAPFCO, http://www.aapfco.org/) provides networking opportunities, conferences, and labeling guidelines for those who wish to register biofertilizer or fertilizer products in the USA. Nonetheless, it is largely still in the best interest of the scientific community to serve as overseers and to test mycorrhizal products for quality purposes. A current challenge for most fungal biotechnology overseers is the lack of effective, affordable, time-efficient, reproducible methods and tools to test for mycorrhizal inocula species, sterility, infectivity, and quality control (Vosátka et al. 2012).

5.3

Conflicting Definitions of Mycorrhizae in the Regulatory Sphere

Another set of challenges a mycorrhizal inoculant producer who wishes to register their product may face is understanding how mycorrhizae are defined in a particular region. Several US states, for example, consider mycorrhizal inoculants as a fertilizer or biofertilizer, while others consider them as a soil amendment or microbial inoculant. Some US states and countries do not have specific definitions of what mycorrhizae are or have yet to define them. These confusing definitions make the situation even more vague and possibly difficult for producers and legislators alike to register and regulate mycorrhizal products. While certain regulatory agencies may require a small one-time registration fee and paperwork defining the safety and efficacy of the mycorrhizal products, others may ask for repeated external laboratory-based testing of various company-produced samples and extensive efficacy testing from successful field trials which can take months, if not years, to execute.

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As widespread cognizance is still in its infancy of how specialized AMF inoculation can benefit plant production operations, navigating local and international registration processes can be more difficult than traditional and more straightforward approaches required of conventional fertilizers or pesticide registration. With obscure and at times confusing legislative standards, it could be advantageous for both new and established mycorrhizal inoculant producers to obtain on-site or external consultants in order to successfully navigate a targeted region’s registration jungle. Labor and capital may need to be allocated in order to hire specialized in-country consultants who are experts in the respective region’s agricultural product legislation, marketing, and registration needs.

6 Challenges of New Mycorrhizal Ventures 6.1

Market Demonstration and Education of Public and Target Market

Producers or distributors of beneficial fungal inoculants must work towards educating the potential customer base regarding the harmless, ubiquitous role of many fungal species in nature. From there, convincing the market base of how the respective mycorrhizal fungi inoculant can benefit plant productivity is the second goal. These two educational deficits are principal challenges for producers. The term “fungi” often has a negative connotation for agriculturalists. Many have learned to associate fungi exclusively with pathogenic species and with harmful instead of beneficial effects. Luckily, as the scientific community continues to dedicate extensive research efforts on how mycorrhizae function, under which types of conditions, and for which types of plants, the agricultural industry is taking notice. Setting up market demonstration opportunities at various outlet types is highly beneficial both for the education of the general public and target market, along with creating effective product marketing. Market demonstration opportunities include but are not exclusive to conference presentations, trade shows, written and visual media coverage of successful field trials or collaborations, video demonstrations, on-site field or laboratory tours, on-site lectures, radio coverage, company and product social media coverage, discounts and free sample distribution, and much more. Currently, one of the more successful avenues to acquire a client base is through extensive local and international networking efforts in the agricultural inoculant and fertilizer industry. Providing customers with samples to try on their specific field and plant types is also generally a harmless approach. With the advent of a host of new software and sensor-based technologies for identifying mycorrhizae, soil nutrient status, crop sensitivity, and more, Rillig et al. (2016) discussed the future of mycorrhizal market demonstration. Marketing the usefulness of “knowledge per acre,” computer-based technologies could be an essential component for

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mycorrhizal inoculant technology and demonstrations. These new technologies could act as two-way communication tools with stakeholders and on-site farmers, as information could be displayed in real time and be provided in many versatile formats including workshops, hands-on demonstrations, and data-driven phone or computer applications. Mycorrhizal inoculant producers and distributors should consider the approach of providing simple phone or tablet-based applications which would offer the user (farmer) to input field- and crop-specific information based on user and sensor contributions. From there, the application could make evidence-based suggestions for appropriate mycorrhizal application types, rates, and methods. These types of applications could instantly enhance networking and post-sales service efforts between farmers, mycorrhizal consultants, and customers.

6.2

Acknowledgment of Consumer Considerations in Product Selection

The mycorrhizal inoculant producer or distributor must acknowledge consumer points of view and variability in marketing their products. The vendor must understand and highlight which climatic and soil conditions, crop type, and field area, along with applied pesticide or growth regulators (along with the application method, concentration, and application frequency), the farmers generally encounter or utilize. Along with these varying parameters, vendors can be limited in the amount of product they can sell as the sales cycle for plants generally depends on the season and plant type. Emphasizing and demonstrating the mycorrhizal inoculant’s return on investment potential in the arenas mentioned above would also be advantageous.

6.3

Wallet Share and Competition

How the mycorrhizal inoculant vendor addresses customers’ questions and concerns about their mycorrhizal inoculants helps dictate how the customer gauges the potential benefits of the mycorrhizal inoculant. Indirect competition to the sale of mycorrhizal products includes synthetic fertilizers, biofertilizers, bio-stimulants, growth regulators, and other types of microbial products. Overall, the vendor needs to provide extensive, professional, and qualified evidence to the general public, to the target customer base, and to prospective legislative bodies on the wide array of their mycorrhizal inoculant benefits. A vendor’s claims to product purity, efficacy, positive biotic and abiotic effects, enhanced plant nutrient uptake, and crop yield enhancement should be provided in as many visual, oral, and documented formats as possible. Adequate professional and credible support for marketing messages is key to product differentiation, especially in a market inundated with “snake oil” products.

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Emphasizing value to potential customers is also critical. Distributing case and field trial evidence of return on investment opportunities provided by mycorrhizal inoculants and maintaining a lower than average price point yet high and effective microbial concentration ratio compared to the competition are necessary emphases. Staying abreast and addressing or incorporating current agricultural trends and scientific and technological developments in the mycorrhizal technology arena could also ensure a more profitable experience for the mycorrhizal developer and distributor.

7 Conclusions In both academic and commercial arenas, application of suitable mycorrhizal inoculants has demonstrated impressive yield and nutrient gains while also assisting in biocontrol efforts across a wide variety of crops and soil types worldwide. However, mycorrhizal inoculant producers and distributors globally face similar challenges. These challenges include the necessity to tailor products, increase market awareness, and create more effective distribution efforts to meet the needs of a diverse customer base. Effective education and marketing of mycorrhizal benefits to this diverse customer base, along with pre- and post-sales support on the appropriate mycorrhizal formulation, application options, and knowledge on the respective host plants and aftercare, are crucial challenges to be met by mycorrhizal inoculant providers who aim to be successful. In both developed and developing regions, novel protocols and tools are needed in order to create cost-effective, simple, streamlined mycorrhizal production methods. Reliable, rapid, and robust techniques to check for mycorrhizal identity, quality, and quantity are also necessary to advance AMF quality control efforts. New social media, sensor, phone application- and computer-based technologies could aid in effective mycorrhizae and soil monitoring. Social media can also assist with consumer education and with customer relations.

References Abd-Alla MH, Omar SA, Karanxha S (2000) The impact of pesticides on arbuscular mycorrhizal and nitrogen-fixing symbioses in legumes. Appl Soil Ecol 14:191–200. https://doi.org/10.1016/ S0929-1393(00)00056-1 Abdel Latef AAH, Chaoxing H (2011) Effect of arbuscular mycorrhizal fungi on growth, mineral nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress. Sci Hortic 127:228–233. https://doi.org/10.1016/j.scienta.2010.09.020 Adholeya A, Tiwari P, Singh R (2005) Large-scale inoculum production of arbuscular mycorrhizal fungi on root organs and inoculation strategies. In: Declerck S, Strullu D-G, Fortin A (eds) In vitro culture of mycorrhizas. Springer, Heidelberg, pp 315–338 Affokpon A, Coyne DL, Lawouin L et al (2011) Effectiveness of native west African arbuscular mycorrhizal fungi in protecting vegetable crops against root-knot nematodes. Biol Fertil Soils 47:207–217. https://doi.org/10.1007/s00374-010-0525-1

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Chapter 10

Molecular and Genetic Strategies for Enhanced Production of Heterologous Lignocellulosic Enzymes Sophie A. Comyn and Jon K. Magnuson

1 Filamentous Fungi as Expression Hosts The fungal kingdom, estimated to number more than two million species, is subdivided into three major non-monophyletic morphological or functional groups: unicellular yeasts, macroscopic filamentous mushrooms, and multicellular filamentous molds (Hawksworth and Lucking 2017; Wakai et al. 2017). Filamentous fungi have a global distribution and are found in a range of environments including soil and plant material. This environmental flexibility is a direct result of their saprophytic lifestyle, whereby nonliving organic matter is consumed through the absorption of plant-derived polysaccharides, proteins, lipids, and lignin, which are first processed using secreted extracellular enzymes. Most are hydrolytic enzymes, but lyases and oxidoreductases are also involved. By decomposing organic matter, filamentous fungi play an essential role in nutrient cycling in terrestrial ecosystems. The capacity to secrete significant quantities of digestive enzymes into their extracellular environment has led filamentous fungi to be exploited for the manufacturing of homologous and heterologous proteins and enzymes for the food, feed, chemical, and biofuel industries. Filamentous fungi also produce a number of primary metabolites (citric, lactic, and malic acids), secondary metabolites (itaconic acid, penicillin, lovastatin, and paclitaxel), and secreted enzymes (amylases, cellulases, pectinases, proteases, and lipases) that are manufactured at industrial scales (Soares et al. 2012; Ward 2012). Two widely cited examples of protein production ability are glucoamylase from Aspergillus niger (25–30 g/L) and cellulase from Trichoderma reesei (100 g/L) (Durand et al. 1988; Gouka et al. 1997a; Demain and

S. A. Comyn · J. K. Magnuson (*) Chemical & Biological Process Development Group, Pacific Northwest National Laboratory, Richland, WA, USA Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_10

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Vaishnav 2009). The characteristics of an ideal microbial expression host for industrial enzyme production include genetic tractability, an ability to express an assortment of non-native enzymes, high growth rates, and resistance to high-stress manufacturing conditions (Fletcher et al. 2016). One such example of a microbial host is Aspergillus, which has been used extensively for industrial purposes for over 100 years (Currie 1917). Aspergillus is a genus of filamentous fungi with approximately 250 recognized species that include industrially relevant (A. niger, A. oryzae, and A. terreus) and pathogenic (A. fumigatus and A. flavus) members (Geiser et al. 2007). Arguably the most economically valuable genera of the filamentous fungi, Aspergillus is the primary source of citric acid production (A. niger), is used extensively in the production of fermented foods (A. oryzae), and is the source of therapeutic agents such as lovastatin and enzymes used in the pulp and paper industry (A. terreus and A. niger, respectively) (Alberts et al. 1980; Geiser et al. 2007). When used for food production and additives, A. niger and A. oryzae are classified as Generally Recognized as Safe (GRAS) by the American Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act. As a microbial expression host, Aspergillus is especially suited to tolerate a range of culture conditions such as temperature (10–50  C), pH (2–11), salinity (0–34%), and organic acids (Kis-Papo et al. 2003; Meyer et al. 2011b). One of the challenges to commercializing fungal-derived enzymes is that their yield, titer, and activity levels are often insufficient for economical production at industrial scales (Fletcher et al. 2016). A desire to move toward more sustainable feedstocks has created an emerging market for enzymes involved in the deconstruction of lignocellulosic biomass, especially the hydrolysis of polysaccharides. However, the cost and availability of the enzyme cocktails required for efficient biomass conversion are a barrier to the commercialization of lignocellulosic feedstocks to produce biofuels and other bio-based products. In this review we will cover the molecular and genetic strategies used for heterologous enzyme production in filamentous fungi with an emphasis on the enzymes involved in valorizing lignocellulosic biomass.

2 Lignocellulosic Biomass Rising global energy use, climate change, and a growing demand for sustainable sources of energy are important factors motivating the search for alternative feedstocks. Toward this end, biomass has the potential to replace a portion of our petroleum usage for the production of fuels and chemicals through the initial conversion of plant-derived feedstocks into fermentable sugars (Yang and Yu 2013). Lignocellulosic biomass in particular is an attractive option due to its abundance, low cost, and high sugar content (Kim 2013; Yang and Yu 2013; Fletcher et al. 2016). Moreover, lignocellulosic biomass can be sourced from dedicated nonfood crops or waste streams, thereby alleviating some of the public’s concerns surrounding the

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feedstocks used to produce first-generation biofuels, bioethanol, and biodiesel (Kricka et al. 2014). Lignocellulose is the most abundant, renewable, bio-based carbon source on Earth and is composed of three major polymers: cellulose, hemicellulose, and lignin. Its function is to provide structural integrity to the plant cell wall as well as to confer resistance to herbivores and plant pathogens (Cragg et al. 2015). While the composition varies by plant and cell type, growth conditions, and plant age, typical ratios are 30–50% cellulose, 20–30% hemicellulose, and 15–25% lignin (Yang and Yu 2013). Cellulose, the primary structural component of the plant cell wall, is a linear polysaccharide composed of D-glucose monomers linked by beta 1,4-glycosidic bonds (Wang et al. 2012; Kim 2013). Chains consisting of hundreds to thousands of D-glucose molecules form insoluble crystalline fibrils, which are stabilized by interand intramolecular hydrogen bonds and are in turn protected by a matrix of pectin, hemicellulose, and lignin molecules. The crystalline nature of cellulose makes it resistant to degradation and nearly impermeable to enzyme hydrolysis (Wang et al. 2012; Jonsson and Martin 2016). Hemicellulose is a family of branched heteropolymers that vary in their monosaccharide composition depending on the source of the plant. The branching leads to an amorphous structure, and, unlike cellulose, they are easily hydrolyzed by acids or enzymes into their composite sugar monomers (Kim 2013). Lignin, the other major component of lignocellulosic biomass, is a highly cross-linked insoluble polymer of aromatic alcohols (Jonsson and Martin 2016). Linked to hemicellulose and together providing a matrix surrounding the cellulose microfibers, lignin helps to impart rigidity to the plant cell wall (Labeeuw et al. 2015). Due to its branched structure, the heterogeneity of the bonds contained within it, and the lack of easily hydrolyzed bonds, lignin is highly resistant to chemical and biological degradation (Hatti-Kaul and Ibrahim 2013). Lignin recalcitrance represents one of the major scientific and economic barriers to the industrial implementation of lignocellulosic biomass as a feedstock. As a result, frequently in the pulp and paper industry today, lignin is separated during the pulping process and burnt to generate energy (Hatti-Kaul and Ibrahim 2013). Furthermore, despite lignin being a primary source of renewable aromatic compounds, unlike the other components of lignocellulosic biomass, lignin hydrolysis does not generate fermentable sugars. Physical and chemical pretreatment is required to disrupt the associations between cell wall components, thereby removing the physical and chemical barriers that block access to the sugars in lignocellulosic biomass and enhancing the efficiency of the downstream enzymatic saccharification process (Kricka et al. 2014; Xue et al. 2018). For interested readers, a number of excellent reviews have been written on the topic (Mosier et al. 2005; Hendriks and Zeeman 2009; Kumar et al. 2009; Jonsson and Martin 2016). In order for the commercialization of lignocellulosic biomassderived products to be realized, a number of engineering bottlenecks in biomass conversion need to be addressed in order to maximize yields and improve economics. One approach to address this problem is to develop and optimize enzyme cocktails used in the deconstruction of lignocellulosic biomass.

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3 Cellulases The breakdown of lignocellulosic biomass in natural soil environments is a sequential process involving the collective contribution of numerous microorganisms. Filamentous fungi play an important role in carbon and nutrient cycling, due in part to their growth morphology whereby expanding hyphae penetrate organic material exposing large surface areas to secreted digestive enzymes. During the biomass conversion process, complex polysaccharides found in lignocellulose are converted into monomeric fermentable sugars through the combined activities of a diverse set of enzymes secreted into the environment. Specifically, simple sugars are released through the enzymatic hydrolysis of cellulose and hemicellulose by cellulases and hemicellulases, respectively. The synergistic activity of a class of three types of enzymes is necessary to degrade cellulose: cellobiohydrolases, endoglucanases, and beta-glucosidases (Lambertz et al. 2014). Cellobiohydrolases are exoglucanases that hydrolyze beta1,4-glucosidic bonds to release the disaccharide cellobiose from the termini of cellulose chains. Endoglucanases cleave internal bonds of amorphous cellulose to produce oligosaccharides. Finally, beta-glucosidases are the key enzyme in cellulose degradation as they release fermentable glucose monomers by the hydrolysis of cellobiose and some soluble short oligosaccharides. Launched in 1999, the carbohydrate-active enzymes (CAZy) database (www.cazy.org) is an extensive online resource of genomic, structural, and biochemical information for enzymes involved in the synthesis, metabolism, and recognition of complex carbohydrates such as cellulose (Lombard et al. 2014). Discovered in the last decade, copper-containing lytic polysaccharide monooxygenase (LPMO) enzymes play a major role in degrading recalcitrant polysaccharides such as chitin and cellulose (Johansen 2016). Their importance in biomass conversion is underscored by their widespread abundance in fungal and bacterial genomes (Kohler et al. 2015; Johansen 2016). LPMOs have a boosting effect on cellulase activity, and consequently their inclusion in commercial cellulolytic enzyme cocktails reduces the enzyme loading required for the saccharification step of the pretreatment process (Harris et al. 2010). A number of enzyme formulations are commercially available from several fungal sources, including Aspergillus (Novozymes Carezyme and Novozyme 188 Cellobiase) and Trichoderma (Genencor ACCELERASE and Novozymes Celluclast). Fungal-derived cellulase cocktails are frequently used in the deconstruction of lignocellulosic biomass for bioethanol production with the most notable product being Novozyme’s Cellic, Ctec, and Htec series.

4 Lignin-Modifying Enzymes (LME) The capacity to completely depolymerize lignin is primarily attributed to the white rot basidiomycetes and, more recently, a few ligninolytic bacteria (Cragg et al. 2015). Traditionally, lignocellulose degradation by the basidiomycete fungi was

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divided into two distinct groups: white rot and brown rot. White rot fungi (e.g., Phanerochaete chrysosporium and Trametes versicolor) can degrade all components of lignocellulose, whereas brown rot fungi are unable to degrade lignin but can degrade cellulose and hemicellulose. More recently, genome sequencing and phylogenetic analyses have challenged this historical classification and demonstrated that brown rot fungi have evolved a number of times from the white rots, contributing to the diversity in fungal wood-decaying mechanisms (Riley et al. 2014). Four types of enzymes have been reported to be involved in fungal lignin depolymerization, laccases, lignin peroxidases (LiP), manganese peroxidases (MnP), and versatile peroxidases (VP), along with a number of hydrogen peroxide-generating enzymes needed as a co-substrate by many of these lignin-deconstructing enzymes (Pollegioni et al. 2015). Fungi differ with regard to the number and type of these lignin-modifying enzymes encoded in their genomes. For example, Phanerochaete chrysosporium primarily utilizes peroxidases, while Pycnoporus cinnabarinus employs laccases (Hatti-Kaul and Ibrahim 2013). As more fungal genomes are sequenced and analyzed, and their degradation strategies studied by a combination of functional genomics techniques, the known diversity of strategies used by fungi for lignin degradation will likely grow. Found in plants, fungi, bacteria, and insects, laccases (EC 1.10.3.2) are multicopper oxidase enzymes that can oxidize a range of aromatic compounds and the phenolic components of lignin. Fungal laccase activity is associated with a number of physiological functions such as development, morphogenesis, pigmentation, and plant pathogenesis (Morozova et al. 2007). A number of laccase isoforms are secreted by fungi with the exact complement influenced by factors such as species, growth conditions, and the presence or absence of inducers (Morozova et al. 2007). Phylogenetic studies examining amino acid sequences suggest that laccases from ascomycetes and basidiomycetes belong to two distinct clusters (Castilho et al. 2009). First described in the late 1880s as a component in lacquer, laccases are used in a number of industrial applications such as bioremediation, biosensors, dye decolorization, wastewater treatment, and wine and food production (Yoshida 1883; Cameron et al. 2000; Mougin et al. 2000; Pointing and Vrijmoed 2000; Raghukumar 2000; Servili et al. 2000; Soares et al. 2001). Laccases from a number of fungal sources, including Phanerochaete flavidoalba, Trametes versicolor, and Pycnoporus cinnabarinus, have been expressed successfully in filamentous fungi such as T. reesei, A. nidulans, and A. niger (Record et al. 2002; Sigoillot et al. 2004; Hoshida et al. 2005; Bohlin et al. 2006; Benghazi et al. 2014) (Table 10.1). Lignin peroxidases are heme-containing glycoproteins that are categorized into three subclasses based on amino acid sequence and catalytic characteristics (Janusz et al. 2017). Lignin peroxidases (LiP; EC 1.11.1.14) have a high redox potential and as a consequence can directly oxidize non-phenolic components of lignin (Sigoillot et al. 2004). An identifying property of the LiP subclass is a catalytic tryptophan residue on the enzyme surface, which participates in long-range electron transfer with heme. Manganese peroxidases (MnP; EC 1.11.1.13) are the most common class of lignin-modifying enzymes in the white rot fungi and were the first lignin peroxidases to be identified over 30 years ago (Tien and Kirk 1983; Glenn

Enzyme class Laccase

Aspergillus oryzae

Host Aspergillus awamori Aspergillus nidulans Aspergillus niger

amyB

lcc1

lac1

laccase

Myceliophthora thermophila Pycnoporus cinnabarinus Pycnoporus coccineus

gpdA TAKA amylase TAKA amylase TAKA amylase gpdA

lcc1, lcc2 laccase IV lcc1

glaA

laccase

Coprinus cinereus

gpdA

pel3

lac1

laccase

gpdA

gpdA

lcs1

lac1

TAKA amylase gpdA

laccase

Stachybotrys chartarum Ceriporiopsis subvermispora Phanerochaete flavidoalba Pleurotus eryngii

Pycnoporus cinnabarinus Pycnoporus cinnabarinus Stachybotrys chartarum Trametes versicolor Trametes versicolor

alcA

Gene lcc1

Origin Trametes versicolor

Promoter glaA

Native

GlaA

Native

Native

Native Native

Native, GlaA Native

Native, GlaA GlaA

GlaA

Native

Native

Signal peptide Native

Table 10.1 Examples of heterologous expression of lignin-modifying enzymes in filamentous fungi

No

No

No

No

No No

No

No

No

No

No

No

No

Codon optimization No

No

No

No

No

No No

No

No

CBM

No

No

No

No

Carrier protein No

Hoshida et al. (2005)

Sigoillot et al. (2004)

Berka et al. (1997)

Bohlin et al. (2006) Téllez-Jurado et al. (2006) Yaver et al. (1999)

Mander et al. (2006)

Larrondo et al. (2003) Benghazi et al. (2014) Rodriguez et al. (2008) Ravalason et al. (2009) Record et al. (2002)

Reference Valkonen et al. (2003) Mander et al. (2006)

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Peroxidase

Aspergillus oryzae Trichoderma reesei

Aspergillus niger

Aspergillus awamori Aspergillus nidulans

Trichoderma atroviride Trichoderma reesei

Aspergillus sojae

cbh1 gpdA exlA

laccase lacA peroxidase mnpl2 vpl2 mnp1

lgp3 DyP

Pleurotus eryngii

Pleurotus eryngii Phanerochaete chrysosporium Phanerochaete chrysosporium Phlebia radiata

Pleurotus sapidus

mnp1

cbh1

laccase

cbh1

TAKA amylase cbh1

alcA gpdA

alcA

cbh1

pki1

TAKA amylase Tannase

lac1

lac1

laccase

lcc1

Melanocarpus albomyces Pycnoporus sanguineus Stachybotrys chartarum Trametes sp. AH28–2 Arthromyces ramosus

Schizophyllum commune Trametes sanguineus

Trametes villosa

Native

TAKA amylase Native

Native Native

Native

Cbh1 ExlA

Native

Native

Native

Native

TAKA amylase Native

No

No

No

No No

No

No Yes

No

Yes

No

No

No

No

No

No

No

No No

No

No No

No

No

Hfb1

No

No

No

Saloheimo et al. (1989) Lauber et al. (2017)

Stewart et al. (1996)

Ruiz-Duenas et al. (1999) Eibes et al. (2009) Conesa et al. (2002)

Zhang et al. (2012) Lokman et al. (2003)

Mander et al. (2006)

Hatamoto et al. (1999) Balcazar-Lopez et al. (2016) Kiiskinen et al. (2004) Zhao et al. (2018)

Yaver et al. (1996)

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and Gold 1985). Mn(III) is generated from the oxidation of Mn(II) in the Mn (II) binding site of MnP. The Mn(III) is chelated by dicarboxylic acids (oxalic or malonic) then diffuses into the lignocellulose substrate to act as a low molecular weight oxidizer (Goszczynski et al. 1994). Due to their low redox potential, MnP cannot oxidize non-phenolic components of lignin. Versatile peroxidases (VP; EC 1.11.1.16) are a hybrid of the MnP and LiP having both manganese and a catalytic tryptophan residue. Capable of oxidizing a range of substrates with medium to high redox potentials, VP can also oxidize non-phenolic lignin compounds (García-Ruiz et al. 2010). Unfortunately, in contrast to cellulases and laccases, lignin peroxidases are not available commercially, and their industrial application is limited by low-expression titers (Hatti-Kaul and Ibrahim 2013). Several groups have reported on the heterologous expression of MnP from P. chrysosporium in A. niger; however, yields are significantly lower than those obtained for fungal laccases (Aifa et al. 1999; Conesa et al. 2000, 2002). More recently, a new superfamily of heme peroxidases called dye-decolorizing peroxidases (DyP) have been described. Prominent in bacterial genomes, DyP are phylogenetically unrelated to fungal lignin-modifying peroxidases (Colpa et al. 2014; Zamocky et al. 2015). However, despite a lack of structural similarity with the fungal peroxidases, DyP exhibit similar catalytic activity and redox potentials (Liers et al. 2014). While lignolytic activity by DyP has been described in some fungi, most of the recent attention has been focused on their lignin-degrading capacity in bacteria (Johjima et al. 2003; Liers et al. 2010; Singh and Eltis 2015; Yoshida and Sugano 2015). Going forward, a number of fundamental challenges need to be addressed before lignocellulosic biomass can be adopted as a feedstock for use at industrial scales. Economic viability will depend upon the maximum valorization of all lignocellulosic biomass components. Not only will this require advances in process engineering but also significant improvements to the enzymes and microbial expression hosts used in biomass conversion. Specifically, identifying, characterizing, and increasing the expression levels, yields, and activities of lignin-modifying enzymes will be a critical part of the successful integration of lignocellulosic biomass into the new bio-economy.

5 Engineering Strategies for Improving Heterologous Protein Production In filamentous fungi, yields of heterologous proteins of distantly related fungal, bacterial, or mammalian origin are significantly lower than those obtained from homologous proteins or closely related fungal species (Tanaka et al. 2014). While the factors limiting production of heterologous proteins are often gene specific, bottlenecks can exist at any stage (or combination thereof) from transcription, mRNA stability, translation, folding, glycosylation, secretion, to extracellular

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protease-dependent degradation. In the following sections, we will describe a number of strategies that have been developed over the years to improve heterologous protein production in filamentous fungi through modification of the coding sequence, protein, and host.

5.1

Remodeling the Gene: Codon Optimization

Codon optimization is an engineering approach used to increase protein expression levels by altering the nucleotide sequence of a heterologous gene, without changing the amino acid sequence, in order to match the codon usage patterns of the expression host organism. In recent years the cost of gene synthesis has been decreasing, enabling codon optimization to become a tool accessible to the broader research community. Most amino acids are encoded by more than one synonymous codon. However, not all of these codons are equally abundant, resulting in a phenomenon called codon usage bias. Codon usage bias is a feature of both prokaryote and eukaryote genomes and shows species-specific differences (Agashe et al. 2013; Zhou et al. 2016). Therefore, it is generally thought that heterologous protein expression is impeded by divergent codon usage between the organism of origin and expression host species (Maertens et al. 2010). Disparate codon usage can have important evolutionary consequences, as shown in a recent report that found a correlation between codon optimization and fungal parasite host range with differences observed between generalist and specialist species (Badet et al. 2017). Bias in synonymous codon usage is most pronounced in highly expressed protein coding genes, within evolutionarily conserved regions, and has been correlated with tRNA abundance (Tanaka et al. 2014). Since protein translation is an energetically costly process for a cell, it was thought that codon bias evolved through selection for efficient and accurate protein translation and folding (Zhou et al. 2016). Therefore, heterologous protein production should be negatively influenced by ribosome pausing at rare codons during translation elongation. This hypothesis is supported by experimental and genome-wide bioinformatic analyses using the filamentous fungus Neurospora crassa. Zhou et al. (2016) demonstrated a correlation between codon usage and predicted protein structures. Intrinsically disordered regions were enriched for nonoptimal codons, whereas structured protein domains showed a preference for optimal, higher-frequency codons (associated with higher tRNA abundance). Experimentally, using a cell-free translation system that reflects protein translation in vivo, Yu et al. (2015) showed that nonoptimal codon usage reduced the rate of translation elongation and affected co-translational protein folding. A number of recent studies have challenged the traditional, evidence-based hypothesis that codon optimization improves protein expression through enhanced translation. Instead, data from organisms ranging from E. coli and zebrafish to humans and fungi suggest that tailoring codon usage to the host organism leads to increased steady-state mRNA levels (Tokuoka et al. 2008; Fath et al. 2011; Boel et al. 2016; Harigaya and Parker 2016; Mishima and Tomari 2016). Expressing a

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heterologous protein in A. oryzae, Tokuoka et al. (2008) demonstrated that codon optimization prevented premature polyadenylation and truncation of the mRNA transcript and instead led to increased steady-state mRNA levels and higher protein expression. First reported in A. awamori, codon optimization in filamentous fungi often raises the GC content of the transcript and eliminates truncated products (Gouka et al. 1997b). The increase in transcript stability may be attributed, at least in part, to the predominance in filamentous fungi of A or U bases in the third position of rare codons (Tanaka et al. 2014). While codon-optimized gene synthesis is an ideal starting point for anyone wanting to express heterologous proteins in filamentous fungi, a number of factors remain unresolved and limit our understanding of the process. For example, tuning the heterologous sequence to match the host’s codon bias does not always maximize protein levels (Maertens et al. 2010). Moreover, producing transcripts encoded exclusively by abundant codons does not guarantee universally higher protein yields (Welch et al. 2009). Until we have a greater understanding of the role of codon bias at the cellular level, we cannot fully appreciate how altering the local sequence context may best influence protein production (Agashe et al. 2013).

5.2

Remodeling the Protein: Signal Peptide and Carrier Protein

At the protein level, numerous posttranslational bottlenecks exist to limit heterologous protein production. They include translation initiation and elongation, folding, posttranslational modifications, and secretion. Given the number of potential checkpoints acting on polypeptides, the second most commonly used strategy to increase heterologous protein yields, after codon optimization, is to modify the protein sequence. Nascent polypeptides of proteins destined for secretion are processed sequentially at two steps along the secretory pathway, first in the endoplasmic reticulum (ER) and then in the Golgi (Heiss et al. 2015). An N-terminal hydrophobic signal peptide (SP) sequence targets nascent polypeptides exiting the ribosome to be translocated into ER lumen. The SP is then removed by signal peptidase, an integral ER membrane protein. There is no known consensus sequence for SPs. However, they share a common structure with a short positively charged N-terminus, a central hydrophobic region, and a polar C-terminus (Heiss et al. 2015). Replacing the native SP of a heterologous protein with one of a highly secreted protein from the fungal host has been a successful strategy used for expressing fungal laccases in A. niger. Laccases from P. flavidoalba and P. cinnabarinus were heterologously expressed by substituting their native SP with the first 24 amino acids of the glucoamylase gene glaA from A. niger (Record et al. 2002; Benghazi et al. 2014) (Fig. 10.1). Nascent polypeptides of secreted proteins may also contain a short propeptide sequence immediately downstream of the SP. This sequence is thought to act as a chaperone and sorting mechanism for the protein as it moves through the Golgi

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Fig. 10.1 Schematic of the A. niger glucoamylase (GlaA) N-terminus containing a signal and propeptide region. Cleavage sites are indicated with arrows

network and is removed in the late Golgi prior to secretion (Heiss et al. 2015). The carrier fusion approach, a strategy similar to native propeptides, is commonly used for heterologous protein expression. In this method, the heterologous protein of interest is expressed as a C-terminal fusion with a homologous secretory protein. The N-terminal carrier protein is thought to improve translocation of the nascent polypeptide into the ER, aid in ER folding, and shield the protein from premature degradation (Gouka et al. 1997b). In Aspergillus, the carrier fusion strategy is often applied using highly expressed secretory proteins such as glucoamylase from A. niger or cellobiohydrolase I from T. reesei. Ward et al. (1990) were the first to demonstrate that an N-terminal GlaA fusion could increase heterologous protein production in Aspergillus. Using the full-length glucoamylase sequence from A. awamori fused to bovine prochymosin B cDNA, they obtained higher yields than a fusion-free control. Moreover, the proteins produced were free of aberrant glycosylation products. The GlaA protein has three distinct regions: an N-terminal catalytic domain, a central linker region, and a C-terminal starch binding domain (Cornett et al. 2003). Later iterations of the GlaA carrier approach have used a truncated GlaA protein lacking the starch binding domain. It is thought that the presence of the linker region allows the GlaA catalytic domain to fold independently of the C-terminal heterologous protein. This approach has been used most notably in the expression of human interleukin-6 and hen egg-white lysozyme in Aspergillus (Broekhuijsen et al. 1993; Jeenes et al. 1993). The introduction of a cleavage recognition site between the fusion proteins results in the secretion of two independent proteins. The NVISKR motif, derived from the GlaA propeptide sequence, is commonly used in A. niger. This site is recognized by the Kex2 endopeptidase that cleaves at the C-terminal end of lysine-arginine or arginine-arginine residues. Kex2 is an integral membrane protein thought to be active in the late Golgi compartment (Zhang et al. 2007). The carrier fusion approach has had mixed success for the expression of ligninmodifying enzymes in Aspergillus. Ravalason et al. (2009) fused the linker region and carbohydrate-binding module of cellobiohydrolase B from A. niger to the P. cinnabarinus laccase lac1 gene. Using A. niger as an expression host, their heterologous protein product demonstrated an increase in biobleaching efficiency for softwood kraft pulp as well as an ability to bind both cellulose and softwood kraft pulp substrates. By contrast, results from lignin peroxidases have been varied. Using a protease-deficient A. niger strain, Conesa et al. (2000) tested for the expression and

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activity of lignin peroxidase H8 (LiPA) and manganese peroxidase H4 (MnP1) from P. chrysosporium. For each peroxidase, three different expression constructs were tested: native SP, GlaA propeptide, and a GlaA carrier fusion. While MnP1 activity could be detected by o-anisidine or ABTS oxidation, no activity was observed for any of the LiPA constructs. Surprisingly, GlaA::LiPA protein was successfully secreted and detected in the culture medium by Western blot. By contrast, for the GlaA::MnP1 construct, neither activity nor protein was detected. The GlaA carrier protein, however, was produced and secreted in this strain. This is the first reported failure of the GlaA fusion approach and suggests that the system is perhaps less universally applicable than originally believed.

5.3

Remodeling the Host: Secretory Pathway

Industrially produced enzymes are predominantly secreted proteins derived from native fungal hydrolytic enzymes or from heterologous proteins expressed in fungal hosts. Manufacturing proteins in secreted versus intracellular form has many benefits as it can allow for simplified downstream purification steps and capitalize on the endogenous protein quality control machinery of the secretory pathway. However, despite these advantages, mechanisms inherent to the secretory pathway frequently restrict heterologous protein yields. Protein secretion is a highly ordered and sequential process that starts with a polypeptide entering into the ER and ends with secretory vesicles fusing with the plasma membrane (Fig. 10.2). Translocation into the ER occurs mostly co-translationally and is directed by the interaction between the signal peptide sequence on the polypeptide and a multicomponent signal recognition particle (SRP). Once in the ER, proteins are folded, posttranslationally modified, and assembled with other subunits if necessary. Proteins are then transported by vesicles from the ER to the Golgi where they are further processed before being targeted and packaged into secretory vesicles that fuse with the plasma membrane, thereby releasing their contents. Quality control mechanisms exist at every stage of the process to monitor and control protein entry and progression. Passage of heterologous proteins through the secretory pathway is often obstructed at these quality control checkpoints, the most important of which include protein folding and glycosylation. These stages represent key targets for initial strain engineering efforts to increase heterologous protein production.

5.4

Remodeling the Host: ERAD and UPR

Correct and timely protein folding is one of the most critical factors in determining whether a protein will transit from the ER to the Golgi. As an estimated one third of all eukaryotic proteins are predicted to be membrane bound or secreted, it is

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Fig. 10.2 Schematic of the secretory pathway in filamentous fungi. Secreted proteins are first transcribed in the nucleus and then translocated into the lumen of the ER with the assistance of a signal peptide. Once properly folded, proteins are packaged into vesicles and transported through the Golgi network. Proteins are delivered to the Spitzenkörper before vesicles fuse with the plasma membrane, and the protein contents are released into the extracellular space

unsurprising that rigorous quality control mechanisms have evolved to monitor and facilitate the large flux of proteins through the ER (Vembar and Brodsky 2008). Proteins that fail to attain their native conformation or posttranslational modifications are targeted for degradation by the ER-associated degradation (ERAD) pathway. ER proteins targeted for degradation must be retrotranslocated across the ER membrane into the cytoplasm in an ATP-dependent manner before they can be degraded by the ubiquitin proteasome system. Expressing heterologous proteins at high levels can exceed the folding capacity of the ER and lead to protein accumulation and/or degradation. Therefore, for any given heterologous protein, one of the first engineering strategies applied is to raise the expression levels of ER-resident chaperones. For example, in an attempt to increase production of manganese peroxidase (MnP) from P. chrysosporium in A. niger, Conesa et al. (2002) examined the effects of overexpressing two key chaperones of the ER lumen: binding protein (BiP) and calnexin. Whereas calnexin overexpression resulted in a four- to fivefold increase in MnP production, overexpressing BiP actually lowered extracellular MnP levels. Interestingly, the beneficial effect of calnexin overexpression was eliminated when the culture medium was supplemented with heme. If misfolded proteins are left to accumulate in the ER, a signal transduction pathway called the unfolded protein response (URP) is activated. The UPR controls expression of over 200 genes involved in protein folding, glycosylation, vesicle trafficking, degradation, cell wall biogenesis, and lipid metabolism (Travers et al. 2000). Rather than indiscriminately increasing the expression of all ER and secretory pathway components, the UPR triggers specific remodeling of the secretory pathway with about half of the functionally assigned genes associated with protein secretion (Travers et al. 2000). In yeast, the accumulation of unfolded proteins in the ER prompts the dissociation of BiP from the ER transmembrane protein Ire1, leading to

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the activation of Ire1 kinase and endoribonuclease functionality (Sidrauski and Walter 1997; Oikawa et al. 2009). The pivotal step in UPR initiation is the splicing of a nonconventional intron from HAC1 mRNA by Ire1, thereby instigating translation of the Hac1 transcription factor. Filamentous fungi including T. reesei, A. nidulans, and A. niger have all been demonstrated to undergo similar splicing of nonconventional introns (Saloheimo et al. 2003). In A. niger, however, translational blockage of hacA is relieved in a slightly modified fashion. Not only is a 20-nucleotide nonconventional intron spliced, but so too is a 230-nucleotide 5’UTR flanking region containing an open reading frame encoding a 44 amino acid peptide (Mulder and Nikolaev 2009). Overexpression of the spliced form of hacA in A. niger mimics UPR and results in upregulation of the ER-resident proteins BipA (a chaperone) and PdiA (a disulfide isomerase), as well as its own mRNA (Mulder et al. 2004). It is possible, therefore, to modify heterologous protein production by engineering strains to constitutively overexpress the processed form of hacA. One such example is the work by Valkonen et al. (2003) which examined the effect of constitutive UPR activation on T. versicolor laccase 1 production in A. awamori. After 5 days of cultivation, laccase 1 levels in the hacA overexpression strain were three- to eightfold higher than the control. Furthermore, a range of hacA expression levels were tested and found to correlate with those of the ER chaperone bipA. Increased protein production was not universal, however, as the hacA overexpression strain produced less total protein and three- to sevenfold less alpha-amylase than the control (Valkonen et al. 2003). Similar to ERAD, UPR can be induced by the expression of heterologous proteins. Using a glucoamylaseglucuronidase (GlaGus) protein as a model substrate, Carvalho et al. (2011) demonstrated that UPR induction depended on the expression level of the GlaGus protein. They then tested the effect of deleting five different genes associated with the ERAD pathway and confirmed previous reports indicating that loss of ERAD leads to further UPR induction (Travers et al. 2000; Carvalho et al. 2011).

5.5

Remodeling the Host: Glycosylation

The Golgi is a dynamic organelle which is central to the secretory pathway. Proteins that pass the quality control checks in the ER are then transported via vesicles to the Golgi. Once in the Golgi, secreted proteins are further processed by glycosylation and sorted before being trafficked to the plasma membrane. Glycosylation is a ubiquitous posttranslational modification that can influence a number of protein characteristics such as solubility, activity, half-life, and resistance to proteolytic attack (Shental-Bechor and Levy 2008). Covalent attachment of glycans to the beta-amide group of asparagine (N-linked) and the beta-hydroxyl group of serine or threonine (O-linked) side chains are two of the most common forms of glycosylation (Beckham et al. 2012). Unfortunately, the protein glycosylation patterns found in filamentous fungi do not match those of humans, making them unsuitable expression hosts for recombinant proteins to be used in human therapeutics.

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Consequently, engineering filamentous fungi to produce appropriate and tunable glycosylation patterns is of upmost importance. For example, most fungal cellulases are O-glycosylated in their linker regions, and deglycosylation of the T. reesei Cel7A cellulase results in decreased solubility and could potentially lead to aggregation under conditions used for biomass conversion (Gupta et al. 2011). Furthermore, in A. niger, deletion of the alg3 gene (encoding a key enzyme in the early stages of the N-linked glycosylation pathway) produces a lower molecular weight species of Cel7A, as would be expected if glycosylation patterns were disrupted (Dai et al. 2013). Hyperglycosylation is a common feature of heterologously expressed laccases in filamentous fungi. In A. oryzae, laccases from T. villosa and Myceliophthora thermophila were both produced as hyperglycosylated forms (Yaver et al. 1996; Berka et al. 1997). In A. niger the picture is somewhat mixed. While some studies report the production of laccases comparable to those of the homologous protein, others demonstrate aberrant hyperglycosylation patterns (Record et al. 2002; Bohlin et al. 2006; Ravalason et al. 2009; Liu et al. 2014; Mekmouche et al. 2014). While developing a fully humanized glycosylation system in a fungal expression host may be years away, important advances toward improving heterologous protein expression can be made by engineering the secretory pathway. The ERAD and secretory systems of S. cerevisiae have been studied extensively. However, until recently, the tools available for filamentous fungi have limited our understanding of the process. Hopefully, with the introduction of systems biology tools and more amenable genetic engineering techniques, researchers will be better equipped to characterize the system in filamentous fungi.

5.6

Remodeling the Host: Proteases

Heterologous protein yields are often negatively influenced by high levels of proteases produced by the fungal host. Proteolysis is a significant barrier to protein production as it reduces yields due to degradation, decreases enzyme activity by creating truncated polypeptides, and lowers the purity of the final product by generating degradation fragments (Zhang et al. 2007). It is not uncommon for heterologous proteins produced by filamentous fungi to be degraded once secreted into the culture medium. The proteases responsible can be extracellular, plasma membrane bound, intracellular proteases released through cell lysis, or a combination of the above (Zhang et al. 2007). One approach frequently used to address this problem is to genetically engineer the fungal host to decrease protease activity. Originally, protease-deficient Aspergillus strains were generated through UV mutagenesis and screening on milk plates, whereby halo size correlates with protease production (Mattern et al. 1992; van den Hombergh et al. 1995). Genetic deletion cassettes were then used to specifically target individual, or multiple, protease genes in tandem (Berka et al. 1990; Mattern et al. 1992; van den Hombergh et al. 1997). As far back as the 1990s, deletion strains of the major extracellular protease PepA were

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built, first in A. awamori and then in A. niger (Berka et al. 1990; Mattern et al. 1992). Deletion of the intracellular acid protease PepE in A. niger resulted in a concomitant decrease in the activity of the intracellular serine proteases and serine carboxypeptidases, without altering their transcript levels (van den Hombergh et al. 1997). More recently, A. niger strains containing a mutation in, or deletion of, the zinc transcription factor prtT have been widely adopted (Mattern et al. 1992; Punt et al. 2008; Kamaruddin et al. 2018). Disruption of prtT, a protease regulator, results in reduced expression of several proteases (PepA, PepB, PepD, and PepF) and decreases total protease activity to levels as low as 20% of wild type (Punt et al. 2008; Kamaruddin et al. 2018). Protease-deficient A. niger prtT mutant strains have been used extensively and are instrumental for the heterologous expression of lignin-modifying laccase enzymes (Record et al. 2002; Bohlin et al. 2006; Ravalason et al. 2009; Benghazi et al. 2014). Culture conditions and morphology also play a role in regulating protease expression and activity in filamentous fungi. The progressive acidification of culture medium during exponential growth is a characteristic of A. niger and is caused by the secretion of gluconic, oxalic, and citric acids (Niu et al. 2015). An acidic growth environment in turn induces expression of the two major extracellular proteases PepA and PepB in A. niger (Jarai and Buxton 1994). Moreover, protease activity is reversely correlated with ambient pH. As the pH value decreases below 4 during exponential growth, protease activity increases, and conversely, as the pH level rises above 5–6, protease activity diminishes (Braaksma et al. 2009). In A. niger, heterologous protein production is directly affected by the pH of the culture medium. O’Donnell et al. (2001) demonstrated that GFP production in medium maintained at pH 6 resulted in tenfold higher yields than when grown at pH 3. In contrast to pH, growth on glucose or other favored carbon or nitrogen sources represses the expression of extracellular proteases (Jarai and Buxton 1994). This allows for the convenient supplementation of production medium to limit protease activity. Industrial culture practices using filamentous fungi can result in a number of growth morphologies. Xu et al. (2000) examined the relationship between cultivation morphology and protease production and observed decreased protease activity with pelleted growth and higher levels with dispersed mycelia. The mechanism by which protease production is decreased remains to be elucidated. However, it is thought to be in response to nutrient and/or oxygen availability. While restricting protease activity can significantly enhance heterologous protein production, a number of further improvements could be achieved through genetic engineering efforts. For example, protease-deficient strains, despite having notably lower proteolytic activities compared to wild type, still possess residual protease activity. Bioinformatic analysis of the A. niger CBS 513.88 genome identified approximately 200 protease genes, of which 32 are predicted to be secreted into the extracellular environment (Pel et al. 2007). With such a high number of predicted genes, it is hard to imagine building a strain that eliminates all extracellular proteolytic activity. Moreover, our current understanding of the proteolytic system in Aspergillus is much less developed than that for other fungal groups such as Saccharomyces. Hopefully, future studies using systems biology and omics

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technologies will lead to a greater understanding of the regulation and interplay between proteases in the various cellular compartments of filamentous fungi.

6 New Tools and Approaches for Strain Engineering: Systems Biology Systems biology is a quantitative approach used to describe and understand the complexity of biological systems (Fletcher et al. 2016). In filamentous fungi, genetic studies have traditionally focused on manipulating and deciphering the function of single genes or pathways. Systems biology approaches, however, work on a larger scale to examine the mechanisms underlying cellular behavior. Genomics, transcriptomics, proteomics, metabolomics and other technologies can be used to make systems-level observations and to measure the effects of various abiotic or biotic factors (Brandl and Andersen 2017). A number of recent studies have applied systems biology tools to examine lignocellulosic biomass conversion in terms of enzyme discovery and physiological responses to various feedstocks.

6.1

Genomics and Transcriptomics

Collaborative fungal genome sequencing efforts and accessible tools such as MycoCosm are invaluable resources for comparative and functional genomics in filamentous fungi (Grigoriev et al. 2014). For example, sequencing and analysis of the 33.9 megabase genome of the industrially relevant A. niger CBS 513.88 strain revealed key components of the secretory pathway, as well as the identity of 198 predicted proteolytic proteins (Pel et al. 2007). Comparing the transcriptomes of the enzyme-producing strain CBS 513.88 with that of the citric acid-producing ATCC 1015 strain showed that CBS 513.88 has increased levels of all tRNA synthases, suggesting a possible mechanism behind its enhanced glucoamylase secretion ability (Andersen et al. 2011). Comparative genomics studies have been especially insightful for understanding the various lignin-degrading systems employed by wood-decaying fungi. Ruiz-Duenas et al. (2013) examined the hemecontaining peroxidases from ten basidiomycete species and confirmed the absence of manganese, lignin, and versatile peroxidases from brown rot fungal genomes. A more recent study by Riley et al. (2014) comparing 33 basidiomycete genomes challenged the findings of Ruiz-Duenas et al. (2013) and instead found that brown and white rot categorization is incorrect and should instead be considered as a continuum. Their findings are based on principal component analysis of both carbohydrate- and lignin-depolymerizing enzymes and demonstrate a clear separation between brown and white rot species along the first, but not the second, principal component axes.

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Transcriptomics provides a systems-wide view of gene expression under specific growth conditions and can be used to examine the effects of experimental perturbations. For example, Delmas et al. (2012) examined the physiological response of A. niger to growth on lignocellulose. Following 24 h of growth in media containing wheat straw, lignocellulosic-degrading enzymes represented ~20% of total mRNA. Interestingly, they observed that wheat straw was not initially recognized as a carbon source and starvation induced the secretion of a small subset of degrading enzymes, which they describe as scouts. Combining transcriptomics and proteomics datasets can be a useful tool for identifying target genes for genetic engineering. Comparing the transcriptional and proteome responses of A. niger expressing three different heterologous proteins, Jacobs et al. (2009) selected as leads for generic strain improvement genes demonstrating coordinated upregulation at both transcript and protein levels. Targets included proteins involved in protein folding, carbon and nitrogen metabolism, as well as ER degradation and oxidative stress.

6.2

Proteomics

Proteomics, the systems-level study of proteins, can be used to examine a number of parameters such as protein abundance, localization, modification, and interactions (de Oliveira and de Graaff 2011). As with transcriptomics studies, reproducible sample culturing, preparation, and biological replicates are crucial for enabling statistical analysis and for avoiding the introduction of bias into proteomics datasets. A robust cell wall, secreted proteases, and low concentrations of secreted proteins mixed with fungal metabolites in culture media all pose unique challenges to sample preparation for proteomics studies using filamentous fungi (Bianco and Perrotta 2015). With respect to lignocellulosic biomass conversion, most proteomics studies have examined the secretome, often measuring the change in protein profiles as a response to growth on various carbon sources (Medina et al. 2005; Tsang et al. 2009; Adav et al. 2013; Jun et al. 2013). The secretome reflects the complement of proteins released into the culture medium, bound to the cell wall, or forming part of the endoand exocytosis pathways (Tjalsma et al. 2000). Performing mass spectrometry on enriched subcellular microsomal fractions, de Oliveira and de Graaff (2011) examined the effect of xylose induction of cellulase and hemicellulase enzymes on the composition of secretory organelles. They showed that xylose induction increased the expression of small GTPases associated with exocytosis and growth. Most notably, and specific to the induced samples, they also observed recruitment of 20S proteasome subunits to the secretory organelles. Enzymatic pretreatment of lignocellulosic biomass is often performed at temperatures above the natural growth range of fungi, and, as a result, most cellulases are not stable at high temperatures (Adav et al. 2013). Using an A. fumigatus strain isolated from compost, Adav et al. (2013) examined lignocellulolytic enzyme profiles at different growth temperatures. They observed temperature-dependent expression during fermentation on sawdust, with maximum enzyme activities at a temperature range of 40–50  C. Proteomics is

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especially well suited for use in identifying bottlenecks to heterologous protein expression. Components of amino acid metabolism, ribosome biogenesis, translation, and ER stress were all enriched in A. nidulans strains overexpressing arabinofuranosidase and cellobiohydrolase enzymes (Zubieta et al. 2018). Approximately 33% of all proteins identified were shared between the overexpression strains, suggesting a common cellular response to heterologous protein production. It will be interesting to see if in further studies a similar response is observed for different classes of enzymes or fungal sources. Using systems biology tools to understand how cells respond to heterologous protein expression will permit the discovery of bottlenecks in engineered strains and, going forward, will allow for the implementation of more informed engineering strategies.

7 New Tools and Approaches for Strain Engineering: Synthetic Biology Synthetic biologists apply principles from engineering and biology to remodel existing biological systems or to design and build novel biological functions (Fletcher et al. 2016). Effective application of synthetic biology requires methods for precise genetic manipulation, tunable expression, and high-throughput screening. While the synthetic biology tools available for filamentous fungi do not match those of bacteria or yeast, a number of recent advances in genetic methods have significantly broadened the molecular toolkit of these organisms.

7.1

CRISPR

Effective genome editing tools enable DNA to be inserted, modified, or removed from the genome in a targeted, sequence-specific manner (Zheng et al. 2017). In filamentous fungi, genome manipulation is severely limited by low homologous recombination (HR) frequencies (~2% in wild type), which results from the nonhomologous end-joining pathway (NHEJ) being the predominant mechanism for DNA repair (Weld et al. 2006; Liu et al. 2015). Consequently, strain engineering in these organisms requires laborious and time-consuming phenotypic or PCR-based screening to identify successful transformants (Wagner and Alper 2016). Traditionally, the rate of homologous integration has been enhanced through the use of NHEJ pathway deletion strains such as ku70Δ or ku80Δ (Wagner and Alper 2016). The kusAΔ strain in A. niger, an ortholog of ku70, increases the frequency of homologous recombination mediated integration from 7% in wild type up to ~80% (Meyer et al. 2007). Despite the gains in integration frequency, extensive screening is still required in some cases as well as the use of long (~1000 bp) homology sequences flanking the integration cassette to ensure HR at the desired locus (Wagner and Alper 2016). Moreover, deletions can have

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deleterious physiological effects as seen with the kusAΔ strain, which is sensitive to UV exposure or X-ray irradiation (Meyer et al. 2007). The adoption of clustered regularly interspaced short palindromic repeats (CRISPR) has the potential to revolutionize synthetic biology in filamentous fungi by increasing the efficiency of genome editing and facilitating the simultaneous editing of multiple genes. The type II CRISPR system from Streptococcus pyogenes has two components: a Cas9 nuclease and a Cas9 binding single-guide RNA (sgRNA), which is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Shi et al. 2017). A 20-nucleotide target recognition sequence in the sgRNA directs Cas9 to a specific genomic locus, whereupon it creates a double-strand break. DNA repair by NHEJ is error-prone and often leads to mutations such as deletions or substitutions (Nodvig et al. 2015). Supplying an expression cassette containing gene targeting homology regions along with the sgRNA activates the homology directed repair pathway, increasing gene targeting efficiency (Zheng et al. 2017). CRISPR-Cas9 has been used to edit a number of filamentous fungi, namely, A. niger, A. oryzae, A. fumigatus, T. reesei, and N. crassa (Fuller et al. 2015; Liu et al. 2015; Matsu-Ura et al. 2015; Katayama et al. 2016; Kuivanen et al. 2016; Leynaud-Kieffer et al. 2019). Moreover, the system has been developed for use in nonconventional fungal species such as Nodulisporium sp. (Zheng et al. 2017). The CRISPR protocol developed by Nodvig et al. (2015) was used to successfully induce mutations into six fungal species, one of which, A. brasiliensis, has never been genetically modified. Their method uses an A. niger codon-optimized Cas9 nuclease and guides RNA supplied by a single plasmid containing an AMA1 sequence. Four different plasmids were constructed, each with a commonly used fungal selection marker (pyrG, argB, bleomycin resistance, and hygromycin resistance). Multiple gene modifications using traditional approaches typically require successive rounds of marker integration and excision. Liu et al. (2015) have demonstrated multiple simultaneous gene editing by CRISPR in T. reesei. Employing an in vitro synthesized sgRNA and a codon-optimized Cas9 nuclease, they achieved homologous recombination frequencies of ~100% for single-gene, ~45% for double-gene, and ~4% for triple-gene deletions. The introduction of multiplexed gene editing with CRISPR-Cas9 technologies is a remarkable improvement of previously available genome editing tools for filamentous fungi and has the potential to massively expand the genetic engineering capabilities of these organisms.

7.2

Tunable Promoters

Promoters play a central role in eukaryotic gene expression and as a consequence are one of the most characterized synthetic parts in expression cassettes (Wagner and Alper 2016). Filamentous fungi, however, lag behind more established synthetic biology model organisms such as yeast and bacteria with respect to the range of available gene expression systems. Developing inducible and tunable promoters for controlled gene expression in filamentous fungi would greatly expand the synthetic biology toolkit for these expression hosts. Ideally, promoter systems should be

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available that can be controlled both temporally and over a range of expression levels in response to specific culture conditions (e.g., depletion of a nutrient), physiological states, or using cost-effective inducing agents (Wagner and Alper 2016). This is especially important for heterologous protein expression in cases where intermediates or final products are toxic to the cell (Kluge et al. 2018). Most inducible promoters fall into two broad categories: physiological and chemical. Physiological promoters are regulated by abiotic environmental factors such as carbon or nitrogen sources (Kluge et al. 2018). One classic example is the glucoamylase A promoter from A. niger, which is used widely in heterologous protein production. Gene expression under the control of the glaA promoter is repressed in growth media containing xylose and activated in the presence of maltose, glucose, or starch. Another such example is the 1,3-beta-glucanosyltransferase promoter from A. niger, which is pH inducible and most active at pH 2.0 under citric acid-producing conditions (Yin et al. 2017). In A. oryzae, addition of thiamine to growth media can be used in a dosage-dependent manner to tune expression of genes under the control of the thiamine promoter (Shoji et al. 2005). While widely adopted, metabolismdependent promoters can have pleiotropic effects and limit the types of media that can be employed. Chemically regulated promoters induced in the presence, and repressed in the absence, of a specific chemical compound can mitigate many of the issues associated with physiological promoters (Kluge et al. 2018). For example, the Tet-on/Tet-off system is an expression method commonly used to study essential gene function in yeast and has now been adopted for tightly controlled, titratable induction of gene expression in Aspergillus. Addition of the tetracycline derivative doxycycline to culture media activates gene expression within minutes through the release of a negative regulator, resulting in tetracycline resistance. Meyer et al. (2011a) established a Tet-on system in A. niger using luciferase as a marker. They found that expression levels could be tuned by copy number and doxycycline concentration and were able to reach levels similar to that of the commonly used constitutive gpdA promoter. Moreover, they created conditional knockout mutants of the essential actA gene demonstrating that the Tet-on system can be used to generate wild-type, knockout, or overexpression phenotypes from a single strain by varying doxycycline concentrations. These results support the use of the Tet-on/Tet-off system as an appropriate tool for inducible, tunable gene expression and for the study of essential genes in filamentous fungi. However, for production of industrial enzymes, the use of expensive chemical inducers would not be cost-effective. Therefore, promoters responsive to carbon sources or nutrients found in the biomass feedstock would be much preferred types of chemically regulated promoters.

7.3

Polycistronic Gene Expression

Until recently, creating strains of filamentous fungi for the concurrent expression of several genes has been a laborious and time-consuming task. Being able to co-express entire pathways is of great interest, however, as fungal secondary metabolites are

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Fig. 10.3 Schematic illustration of the viral 2A peptide used for polycistronic gene expression. The Porcine teschovirus P2A sequence, codon optimized for A. niger, is shown as per Schuetze and Meyer (2017)

frequently produced from biosynthetic gene clusters requiring the simultaneous expression of multiple genes. Unfortunately, many of these biosynthetic clusters are not expressed under laboratory conditions, thereby limiting their potential application in the pharmaceutical and/or biotechnology industries. Replacing traditional cloning techniques with polycistronic gene expression systems using self-cleaving viral 2A peptides can help to address this technical challenge. Moreover, this method has many advantages for fungal synthetic biology, namely, reduced construct size, fewer construct parts, and potentially 1:1 protein stoichiometry. The viral 2A peptide method produces equimolar amounts of multiple proteins from a single open reading frame. Expression cassettes are designed such that an 18–22 amino acid viral 2A peptide sequence is placed between each individual gene (Schuetze and Meyer 2017) (Fig. 10.3). During translation, the viral 2A peptide prevents peptide bond formation between the last two amino acids of the 2A peptide (glycine and proline) without affecting translation of the downstream polypeptide (SzymczakWorkman et al. 2012). This technique has been used in A. niger for the successful expression of genes necessary for the production of the secondary metabolites enniatin and melanin (Geib and Brock 2017; Schuetze and Meyer 2017). In T. reesei, Subramanian et al. (2017) adapted the 2A peptide from the foot and mouth disease virus to co-express intracellular eGFP and secreted cellobiohydrolase Cel7A. Protein function was tested by measuring fluorescence in the case of eGFP and by growth on pretreated corn stover as a complex carbon source for Cel7A. While polycistronic gene expression using the viral 2A peptide is a promising technology for use in producing enzyme cocktails for lignocellulosic biomass conversion, a number of technical challenges remain. For example, for each secreted protein, a signal peptide sequence must be inserted downstream of the 2A peptide in the expression cassette to ensure proper processing and targeting (Yan et al. 2010). Furthermore, in some cases, gene order within the polycistronic mRNA has been shown to influence enzyme activity (Schuetze and Meyer 2017; Subramanian et al. 2017). For applications that require precise ratios of proteins or activities, preliminary experiments should be conducted first to test for any positional effects.

7.4

Flow Cytometry

The development of synthetic biology tools for filamentous fungi has expedited the production of expression cassettes and genetically engineered strains. However, in

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order to leverage these new resources, they need to be combined with suitable highthroughput screening methods. Flow cytometry is a technique used for the rapid analysis of physical and physiological characteristics of cells based on optical parameters (Bleichrodt and Read 2018). It confers a number of advantages over many traditional biochemical methods that involve labor-intensive steps, require cell lysis, and limit the potential throughput of screening activities. Using fluorescently tagged proteins or dyes, measurements can be performed in vivo, and, for most applications, minimal or no additional processing or cell lysis is required. The method has a large dynamic range and is quantitative with data collected in real time. Fluorescence-activated cell sorting allows for rapid sorting and collection of user-determined subpopulations and has been used extensively in yeast for highthroughput screening. The formation of hyphae and aggregate mycelium by filamentous fungi has limited the use of flow cytometry in these organisms as these structures are often too large, precluding their use with flow cytometer machines. Bradner and Nevalainen (2003), however, have demonstrated that filamentous fungi can be analyzed by flow cytometry during a 12–13 h window following germination and in the early stages of hyphal development. For example, expressing green fluorescent protein (GFP) under the control of the cellobiohydrolase I promoter and terminator in T. reesei, Throndset et al. (2010) performed a successful fluorescence-activated cell sorting (FACS)-based screen for increased protein production and cellulase activity. In their study, germinating spores were UV mutagenized and then enriched for increased GFP expression through multiple rounds of FACS. Cellobiohydrolase expression was measured in the strains with the highest GFP expression, and the top strain was then tested and compared against wild type for biomass conversion capability. More recently, single-spore microencapsulation in alginate microparticles has been used in conjunction with large particle flow cytometry to screen auxotrophic and conditional mutants (DelgadoRamos et al. 2014). Studies such as those detailed above highlight the feasibility of using flow cytometry as a screening technology for filamentous fungi and hint at the possibility of implementing high-throughput screening efforts similar to those used with bacteria and yeast.

8 Conclusion There is a growing desire to move away from fossil fuels toward renewable feedstocks for the production of fuels and chemicals. Nonfood-based plant biomass such as agricultural waste or energy crops grown on marginal lands are an ideal source of lignocellulosic biomass, as they do not compete with critical food crops. However, the economical conversion of plant biomass into fermentable sugars is restricted by the recalcitrant nature of lignocellulose. Moving forward, the economic viability of lignocellulosic biomass-based biofuels and biochemicals will depend upon the maximum valorization of all lignocellulosic biomass components.

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Addressing this issue, advances in process engineering have resulted in mechanical and thermochemical pretreatments for plant biomass that improve access to cellulose for subsequent enzyme conversion. Significant improvements to the microbial expression hosts and enzymes used for the hydrolysis of lignocellulosic biomass will also be required. In this review, we have summarized the molecular and genetic strategies used for heterologous enzyme production in filamentous fungi, with an emphasis on the enzymes involved in valorizing lignocellulosic biomass. Efficient heterologous protein production often requires multilevel optimization strategies for both the protein and the expression host. Decisions can be made whether to use codon optimization, signal peptides, carrier proteins, inducible promoters, or proteasedeficient strains, to name just a few. Traditional rational genetic engineering approaches are limited to the manipulation of genes from a small subset of pathways. The recent adoption of systems biology methods in filamentous fungi now means that genomics and proteomics tools can be used to unravel the complex cellular mechanisms underlying heterologous protein production. Combining this expanding base of knowledge with new synthetic biology tools will facilitate the rapid development of new efficiently designed genetically engineered protein production strains. Looking forward, these techniques are all critical for identifying, characterizing, and increasing the expression levels, yields, and activities of lignin-modifying enzymes for the successful integration of lignocellulosic biomass into the new bio-economy.

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Part IV

Branching Out: Emerging Opportunities

Chapter 11

Horizontal Gene Transfer in Fungi Erin L. Bredeweg and Scott E. Baker

1 Introduction Nucleotides are one of the major forms of biological polymers, among carbohydrates and amino acids. DNA and RNA both function to hold and convey information but differ in their chemistry and stability. DNA’s stability has led to its longer retention time in the environment, from crime scenes, to soil microbiomes, to ancient organism identifications. Acceleration of genome sequencing and assembly of fungal genomes (Baker et al. 2008; Grigoriev et al. 2011, 2013; Martin et al. 2011), both in depth and breadth of species and group coverage, has enabled analysis of many types of interspecies DNA transfer. Lineage-specific genes, found by comparison between related species, followed by comparison to wider groups of organisms, can uncover evidence of genome dynamics not previously accessible. To serve as a contrast to DNA movement in eukaryotic genomes, prokaryotic genomes can be referred to as “pan-genomes” (Medini et al. 2005), defined by “core” shared genes and accessory genes that vary between strains of the same species. Eukaryote genomes increasingly are found to have a shared core and varying accessory gene structure, including fungi (McCarthy and Fitzpatrick 2019). Species definition and genome content can be passed horizontally through conjugation and environmental uptake as well as vertically through cell division. It has been suggested that this method of promiscuous DNA exchange serves as a source of variation, similar to meiotic recombination in sexual reproduction. Bacterial pan-genomes may also be responsive to sequence-specific DNA uptake preferences, which is consistent with innate surveillance mechanisms such as genome defense by sequence-specific restriction enzymes. DNA can be taken up from the environment via a type IV pilus or E. L. Bredeweg · S. E. Baker (*) Biosystems Dynamics and Simulation Group, Environmental Molecular Science Division, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA e-mail: [email protected]; [email protected] © Battelle Memorial Institute 2020 H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_11

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be injected from one organism to another through a type II secretion system (Mitrikeski 2015). Eukaryotic systems increase genetic diversity in their DNA sequences from sexual reproduction or vertical inheritance, which serves to reshuffle gene combinations during meiosis. Additional DNA movement by transposable elements or “jumping genes,” viral infection, and DNA repair mechanisms may serve to transport or incorporate a new horizontal gene transfer (HGT). Transposons can move themselves or initiate movement of other DNA-/RNA-based transposable elements through a variety of mechanisms, including long terminal repeats. The epigenetic scaffold and sequence context of DNA is notable in gene clusters gained in subtelomeric regions which are typically repeat- and transposon-rich. To minimize transposon movement and disruption, eukaryotic genomes have utilized a number of defenses like epigenetic and silencing mechanisms which control access to DNA (meiotic silencing, RNA silencing, repeat-induced point mutation or RIP, and heterochromatin formation); these may in turn influence rates of HGT. These topics are all accessible in depth in other reviews (Gladyshev 2017; Hammond 2017). From an evolutionary standpoint, genome dynamics—losses, gains, reorganizations, defense, etc.—of related fungi may explain the wide variety of genome sizes (Szollosi et al. 2015) obtained from sequenced Dikarya. Horizontal gene transfer is one possible source of DNA sequence expansion, among genome duplications (e.g., yeast, Wapinski et al. 2007), transposon activity, species fusion, and other phenomena. Recent work on four model fungi suggests phenomena alternative to HGT may underlie the development of the pan-genome for each species (McCarthy and Fitzpatrick 2019). A pan-genome consists of two main components—strainspecific genes that vary across members of the same species and a commonly occurring core genome (Tettelin et al. 2005). However, there is strong evidence for cases of horizontal gene transfer, including ample opportunity for neighboring organisms to interact. Incidence of HGT in arbuscular mycorrhizal fungi (AMF) has been under recent scrutiny, with detections of both bacterial and plant-origin genes in fungal genomes (Li et al. 2018; Sun et al. 2019). Beyond movement of small autonomous DNA elements (plasmids, transposons), eukaryotic genomes within fungi have been noted to both acquire and exchange entire chromosomes. Instead of requiring insertion, DNA segments may contain sequence elements for independent propagation (e.g., the point centromere of S. pombe (Pluta et al. 1995), as have been seen in the grass pathogen Zymoseptoria tritici and in pathogenic strains of Fusarium oxysporum. However, the molecular route these accessory chromosomes utilize between strains is somewhat mysterious and outside the bounds of the vertical inheritance. Bioinformatics-based evidence for gene exchange between different kingdoms is currently supported by species trees, high-throughput sequence assembly comparison, and comparing the genes in neighboring species. Concepts related to computational methods for detection of HGT have been reviewed elsewhere (Ravenhall et al. 2015) and are under continuous development, for example, Tang et al. (2018) and (Jeong and Nasir (2017).

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Fig. 11.1 Horizontal gene transfer proposed mechanism and detection methods. (a) Sources of environmental DNA in cases discussed in this chapter, selective pressure types, and a proposed mechanism for DNA uptake, including conserved pathways implicated in transformation efficiency in S. cerevisiae. The mechanism for DNA uptake includes tethering of exogenous DNA to the external surface of the cell, a break in the cell wall, and use of endocytosis machinery to allow delivery of DNA to the nucleus. (b) Methods driving horizontal gene transfer detection: observed phenotypic transfer such as strain virulence or host specificity, identification of foreign DNA by genome sequencing and comparison, and phylogenetic detection by surveying traits or genes in related fungal species

Critics suggest that evidence of HGT in eukaryotes is frequently artifact (Dupont and Cox 2017). Proponents point to sequence specificity found and absorbed from “prey” plants or fungi (e.g., consumed by Trichoderma spp.) (Druzhinina et al. 2018), eukaryotic origin intron inclusions, and comparative sequencing analysis. A bioinformatic comparison of gene tree-dependent and gene tree-independent methods (Szollosi et al. 2015) took a theoretical approach, simulating explanation of genome size and content through gene loss and duplication, which they then compared with a model including potential interspecies transfer events. For the ancestral Aspergillus clade gene contents, the duplication-loss model had an inflated content (14, 244 genes) compared to the transfer-enabled ancestral genome (8238 genes) which is much closer to the extant range of genome sizes analyzed (8891–7797 genes). Lifestyles that include engulfment and phagocytosis (Mitrikeski 2015) and parasitism (see Whitaker et al. 2009; Wijayawardena et al. 2013) have frequently been found to be associated with HGT in eukaryotes, supported by an increase in exposure to DNA from nearby organisms. “Highways of gene exchange” in prokaryotes have been hypothesized, driven by a shared ecological niche (Beiko et al. 2005; Duarte and Huynen 2019; Murphy et al. 2018; Szollosi et al. 2015). Correspondingly, genetic transfers between two species that are otherwise distantly related are more likely in a shared environment (Fig. 11.1a). Ecologically co-occurring DNA for eukaryotes is hypothesized here to result in rare but data-substantiated incorporation. The environmental advantage of virulence, increased fitness, or metabolic gains supports the

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propagation of horizontally acquired sequences. Following exposure to DNA, evolutionary retention of horizontally acquired traits coincides with the presence of a selective driver, such as acquisition of anaerobic metabolism (Gojkovic et al. 2004), fungicide resistance (Riccombeni et al. 2012), or species-targeted specific virulence factors (A. alternata, Mehrabi et al. 2011).

2 Modes of Horizontal Gene Transfer What follows is a brief listing of select horizontal gene transfer (HGT) events, size, and sequence characteristics, spanning a transition from phenotypic to genotypic discovery (also see Fig. 11.1b, Table 11.1). Previously unique fungal phenotypes discovered by observation are now accessible through whole genome sequencing.

2.1

Phenotypic Transfer

Early documentation of HGT events was noted by transfer of characteristics such as pathogenic toxicity between fungal species. These events existed as anecdote and hypothesis (Walton 2000) but lacked direct evidence until recently. A classic example is that of ToxA, a virulence protein, which appears to have been transferred from the wheat pathogen Stagonospora nodorum to Pyrenophora tritici-repentis. There are multiple lines of evidence for the HGT event including ToxA sequence diversity in S. nodorum and a derivative sequence and context found as an almost exact match in P. tritici-repentis (Barrus 1942; Friesen et al. 2006; Nisikado 1929; Syme et al. 2018). Further summary of the interaction specifics has been done, which reviews progression of disease caused by this host-specific proteinaceous toxin (Ciuffetti et al. 2010). Trichoderma reesei is notable for its arsenal of high-activity carbon active enzymes, flexible metabolism, and broad host range. An analysis of protein sequence and gene clustering suggests a nitrate assimilation function was acquired by Trichoderma reesei from a heterokont (Slot and Hibbett 2007). Heterokonts are a major line of eukaryotes, many of which are algae; an HGT event for the marineisolated fungus T. reesei QM6A is consistent with organisms in a shared environment. When presenting the gene trees of nitrate assimilation enzymes including other dikarya (ascomycota and basidiomycota), addition of the T. reesei genes disagreed with a monophyletic structure, suggesting HGT. This mirrors discovery of CAZymes unique to the genus Trichoderma, which appear to have been picked up from plant-associated filamentous fungi (Druzhinina et al. 2018). A shift in host specificity has been identified in Trichoderma substantially by detection of HGT from Ascomycota that feed on plants (Druzhinina et al. 2018). The associated gene transfers extended Trichoderma’s host range to plants, insects, and other fungi, enabling endoparasitism on not just Basidiomycetes but also other Ascomycota.

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Horizontal Gene Transfer in Fungi

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Table 11.1 Selected horizontal gene transfer (HGT) events in filamentous fungi Method of detection Phenotype, gene tree comparison Gene tree comparison, ShimodairaHasegawa test Whole genome sequencing, gene tree comparison Comparative genomics

HGT description ToxA transfer

Size 11 kb

Fungal species Pyrenophora triticirepentis

Nitrate assimilation cluster

Variable, 3 genes

Trichoderma reesei

CAZymes for fungal parasitism, from ascomycetes Lineage specific chromosomes

Estimated 123 genes+

Trichoderma reesei, and other species.

4 chromosomes, ~2.4– 6 Mb 14 genes

Fusarium oxysporum f. sp. lycopersici Fusarium oxysporum

Unknown

Phytophthora mirabilis

1 gene

Penicillium simplicissimum

p450nor

1 gene

General fungi from proteobacterium

ZT clade spoilage yeast gene influx Fucose transporter and SM gene clusters Alpha-amylase

17.5 kb, 65 kb

Wine yeast (S. cerevisiae)

0.62 Mb

Colletotrichum

Comparative genomics

Liang et al. (2018)

12 kb

Aspergillus niger and Aspergillus oryzae Aspergillus fumigatus

Comparative genomics Whole genome sequencing, variation of tetranucleotides Comparative genomics Phenotype, gene expression, sequencing Comparative genomics

Andersen et al. (2011) Ma et al. (2010)

>>SIX gene cluster pathogenicity Species hybrid, unstable Vanillyl oxidase

Genome-wide HGT

SM cluster variation Sterigmatocystin cluster

Wallaby

~214 genes 16 genes LS 26 prok 19 euk 6 clusters 54 kb

Aspergillus fumigatus strains Podospora anserina

575 kb

Penicillium sp.

Evolutionary gene tree divergence Virulence transfer Gene trees and regional synteny Gene treespecies tree divergence Comparative genomics

References Friesen et al. (2006) Slot and Hibbett (2007) Druzhinina et al. (2018)

Ma et al. (2010) Laurence et al. (2015) Mehrabi et al. (2011) Gygli et al. (2018) Higgins et al. (2018) Marsit et al. (2015)

Lind et al. (2017) Slot and Rokas (2011)

Cheeseman et al. (2014) (continued)

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Table 11.1 (continued) HGT description LS subtelomeric region

Size 12 kb

Fungal species Fusarium circinatum

Inter-genome transfers Pan-genome

33 genes

ColletotrichumMagnaporthales Zymoseptoria tritici

6600 accessory genes

Gut fungi

2.9–4.1% genome content

Neocallimastigomycota

LS chromosomes