Pharmaceutical Biocatalysis: Drugs, Genetic Diseases, and Epigenetics 981487714X, 9789814877145

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Jenny Stanford Series on Biocatalysis Volume 7

Pharmaceutical Biocatalysis

Jenny Stanford Series on Biocatalysis

Series Editor

Peter Grunwald

Titles in the Series Published Vol. 1 Industrial Biocatalysis Peter Grunwald, ed.

2015

978-981-4463-88-1 (Hardcover)

978-981-4463-89-8 (eBook)

Vol. 2 Handbook of CarbohydrateModifying Biocatalysts Peter Grunwald, ed.

2016

978-981-4669-78-8 (Hardcover)

978-981-4669-79-5 (eBook)

Vol. 3 Biocatalysis and Nanotechnology Peter Grunwald, ed.

2017

978-981-4613-69-9 (Hardcover)

978-1-315-19660-2 (eBook)

Vol. 4 Pharmaceutical Biocatalysis: Fundamentals, Enzyme Inhibitors, and Enzymes in Health and Diseases Peter Grunwald, ed.

2019

978-981-4800-61-7 (Hardcover)

978-0-429-29503-4 (eBook)

Vol. 5 Pharmaceutical Biocatalysis: Chemoenzymatic Synthesis of Active Pharmaceutical Ingredients Peter Grunwald, ed.

2019

978-981-4800-80-8 (Hardcover)

978-0-429-35311-6 (eBook)

Vol. 6 Pharmaceutical Biocatalysis: Important Enzymes, Novel Targets, and Therapies Peter Grunwald, ed.

2021

978-981-4877-13-8 (Hardcover)

978-1-003-04539-7 (eBook)

Vol. 7 Pharmaceutical Biocatalysis: Drugs, Genetic Diseases, and Epigenetics Peter Grunwald, ed.

2021

978-981-4877-14-5 (Hardcover)

978-1-003-04541-0 (eBook)

Forthcoming

Vol. 8 Agricultural Biocatalysis Peter Grunwald, ed. 2022 Vol. 9 Biocatalysis for Sustainable Process Development Peter Grunwald, ed. 2023

Jenny Stanford Series on Biocatalysis Volume 7

Pharmaceutical Biocatalysis Drugs, Genetic Diseases, and Epigenetics

edited by

Peter Grunwald

Published by Jenny Stanford Publishing Pte. Ltd. Level 34, Centennial Tower 3 Temasek Avenue Singapore 039190 Email: [email protected] Web: www.jennystanford.com

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Pharmaceutical Biocatalysis: Drugs, Genetic Diseases, and Epigenetics Copyright © 2021 Jenny Stanford Publishing Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

Cover: Yew needles and berry, by Dr. Gorden Cragg. National Cancer Institute, USA. ISBN 978-981-4877-14-5 (Hardcover) ISBN 978-1-003-04541-0 (eBook)

Contents Preface

1. Fermentative Production of Vitamin B6

xv

1

Jonathan Rosenberg, Björn Richts, and Fabian M. Commichau

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

Introduction De novo Synthesis of Vitamin B6 Control of Vitamin B6 Homeostasis Engineering Microorganisms for the Production

of B6 Vitamers Novel Routes for Vitamin B6 Biosynthesis and

Production Rational Design and Construction of a Vitamin

B6-Producing Bacterium Alternative Approaches for Enhancing Vitamin

B6 Production Conclusions

2. Exploring Alternative Taxol Sources: Biocatalysis of

7-b-Xylosyl-10-Deacetyltaxol and Application for Taxol

Production

1

4

7

10

17

20

23

25

35

Wan-Cang Liu, Bing-Juan Li, Ting Gong, and Ping Zhu

2.1 Introduction 2.2 High-Cell-Density Fermentation of the

Engineered Yeast 2.2.1 General Fed-Batch HCDF Process 2.2.2 HCDF Process Optimization 2.2.2.1 Elimination of pure oxygen

supplement by increasing air

pressure 2.2.2.2 Fermentation using biomass-stat

strategy

35

37

38

38

38

39

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Contents

2.2.2.3 Fermentation using reduced

induction DO value 2.2.2.4 Optimization of initial induction

biomass 2.2.3 Scaling Up HCDF from Pilot Scale to

Demonstration/Commercial Scale 2.3 Biocatalysis of 7-b-Xylosyltaxanes 2.3.1 General Biocatalysis Protocol 2.3.2 Optimization of the Biocatalysis 2.3.2.1 Impact of dry cell amount on

biocatalysis 2.3.2.2 Impact of DMSO concentration on

biocatalysis 2.3.2.3 Impact of substrate concentration

on product yield 2.3.2.4 Effect of antifoam supplement on

biocatalysis 2.3.3 Scale-Up Biocatalysis 2.4 One-Pot Enzymatic Reaction from 7-b-Xylosyl-

10-Deacetyltaxol to Taxol 2.4.1 Reaction System for the Biocatalysis 2.4.2 Protein Engineering of the

10-b-Acetyltransferase 2.4.2.1 l-Alanine scanning mutagenesis 2.4.2.2 Saturation mutagenesis 2.4.2.3 Construction of one-pot reaction

system 2.5 Summary

3. Molecular Farming through Plant Engineering: A

Cost-Effective Approach for Producing Therapeutic

and Prophylactic Proteins

40

41

41

44

45

45

45

45

46

46

47

49

49

50

50

51

53

53

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Prakash Narayana Reddy, Krupanidhi Srirama,

and Vijaya R. Dirisala

3.1 Introduction 3.2 Strategies for Production of Therapeutics in Plants 3.2.1 Stable Expression

57

59

59

Contents

3.2.2 Transient Expression 3.3 Plant-Made Vaccines 3.4 Plantibodies 3.5 Conclusions

4. Microbial Biotransformations in the Production and Degradation of Fluorinated Pharmaceuticals

61

62

68

74

81

Cormac D. Murphy and Aoife Phelan

4.1 Introduction 4.2 Fluorinated Natural Products 4.3 Production of Fluorinated Antibiotics in

Microorganisms 4.4 Biological Production of [18F]-Labelled

Compounds for PET Analysis 4.5 Microorganisms that Enable Fluorinated Drug

Design 4.6 Production of Fluorinated Drug Metabolites in

Microorganisms 4.7 Microbial Degradation of Fluorinated Drugs 4.8 Future Prospects and Challenges

5. Successful Screening of Potent Microorganisms

Producing l-Asparaginase

81

82

84

89

91

94

95

99

105

Archana Vimal and Awanish Kumar

5.1 Introduction 5.2 Purpose of Screening Prospective Source of

l-Asparaginase 5.2.1 High Cost of Treatment 5.2.2 Minimizing Side Effects 5.2.3 Prolongation of Half-Life 5.2.4 Explore the Multifaceted Use of

l-Asparaginase 5.3 Different Methods of Screening Potential Source

of l-Asparaginase 5.3.1 In silico Approach 5.3.2 Dye-Based Method

105

106

107

108

108

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111

vii

viii

Contents

5.4 5.5

5.6

5.7

5.3.3 Assay-Based Method 5.3.3.1 Radioactive isotope assays 5.3.3.2 Indophenol assay 5.3.3.3 Coupled assay 5.3.3.4 Aspartic acid determination

assay 5.3.3.5 Hydroxylamine assay 5.3.3.6 Fluorometric assay 5.3.3.7 Direct nesslerization assay Activators and Inhibitors of l-Asparaginase Various Microbial Sources of l-Asparaginase 5.5.1 Microbial Sources 5.5.1.1 Bacterial source 5.5.1.2 Fungal source 5.5.2 Plant Source 5.5.3 Animal and Other Sources Pharmaceutical Application of l-Asparaginase 5.6.1 Chemotherapy 5.6.2 Infectious Disease 5.6.3 Autoimmune Disorder 5.6.4 Veterinary 5.6.5 Food Additive 5.6.6 Medical/Food Biosensor Conclusion

6. Biotransformation of Xenobiotics in Living

Systems—Metabolism of Drugs: Partnership of Liver

and Gut Microflora

113

114

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114

115

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117

117

120

120

120

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121

121

122

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123

124

124

129

Maja Đanić and Momir Mikov

6.1 6.2

Introduction Liver Metabolism 6.2.1 Phase I Biotransformation 6.2.1.1 Oxidations 6.2.1.2 Reductions 6.2.1.3 Hydrolysis

129

132

132

133

141

143

Contents

6.2.2 Phase II Biotransformation 6.2.2.1 Uridine diphosphate­ glucuronosyltransferases 6.2.2.2 Glutathione S-transferases 6.2.2.3 Methyltransferases 6.2.2.4 N-Acetyltransferases 6.2.2.5 Sulfotransferases 6.3 Metabolism of Xenobiotics in Gut 6.3.1 Luminal and Cell Wall Metabolism of

Drugs 6.3.2 Gut Microflora Implication in Xenobiotic

Metabolism 6.3.2.1 Reduction of drugs by microbiota 6.3.2.2 Microbial metabolism of drugs

by hydrolysis 6.4 Conclusion

7. Degradation of Pharmaceutically Active Compounds

by White-Rot Fungi and Their Ligninolytic Enzymes

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147

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149

150

151

152

153

153

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159

160

167

Muhammad B. Asif and Faisal I. Hai

7.1 Introduction

7.2 PhAC Removal by WRF and Their Ligninolytic

Enzymes 7.2.1 Effect of Fungal Species 7.2.2 Effect of Enzyme Types

7.2.3 Effect of PhAC Properties on Their

Removal

7.2.3.1 Removal of PhACs containing

EDGs 7.2.3.2 Removal of PhACs containing

EWGs 7.2.3.3 Removal of PhACs containing

both EDGs and EWGs 7.2.3.4 Effect of hydrophobicity

7.2.4 Laccase-Redox Mediator System

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Contents

7.3 Impact of Physicochemical Characteristics of

Wastewater 7.4 Treatment of Real Wastewater by WRF and

Ligninolytic Enzymes 7.5 Future Research 7.6 Conclusion

8. Removal of Pharmaceutical Pollutants from

Municipal Sewage Mediated by Laccases

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197

199

200

209

Thomas Hahn, Fabian Haitz, Jan Gajewski, Marius Mohr,

Marc Beckett, and Susanne Zibek

8.1 Introduction 8.2 Political and Societal Framework Conditions 8.2.1 Situation in Germany 8.2.2 Situation in Switzerland 8.3 Elimination of Pharmaceuticals with Physical

and Chemical Methods 8.3.1 Use of Activated Carbon 8.3.1.1 Granulated activated carbon 8.3.1.2 Powdered activated carbon 8.3.2 Use of Ozonation 8.3.3 Combined and Other Treatment Processes 8.3.3.1 Combined ozonation and activated

carbon 8.3.3.2 Combined ozone and hydrogen

peroxide 8.3.3.3 UV light 8.3.3.4 Membrane filtration 8.4 Theoretical Background and Application of

Laccases 8.4.1 Occurrence, Structure and Functionality

of Laccases 8.4.1.1 Origin and characterization 8.4.1.2 Reaction mechanism and

stoichiometry 8.4.1.3 Substrates and products

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Contents

8.5 8.6 8.7 8.8

8.4.1.4 Inhibition of laccase activity 8.4.1.5 Immobilization types for laccases 8.4.2 Laccase-Mediator-System 8.4.3 Industrial Use of Laccase Elimination of Pharmaceuticals by the Use of

Laccase Comparison of Different Technologies for the

Elimination of Pharmaceuticals Assessing the Use of Laccase in Municipal

Wastewater Treatment 8.7.1 Use of Native Laccases 8.7.2 Use of Immobilized Laccase Summary and Conclusions towards Removal of

Pharmaceuticals

9. Mechanism of Drug Resistance in Staphylococcus

aureus and Future Drug Discovery

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228

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233

239

243

243

245

248

259

Felipe Wakasuqui, Ana Leticia Gori Lusa, Sven Falke,

Christian Betzel, and Carsten Wrenger

9.1 9.2 9.3 9.4

Introduction Drugs, Targets and Resistance Mechanism Future Drug Discovery and New Drugs Conclusion

10. Genome Editing and Gene Therapies: Complex and

Expensive Drugs

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261

266

273

281

Peter Grunwald

10.1 Introduction 10.2 Some General Aspects 10.3 Genome Editing Techniques: Fundamentals 10.3.1 Zinc Finger Nucleases 10.3.2 TALENs 10.3.3 CRISPR/Cas Systems 10.3.3.1 Other applications of

CRISPR-systems 10.4 Therapeutic Genome Editing

281

282

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296

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Contents

10.4.1 HDR-Mediated Genome Editing 10.4.2 Ex vivo and in vivo Somatic Genome

Editing 10.4.3 Delivery Strategies 10.4.3.1 Adeno-associated viral vectors 10.4.3.2 Lentiviral vectors 10.4.3.3 Nanocarrier-based gene/drug

delivery 10.4.3.4 Physical methods 10.4.4 Genome Editing and Disease Models 10.4.5 Induced Pluripotent Stem Cells 10.4.5.1 Human diseases: From 2D to

3D iPSC models 10.4.5.2 Genome editing and human

iPSCs 10.4.6 Genome Editing and Diseases 10.4.6.1 Genome editing studies in

non-clinical development and

clinical trials 10.4.6.2 Examples of non-clinical

developments 10.4.6.3 CAR-T cell therapy and CRISPR 10.4.7 Gene Therapies without Modifying the

Existing DNA 10.4.8 Genome Editing-Based Therapeutics in

Clinical Trials and Off-Target Effects 10.4.8.1 Off-target effects 10.4.9 Genome Editing: Commercialization 10.4.10 Ethical Concerns and Regulatory Aspects 10.5 Summary and Outlook

11. Epigenetic and Metabolic Alterations in Cancer Cells:

Mechanisms and Therapeutic Approaches

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334

337

339

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347

350

375

Chi Chun Wong and Jun Yu

11.1 Introduction

11.2 Epigenetic Alterations in Human Cancers 11.3 Metabolic Alterations in Human Cancers

375

376

379

Contents

11.4 Interplay between Epigenetics and Tumor

Metabolism

381

11.4.2 Acetyl-CoA Influences Histone Acetylation

381

11.4.1 Modulation of Epigenetics by Tumor

Metabolism 11.4.3 SAM/SAH Ratio Regulates DNA and

Histone Methylation

11.4.4 TCA Cycle Metabolites Modulate DNA

and Histone Demethylation 11.4.5 Succinate and Fumarate drive

DNA/Histone Methylation

11.4.6 2-Hydroxylglutarate in IDH1/IDH2

Mutant Cancers Drive DNA/Histone

Methylation

11.5 Therapeutic Approaches

11.5.1 Inhibition of Acetyl-CoA Production

Using Glycolysis Inhibitors

11.5.2 Inhibition of Succinate/Fumarate/2­ Hydroxylglutarate Using Glutaminolysis

Inhibitors 11.5.3 Inhibition of 2-Hydroxylglutarate Using

IDH1/2 Inhibitors 11.5.4 Inhibition of One Carbon Metabolism

by Limiting Methionine Cycle 11.5.5 Inhibition of DNA Methylation by

DNMTs

11.5.6 Inhibition of Tumor Metabolism by

HDACi

11.6 Conclusion

Index

381

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393

403

xiii

Preface The relations between biocatalysis and pharmaceutical sciences are manifold. Outstanding aspects are the excellent properties of biocatalysts which are among others their high chemo-, regio-, and enantioselectivities, making them particularly suitable for the synthesis of safe chiral drug intermediates and drugs. Furthermore, several (engineered) enzymes find increasing application for the treatment of important and in part serious diseases, such as fibrinolytic disorders, cancer, or the rare lysosomal storages diseases. Drug discovery also comprises the isolation of therapeutic enzymes and drugs from natural sources such as microorganisms and plants; the blockbuster anticancer drug paclitaxel is an early example (see this volume, Chapter 2). Finally enzymes and microorganisms are indispensable in connection with the degradation of pharmaceuticals, including antibiotics that entered the ecosystem with potentially hazardous consequences. These issues are discussed in volumes 4 to 7 of this Series on Biocatalysis. The present volume 7 focuses on drugs, inherited diseases, and epigenetics. In addition, several chapters are devoted to natural sources of drugs, as well as to their degradation, and to drug resistance mechanisms. Many enzymes involved in different biocatalytic processes require vitamin B6 as cofactor. The first chapter, written by Jonathan Rosenberg, Björn Richts, and Fabian M. Commichau, summarizes recent findings concerning biosynthesis and homeostasis of vitamin B6 metabolism with a focus on prokaryotic microorganisms. Procedures for developing microbial fermentation processes with the aim to produce vitamin B6, novel routes for vitamin B6 biosynthesis and their potential for overproducing the commercially valuable substance, as well as bottlenecks of the vitamin B6 biosynthetic pathways together with strategies to overcome existing limitations are also described. “Exploring alternative Taxol sources: Biocatalysis of 7-β-xylosyl10-deacetyltaxol and application for Taxol production” is the title of Chapter 2, authored by Wan-Cang Liu, Bing-Juan Li, Ting

xvi

Preface

Gong, and Ping Zhu. Taxol is a very successful anticancer drug, and the long-term supply of Taxol is a critical issue due to the fact that it accumulates only in very low concentrations in the natural source of the drug, the bark of yew trees; however, Taxol analogues occur in much higher concentrations. The authors developed a one-pot reaction system for the bioconversion of the analogue 7-β-xylosyl-10-deacetyltaxol isolated from dried stem bark to Taxol. Using fed-batch high-cell-density fermentation and microbial biocatalysis, they achieved a yield of over 600 mg/mL Taxol. Many drugs are of plant origin. In addition, molecular farming through plant engineering is a cost-effective possibility to produce therapeutic and prophylactic proteins. Prakash Narayana Reddy, Krupanidhi Srirama, and Vijaya R. Dirisala comment on this topic in Chapter 3 and explain why plants are ideal for producing safer and effective biobetters due to the possibility of producing therapeutics such as vaccines, antimicrobial peptides or antibodies with specific and mammalian glycoforms using glyco-engineered plant hosts, which is presently not possible by existing conventional cell culture systems. They also address existing public concerns regarding the use of genetically modified organism for generating plant-made pharmaceuticals. A variety of pharmaceuticals, including so-called blockbusters such as fluoxetine, sofosbuvir, and atorvastatin, contain fluorine. The importance of this group of drugs can be derived from the fact that in 2017, 8 out of the 29 small molecules approved by the U.S. Food and Drug Administration contained this element. Cormac D. Murphy and Aoife Phelan discuss the production of fluorinated antibiotics in microorganisms, the biological production of [18F]­ labeled compounds for positron emission tomography analysis, the employment of microorganisms for fluorinated drug design, and the production of fluorinated drug metabolites in microorganisms. They close their informative contribution with a summary of biodegradation studies on fluorinated drugs. Chapter 5, about successful screening of potent microorganisms producing L-asparaginase, has been provided by Archana Vimal and Awanish Kumar. The enzyme has therapeutic application among others in the treatment of varies types of cancer and finds also application as the biological component in biosensors for determining the l-asparagine content in the blood samples of

Preface

leukemia patients. The aspects discussed here include different methods of screening potential sources (bacterial, fungal, animal, etc.) for this protein, the description of a variety of assay-based approaches, activators, and inhibitors of the enzyme, and finally the pharmaceutical application of l-asparaginase. The topic “biotransformation of xenobiotics in living systems”— most drugs are xenobiotics—has been treated by Maja Đanić and Momir Mikov. The authors first discuss biotransformations of xenobiotics/drugs catalyzed in the presence of hepatic enzymes, a process divided into phase I (activation by oxidations, reductions and hydrolysis) and phase II metabolism (conjugation of the activated xenobiotics with an endogenous substrate catalyzed by transferases). In addition, emphasis is put on metabolism at the intestinal level, in particular on the role of the gut microflora in xenobiotic biotransformation. The authors underline the importance of studying the gut microbiome contribution to drug pharmacokinetics as an integral part of drug development processes. This is followed by Chapter 7, where Muhammad B. Asif and Faisal I. Hai tackle the problem that a high amount of pharmaceutically active compounds and their intermediates find their way into different environmental systems through municipal and hospital wastewater, pharmaceutical industry effluents and other sources. The authors describe in detail the state of the art of processes (whole-cell WRF and/or enzyme-based treatment systems) where white-rot fungi (WRF), using their intracellular or extracellular ligninolytic enzymes (laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase) are employed to degrade recalcitrant compounds in waste water including drugs. Special attention is given to the influence of structural properties of the drugs to be degraded and the impact of the physicochemical characteristics of the wastewater on drug removal. The subsequent chapter, written by Thomas Hahn, Fabian Haitz, Jan Gajewski, Marius Mohr, Marc Beckett, and Susanne Zibek, also deals with the environmental problems caused by pharmaceutical pollutions of municipal sewage. Apart from political and societal aspects, the chapter provides an overview of the elimination of pharmaceuticals via physical and chemical methods (use of activated carbon, ozonation, and combination of both) which are then compared with the removal of pharmaceutical pollutions from municipal sewage

xvii

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Preface

mediated by laccases. In addition, the structural and kinetic properties of laccases, strategies for their immobilization (e.g., the immobilization of laccase on activated carbon), and the different laccase-mediator-systems with their pro and cons are described in detail. The World Health Organization (WHO) recently stated that “antibiotic resistance is one of the biggest threats to global health, food security, and development today.” In a contribution authored by Ana Leticia Gori Lusa, Felipe Wakasuqui, Christian Betzel, and Carsten Wrenger, this issue has been taken up with the example of the mechanism of drug resistance in Staphylococcus aureus and its importance for future drug discovery and development. Drugs used to treat Staphylococcus aureus infections are reviewed, together with strategies developed by bacteria to repel their action. The need to discover new drugs involves the use of high-resolution methods to select a target molecule and to identify lead compounds that are successively optimized among others via structural investigations into complexes formed with proteins. The next chapter is about genome editing with engineered nucleases and gene therapies. It contains sections introducing the fundamentals of the genome editing techniques (zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced, short palindromic repeat (CRISPR) technologies), therapeutic genome editing, including different delivery techniques, genome editing and disease models, and induced pluripotent stem cells. Additional aspects treated are genome editing studies in non-clinical development and clinical trials, examples of non-clinical developments (infectious, monogenic, neurodegenerative diseases, CAR-T cell therapy and CRISPR), genome editing-based therapeutics in clinical trials and off-target effects. Recent advances in the field of gene therapy are reviewed. Finally, the commercialization of genome editing, together with ethical concerns and regulatory aspects, is discussed. Epigenetics is the science of heritable changes in gene expression that cannot be traced back to changes in the underlying DNA sequence, and disruptions of epigenetic processes are associated with initiation and progression of cancer. This is the topic of the final chapter authored by Chi Chun Wong and Jun Yu,

Preface

which deals with epigenetic and metabolic alterations in human cancer cells and the interplay between epigenetics and tumor metabolism resulting in epigenetic dysfunction. The authors explain how drugs targeting tumor metabolism can be combined with epigenetic drugs to achieve a synergistic effect in tumor inhibition. Examples of therapeutic approaches are given aiming among others at reversing aberrant histone acetylation and DNA/ histone methylation—both well-established cancer characteristics. The authors emphasize the importance of considering the whole tumor microenvironment, including stromal cells, T-cells, macrophages, and other immune cell types, for developing novel metabolic and epigenetic drugs. This volume of the Jenny Stanford Series on Biocatalysis provides an insight into a variety of important aspects of pharmaceutical biocatalysis beyond the synthesis and application of active pharmaceutical ingredients. These include natural sources of drugs, drug metabolism in the human body, environmental problems caused by their use and possibilities to mitigate them, and the threatening phenomenon “antibiotic resistance.” The information is completed by actual articles about genome editing approaches and the relationship between epigenetics and human cancers. Peter Grunwald

xix

Chapter 1

Fermentative Production of Vitamin B6 Jonathan Rosenberg,a Björn Richts,a and Fabian M. Commichaub aDepartment of General Microbiology, Georg-August-University Goettingen,

Grisebachstr. 8, D-37077 Göttingen, Germany

bBTU Cottbus-Senftenberg, Institut für Biotechnologie, FG Synthetische Mikrobiologie,

Universitätsplatz 1, 01968 Senftenberg, Germany

[email protected]

1.1 Introduction Vitamin B6 has been discovered almost one century ago and is an essential organic micronutrient for organisms from all kingdoms of life (György, 1956; Hellmann and Mooney, 2010; Kraemer et al., 2012; Eggersdorfer et al., 2012). Vitamin B6 collectively designates the water-soluble vitamers pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), and their respective phosphate esters pyridoxal 5-phosphate (PLP), pyridoxine 5-phosphate (PNP), and pyridoxamine 5-phosphate (PMP) (Fig. 1.1A; Rosenberg, 2012; Rosenberg et al., 2017). PLP is the most important vitamer serving as a cofactor for a plethora of proteins and enzymes (Mehta et al., 1993; Jansonius, 1998; Mehta and Christen, 2000; Christen and

Pharmaceutical Biocatalysis: Drugs, Genetic Diseases, and Epigenetics Edited by Peter Grunwald

Copyright © 2021 Jenny Stanford Publishing Pte. Ltd.

ISBN 978-981-4877-14-5 (Hardcover), 978-1-003-04541-0 (eBook)

www.jennystanford.com

2

Fermentative Production of Vitamin B6

Mehta, 2001; Eliot and Kirsch, 2004; Phillips, 2015). Estimations revealed that over 160 enzymes with distinct catalytic activities require vitamin B6 as a cofactor (about 4% of all described catalytic activities) (Percudani and Peracchi, 2009). Most of the PLP-dependent enzymes are involved in biosynthesis of amino acids, decarboxylation and racemization reactions, cleavage of Cα-Cβ bonds, α-, β- and γ-elimination or replacement reactions (John, 1995; Mehta and Christen, 2000; Christen and Mehta, 2001; Eliot and Kirsch, 2004). Moreover, PMP serves as a cofactor for enzymes of deoxysugar biosynthetic pathways (Burns et al., 1996; Romo and Liu, 2011). Furthermore, PLP modulates the activity of DNA-binding transcription factors in eukaryotes and prokaryotes (Oka et al., 2001; Huq et al., 2007; El Qaidi et al., 2013; Belitsky, 2004a; Belitsky, 2014; Tramonti et al., 2015, 2017 2018; Suvorova and Rodionov, 2016; Tramonti et al., 2017). The finding that about 1.5% of the genes of many free-living prokaryotes code for PLP-dependent proteins underlines the importance of the B6 vitamer for the function of proteins and catalytic enzymes (Percudani and Peracchi, 2003). There is also evidence that vitamin B6 is implicated in oxidative stress responses in plants (Bilski et al., 2000; Mooney and Hellmann, 2010; Moccand et al., 2014; Vanderschuren et al., 2013). Thus, vitamin B6 fulfils a variety of vital functions in different cellular processes (Fitzpatrick et al., 2007; Mooney et al., 2009; Vanderschuren et al., 2013; Parra et al., 2018). Animals and humans have to ingest vitamin B6 with their diet because these organisms are unable to synthesize the micronutrient (Fitzpatrick et al., 2007, 2010; Kraemer et al., 2012). Vitamin B6 limitation has been associated with neurological disorders such as epileptic encephalopathy due to inherited errors in the enzymes interconverting B6 vitamers in the socalled “salvage pathway” (Mills et al., 2005; Bagci et al., 2008; di Salvo et al., 2012). Vitamin B6 deficiency can also be caused by interactions between drugs, such as contraceptives, and enzymes of the salvage pathway (Lumeng et al., 1974; Lussana et al., 2003; di Salvo et al., 2011). Therefore, vitamin B6 is of commercial interest for improving the quality of the food and for applications in the pharmaceutical industry (Rosenberg et al., 2017; AcevedoRocha et al., 2019). In the food industry, the hydrochloride salt of

Introduction

the B6 vitamer PN is usually used in combination with other vitamins in a variety of food products (Domke et al., 2005; Eggersdorfer et al., 2012). Vitamin B6 is also added to the food that is used for intensive animal farming to improve animal health and to enhance the yield (Johnson et al., 1950; Verbeek, 1975; Eggersdorfer et al., 2012). Many studies report positive effects of vitamin B6 although a large number of commercial products contain this compound. Only a few studies revealed that vitamin B6 can be toxic. A recent case described photosensitive skin darkening, hyperemesis and diarrhea as toxic effects, which disappeared soon after intoxication stopped (Cupa et al., 2015). Moreover, long-time supplementation of PN in higher doses is known to cause sensory neuropathy (Schaumburg et al., 1983; Albin et al., 1987). This effect is also used as a model for neuropathy (Hong et al., 2009; Potter et al., 2014). So far the B6 vitamers are fully chemically synthesized via five different routes with variations, partly using expensive and/or toxic chemicals such as hydrogen cyanide, phosphorous pentoxide, and 1,4-butenediol (Pauling and Weimann, 1996; Kleemann et al., 2008; Eggersdorfer et al., 2012; Acevedo-Rocha et al., 2019). Several extensive attempts have been made by the biotech industry and academia to engineer microorganisms for vitamin B6 production by classical mutagenesis and by genetic modification (Pflug and Lingens, 1978; Ischikawa et al., 1997; Yocum et al., 2004; Hoshino et al., 2006a,b,c; Commichau et al., 2014, 2015; Rosenberg et al., 2017; Acevedo-Rocha et al., 2019). Unfortunately, none of the attempts were promising enough to establish an effective fermentation process (Rosenberg et al., 2017; Acevedo-Rocha et al., 2019). However, there is still considerable interest on the industrial side to shift from chemical synthesis processes to environmentally sustainable fermentation technologies (Acevedo-Rocha et al., 2019). This chapter summarizes recent findings regarding biosynthesis and homeostasis of vitamin B6 metabolism with a focus on prokaryotic microorganisms (bacteria). We also describe the approaches for developing microbial fermentation processes to produce vitamin B6 using microorganisms. Furthermore, we describe novel routes for vitamin B6 biosynthesis that have been discovered recently and discuss their potential for overproducing

3

4

Fermentative Production of Vitamin B6

the commercially valuable substance. Finally, we highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies that might help to circumvent these limitations to improve vitamin B6 production.

1.2 De novo Synthesis of Vitamin B6

Two non-homologous pathways for de novo vitamin B6 biosynthesis are known (Fig. 1.1B; Mittenhuber, 2001; Tanaka et al., 2005; Fitzpatrick et al., 2007, 2010; Rosenberg et al., 2017; Parras et al., 2018). The longer vitamin B6 biosynthetic pathway that was first discovered in the Gram-negative model bacterium Escherichia coli depends on the sugar deoxyxylulose 5-phosphate (DXP). The DXP-dependent pathway consists of two branches and seven enzymatic steps. In the longer branch of this pathway, the erythrose 4-phosphate (E4P) dehydrogenase (Epd), the 4-phosphoerythronate (4PE) dehydrogenase (PdxB and PdxR in E. coli and in the Gram-negative bacterium Sinorhizobium meliloti, respectively), and the 3-phosphoserine aminotransferase (SerC) convert E4P, which is derived from the pentose phosphate pathway, to 4-phosphohydroxy-L-threonine (4HTP) (Fig. 1.1B; Zhao et al., 1995; Drewke et al., 1996; Yang et al., 1998; Tazoe et al., 2006; Rudolph et al., 2010). 4HTP is then oxidized by the 4HTP dehydrogenase (PdxA) to 2-amino-3-oxo-4­ phosphohydroxy)butyric acid that spontaneously decarboxylates to 3-phosphohydroxy-1-aminoacetone (PHA) (Cane et al., 1998; Laber et al., 1999; Banks and Cane, 2004). The PNP synthase PdxJ produces the B6 vitamer PNP from PHA and DXP, of which the latter substrate is generated by the DXP synthase Dxs from glyceraldehyde 3-phosphate (G3P) and pyruvate in the short branch of the DXP-dependent vitamin B6 pathway (Cane et al., 1999; Laber et al., 1999). The final step is catalyzed by the PNP oxidase PdxH and yields in the biologically active B6 vitamer PLP (Fig. 1.1B; di Salvo et al., 1998, 2002, 2003). The DXPdependent vitamin B6 pathway has been intensively studied in E. coli. However, an in silico analysis revealed that it is only present in α- and γ-proteobacteria, who acquired it with the function of PdxB after the DXP-independent vitamin B6

De novo Synthesis of Vitamin B6

pathway had been lost in their ancestral lineage (Mittenhuber, 2001; Tanaka et al., 2005; Rosenberg et al., 2017). Additionally, the pathway was constituted by members of the α-proteobacteria who acquired pdxR, which is not homologous to pdxB, but fulϐils the same enzymatic function (Tazoe et al., 2006). It is also interesting to note that two enzymes of the DXP-dependent vitamin B6 route are involved in other metabolic pathways. SerC is essential for de novo biosynthesis of serine and the Dxs provides DXP as a precursor for thiamine and isoprenoids to the cell (Dempsey and Itoh, 1970; Sprenger et al., 1997).

Figure 1.1 (A) The B6 vitamers pyridoxal (PL), pyridoxal 5-phosphate (PLP), pyridoxine (PN), pyridoxine 5-phosphate (PNP), pyridoxamine (PM), and pyridoxamine 5-phosphate (PMP). PLP is the physiologically most-relevant B6 vitamer. (B) The deoxyxylulose 5-phosphate (DXP)dependent and DXP-independent vitamin B6 biosynthetic routes and the salvage pathway for the interconversion of the B6 vitamers. Epd, erythrose 4-phosphate dehydrogenase; PdxB (PdxR), 4-phosphoerythronate dehydrogenase; SerC, 3-phosphoserine aminotransferase; PdxA, 4phosphohydroxy-L-threonine dehydrogenase; PdxJ, PNP synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdxH, PNP oxidase; PdxS (PLP synthase subunit) and PdxT (glutaminase subunit) form the PLP synthase complex; PdxK, PL kinase present in B. subtilis and E. coli; PdxY, PL kinase present in E. coli. PdxK from B. subtilis has PN, PL, and PM kinase activity (Park et al., 2004).

5

6

Fermentative Production of Vitamin B6

The DXP-independent vitamin B6 biosynthetic pathway is much shorter because it involves only the PdxST enzyme complex, consisting of 12 PdxS and 12 PdxT subunits (Ehrenshaft and Daub, 2001; Belitsky, 2004b; Burns et al., 2005; Raschle et al., 2005; Strohmeier et al., 2006). PdxT is a glutaminase that converts glutamine to glutamate and ammonium, of which the latter serves as a substrate for the PLP synthase PdxS (Belitsky, 2004b). PdxS catalyses the reaction from ribulose 5-phosphate (Ru5P) and G3P to PLP. Due to triose and pentose isomerase activity, it can use either Ru5P or ribose 5-phosphate (Ri5P) together with either G3P or dihydroxyacetone phosphate (DHAP). Thus, the PdxS enzyme complex unifies three enzymatic activities: triose isomerase and pentose isomerase activity as well as imine formation activity for PLP synthesis (Burns et al., 2005). Even though the DXPindependent vitamin B6 pathway has been discovered a few years ago, it is more abundant in nature than the DXP-dependent route. It is present in archaea, bacteria, fungi, plants, and Plasmodium and in the sponge Suberites domuncula (Seack et al., 2001; Ehrenshaft and Daub, 2001; Fitzpatrick et al., 2007; Guédez et al., 2012; Rosenberg et al., 2017). Moreover, the DXP-independent vitamin B6 pathway emerged earlier and it has been lost several times in the course of evolution (Tanaka et al., 2005). Those organisms that are capable of producing vitamin B6 use either the DXP-independent of the DXP-dependent pathway. However, many organisms that synthesize vitamin B6 or need to ingest it possess a salvage pathway for the interconversion of the B6 vitamers (Fig. 1.1B; Fitzpatrick et al., 2007). For instance, E. coli and the Gram-positive model bacterium Bacillus subtilis synthesize the B6-vitamer kinase PdxK that phosphorylates PN, PM and PL (Dempsey and Pachler, 1966; Yang et al., 1996, 1998; White and Dempsey, 1970; di Salvo et al., 2004; Park et al., 2004). All those organisms carrying only the salvage pathway have to take up the vitamers from the environment. So far, only two vitamin B6 transporters have been in described in eukaryotes: Tpn1p in the fungus Saccharomyces cerevisiae, and PUP1 in Arabidopsis (Stolz and Vielreich, 2003; Szydlowski et al., 2013). The Tpn1p transporters are conserved in humans as well (Hediger et al., 2013). Surprisingly, even though the auxotrophy of bacteria for vitamin B6 can be relieved by supplementation

Control of Vitamin B6 Homeostasis

with non-phosphorylated B6 vitamers, the uptake systems are unknown in bacteria.

1.3 Control of Vitamin B6 Homeostasis

The two pathways for the biosynthesis of vitamin B6 and the salvage pathway have been intensively genetically studied (Rosenberg et al., 2017). Moreover, many of the involved enzymes have been biochemically and structurally studied in the past years (Mukherjee et al., 2011; Rosenberg et al., 2017). Unfortunately, little is known about the control of biosynthesis, recycling and degradation of vitamin B6. However, detailed knowledge about the control of vitamin B6 metabolism is required to facilitate the metabolic engineering of microorganisms for vitamin B6 overproduction. It is also poorly understood how PLP is delivered to the target proteins that require the vitamin for function. However, vitamin B6 biosynthesis has to be controlled because PLP is highly reactive and intermediates of the DXP-dependent pathway are toxic (Shames et al., 1984; Drewke et al., 1993; Farrington et al., 1993; Mizushina et al., 2003; Vermeersch et al., 2004; Commichau et al., 2015). For instance, PLP inhibits enzymes that are involved in DNA metabolism and in central metabolism in eukaryotes (Mizushina et al., 2003; Vermeersch et al., 2004; Lee et al., 2005). For E. coli it has been shown that the addition of vitamin B6 affects multiple metabolic pathways that are involved in amino acid biosynthesis (Vega and Margolin, 2017; Sugimoto et al., 2017). Thus, excess of vitamin B6 can negatively affect different cellular processes. The B6 vitamers PLP and PMP belong to the members of the 30 most damage-prone metabolites (Lerma-Ortiz et al., 2016). PLP is also prone to damage due to side reactions that are catalyzed by promiscuous enzymes or due to spontaneous chemical reaction (Linster et al., 2013). However, PLP is required for growth in only little amounts (Hartl et al., 2017). Therefore, PLP can be synthesized at a minimal necessary rate (Hartl et al., 2017). Probably, the perturbation of essential cellular processes is prevented by the low requirement of PLP and its low cellular concentration. However, PLP metabolism seems to be regulated in some organisms. In E. coli, a positive correlation between the expression of the pdxB and pdxA genes

7

8

Fermentative Production of Vitamin B6

and the growth rate has been reported (Pease et al., 2002). Probably, the amount of PdxB and PdxA enzymes has to be increased to meet the demand of vitamin B6 needed to achieve high growth rates, possibly due to its involvement in amino acid and thus protein biosynthesis. However, the molecular mechanism behind the growth rate-dependent regulation is still unknown. Recently, it has been demonstrated that vitamin B6 biosynthesis is positively controlled by the DNA-binding transcription factor PdxR (not to be confused with the PdxR enzyme from S. meliloti), which belongs to the MocR subfamily of transcriptional regulators. This regulation was shown for the Gram-positive bacteria Bacillus clausii, Corynebacterium glutamicum, Listeria monocytogenes, and Streptococcus mutans (Bramucci et al., 2011; Jochmann et al., 2011; Belitsky, 2014; Liao et al., 2014; Tramonti et al., 2015, 2017, 2018; Suvorova and Rodionov, 2016). At low PLP levels PdxR activates the expression of the divergently transcribed pdxST genes encoding the PLP synthase complex. By contrast, at increased PLP levels, the B6 vitamer acts as an inhibitor of the transcription factor PdxR, which then prevents its own synthesis (Belitsky, 2014). It remains to be elucidated whether de novo synthesis of PLP is controlled in other organisms lacking a homolog of the transcription factor PdxR. An excess of vitamin B6 might also be prevented by dephosphorylation and export. It has been shown that the S. meliloti strain IFO14782 possesses the phosphatase PdxP, which can dephosphorylates PNP and PLP (Tazoe et al., 2005; Nagahashi et al., 2008). Recently, the PLP phosphatase YbhA has been identified in E. coli (Saito et al., 2006; Sugimoto et al., 2017). The enzyme also displays in vitro phosphotransferase and phosphatase activity towards different sugars and sugar phosphates like fructose 1,6bisphosphate (Saito et al., 2006; Kuznetsova et al., 2006). However, the dephosphorylation of fructose 1,6-bisphosphate by YbhA does not seem to be physiologically relevant (Sugimoto et al., 2017). YbhA shows about 31% overall sequence identity with YitU from B. subtilis. YitU is a HAD phosphatase that has a minor activity in dephosphorylating the riboflavin precursor 5-amino6-ribiylamino-2,4(1H,3H)-pyrimidinedione 5-phosphate (Sarge et al., 2015). It will be interesting to evaluate whether the protein may act as a PNP/PLP phosphatase in B. subtilis. The transporter involved in the export of B6 vitamers also remains to be

Control of Vitamin B6 Homeostasis

identified. However, the PNP/PLP phosphatases together with the export system could be major players in controlling the intracellular vitamin B6 levels. Therefore, experiments targeting these important aspects of B6 homeostasis have to be pursued. Genetic screenings for PL-resistant mutants might reveal PL importers, while random mutagenesis approaches with selection for more PL-susceptible strains could shed light on export systems. Control of vitamin B6 homeostasis might also be achieved at the level of enzyme activity (di Salvo et al., 2011). For instance, the PNP oxidase PdxH and PN/PM/PL kinase PdxK from E. coli are regulated by PLP (White and Dempsey, 1970; Zhao and Winkler, 1995; Yang et al., 1996; Yang and Schirch, 2000; Fu et al., 2001; Ghatge et al., 2012). The B6 vitamers PL and PLP tightly bind to the lysine residue at the position 229 of PdxK, which inhibits the kinase (Ghatge et al., 2012; di Salvo et al., 2015). PLP also binds to a non-catalytic site of PdxH (Yang and Schirch, 2000). The PdxK-PLP and PdxH-PLP complexes could act as chaperones transferring the cofactor to PLP-requiring enzymes (Yang and Schirch, 2000; Ghatge et al., 2012). Indeed, PdxK specifically interacts with metabolic enzymes and the kinase can partially be recovered from the PdxK-PLP complex by transferring the PLP to an apo-B6 enzyme (Cheung et al., 2003; Ghatge et al., 2012). Similarly, the B. subtilis aspartate aminotransferase triggers hydrolysis and release of PLP from the PdxST enzyme complex, thus allowing the transfer of the highly reactive cofactor to a vitamin B6-dependent protein (Moccand et al., 2011). Similar to the E. coli biotin protein ligase, which is extraordinarily specific and attaches the cofactor to few proteins, the PLP-synthesizing enzymes might assure that the cofactor PLP is only attached to target proteins (Choi-Rhee et al., 2004). Alternatively, PLP bound to cellular amino acids could be the mechanism that directs the vitamin to the apo-enzymes (di Salvo et al., 2011). It remains to be elucidated whether the PdxHand PdxK-dependent or the amino acid-dependent transfer of vitamin B6 to the target proteins is physiologically relevant. Recently, it has been shown that YggS from E. coli is a PLPbinding protein, which belongs to a highly conserved protein family and exists in almost all kingdoms of life, including bacteria, fungi and animals (Ito et al., 2013). YggS shows about 33% overall sequence identity with YlmE protein of unknown

9

10

Fermentative Production of Vitamin B6

function from B. subtilis (Zhu and Stülke, 2017). The high conservation of this protein indicates that it must fulfill an important cellular function. Indeed, the lack of YggS in E. coli results in imbalance of PLP homeostasis, sensitivity towards PN and perturbation of biosynthesis of branched-chain amino acids (Prunetti et al., 2016). YggS might serve as a carrier that delivers PLP to apo-B6 proteins or fulfils a protective function in preventing inactivation of essential proteins by the modification of lysine residues. Recently, it has been reported that the YggS homolog from Streptomyces coelicolor plays a role in sporulation­ specific cell division (Zhang et al., 2018). However, the precise function of this important and abundant protein in controlling vitamin B6 homeostasis and other cellular processes remains to be determined. Finally, PLP homeostasis could be achieved by its degradation. Several years ago it was reported that bacteria like Mesorhizobium loti, Acetobacter rancens and some pseudomonads degrade vitamin B6 and use it as a carbon and nitrogen source for growth (Rodwell et al., 1958; Yoshikane et al., 2006; Mukherjee et al., 2011). As yet, two vitamin B6 degradation pathways have been described but the involved genes were only identified in the M. loti strain MAFF303099 (Mukherjee et al., 2011). Certainly, other pathways for the degradation of vitamin B6 will be identified in the future.

1.4 Engineering Microorganisms for the

Production of B6 Vitamers

Early attempts for the overproduction of vitamin B6 were performed with wild-type isolates of bacteria and fungi in the genera: Achromobacter, Bacillus, Flavobacterium, Klebsiella, Vibrio, and Kluveromyces, Pichia, and Simuliomyces, respectively (Table 1.1). Previously, a maximum of about 25 mg/L of B6 vitamers were obtained using the yeast Pichia guilliermondii NK-2 without genetic modification of the organism (Nishio et al., 1973). The first strain engineering approach for vitamin B6 production was reported about 15 years ago. The introduction and overexpression of the B. subtilis pdxST genes in E. coli resulted in the production of more than 60 mg/L of B6 vitamers within 48 h of cultivation (Yocum et al., 2004). The vitamin B6 production was further

Engineering Microorganisms for the Production of B6 Vitamers

enhanced to 78 mg/L within 31 h of cultivation by overexpressing the native epd, pdxJ, and dxs genes in E. coli (Hoshino et al., 2006a). The same group demonstrated that the S. meliloti strain IFO14782, a natural overproducer of vitamin B6, produces about 100 mg/L B6 vitamers within 168 h (Hoshino et al., 2006b). The overexpression of the native dxs gene and the E. coli epd gene in the S. meliloti strain increased the production of vitamin B6 to 1.3 g/L (Table 1.1; Hoshino et al., 2006b). Also B. subtilis, a bacterium that is widely used for industrial applications (Schallmey et al., 2004; van Tilburg et al., 2019), has been genetically engineered for vitamin B6 production using a heterologous DXP-dependent vitamin B6 pathway (AcevedoRocha et al., 2019). In a first attempt, the E. coli epd gene and the pdxR, serC, pdxA, and pdxJ genes from the S. meliloti strain IFO14782 strain were codon-optimized, assembled to artificial operons whose expression is driven by constitutively active promoters, and introduced in single copy into the B. subtilis chromosome (Commichau et al., 2014). The heterologous pathway was shown to be fully functional and the engineered bacteria produced about 41 mg/L PN within 72 h of incubation (Table 1.1). Unfortunately, the B. subtilis strain containing the non-native DXP-dependent vitamin B6 pathway was genetically unstable, which might be due to unbalanced expression of the enzymes of the heterologous pathway and accumulation of toxic intermediates (Commichau et al., 2014). A comparison of the kinetic properties of the enzymes of the DXP-dependent pathway shows that PdxA and PdxJ are catalytically inefficient enzymes (Table 1.2). Moreover, a quantitative proteomics analysis revealed that both enzymes were not very abundant in the engineered B. subtilis strain (Commichau et al., 2014). Unfortunately, the enzymes are required in high amounts to synthesize PNP from 4HTP, an intermediate that had been shown to be highly toxic for bacteria because it inhibits threonine and isoleucine biosynthesis (Shames et al., 1984; Drewke et al., 1993; Farrington et al., 1993; Kim et al., 2010; Commichau et al., 2014, 2015). Thus, the overexpression of the highly active Epd enzyme (Table 1.2), which together with the PdxR enzyme and SerC, generates 4HTP might be the reason for a strong selective pressure acting against the heterologous vitamin B6 pathway (Fig. 1.1B; Commichau et al., 2014).

11

pdxST-Bs

Escherichia coli

Pichia guilliermondii isolate NK-2

—

Flavobacterium

isolate

isolate

Vibrio sp. M31

Flavobacterium sp. 238-7

Mut

—

—

Bacillus subtilis

Achromobacter cycloclastes

Saccharomyces microsporus

—

—

Klebsiella sp.

Kluyveromyces marxianus

Genotypea

Enzyme

PL

Vitamin B6

Vitamin B6

Vitamin B6

Vitamin B6

Vitamin B6

Vitamin B6

Vitamin B6

Vitamerb

MM + yeast extract

Vitamin B6

MM + Hydrocarbon, Vitamin B6 yeast extract, Tween80

MM + CAA

Peptone, Glycerol, salts, Mn

Peptone, Glycerol, salts

MM

MM + CAA

MM

MM

MM + CAA

Medium

Table 1.1 Vitamin B6 production by natural isolates and engineered bacteria

>60

25

4

18

5

2–5

3–4

3

2

2

Titer [mg/L]

48

144–216

60

70

100

96

36–42

168

96

60

Time [h]

Yocum et al., 2004

Nishio et al., 1973

Suzue and Haruna, 1970

Tani et al., 1972

Tani et al., 1972

Pflug and Lingens, 1978

Ishida and Shimura, 1970

Scherr and Rafaelson, 1962

Pardini and Argoudelis, 1968

Suzue and Haruna, 1970

Reference

12 Fermentative Production of Vitamin B6

MM + yeast extract epd-Ec, pdxR-, serC-, pdxA-, pdxJSm

pdxA-Ec, pdxJ-Sm MM + amino acid cocktail + 4HT

Bacillus subtilis

Bacillus subtilis

MM + yeast extract

PN

PN

PN

PN

PN

PN

Vitamin B6

Vitamerb

65

41

1300

362

149

103

>78

Titer [mg/L]

72

72

168

168

216

168

31

Time [h]

Commichau et al., 2015

Commichau et al., 2014

Hoshino et al., 2006b

Hoshino et al., 2006c

Nagahashi et al., 2008

Hoshino et al., 2006b

Hoshino et al., 2006a

Reference

B6” refers to a mixture of vitamers (see Fig. 1.1A).

Bacillus subtilis; Ec, Escherichia coli; Sm, Sinorhizobium meliloti; Mut, classical mutagenesis; MM, minimal medium.

b“Vitamin

aBs,

epd-Ec, pdxJ-Sm

Sinorhizobium meliloti IFO14782

MM + yeast extract

Mut, pdxJ-Sm

Sinorhizobium meliloti IFO14782

MM + yeast extract

MM + yeast extract

pdxP-Sm, pdxJ-Ec

—

Sinorhizobium meliloti IFO14782

Sinorhizobium meliloti IFO14782

epd-, pdxJ-, dxs-Ec MM + yeast extract

Escherichia coli

Medium

Genotypea

Enzyme

Engineering Microorganisms for the Production of B6 Vitamers 13

OHPB, Glutamate

4HTP

DXP, PHA

Pyruvate, G3P

Escherichia coli

Escherichia coli

Escherichia coli

PdxA

PdxJ

Dxs

PdxH

Escherichia coli

PNP, PMP

PE

Escherichia coli

SerC

Sinorhizobium meliloti

PE, OG

Escherichia coli

PdxB

PdxR

EP

Escherichia coli

Substrate(s)

Epd

Enzyme Organism

DXP

PLP

PNP

AHP

4HTP

OHPB

OHPB

PE

Mg2+, Mn2+, TPP

FMN

n. a.

NAD, Mg2+

PLP

FAD

NAD

NAD

n. a.

PLP (–)

n. a.

n. a.

n. a.

n. a.

n. a.

n. a.

DXP-dependent pathway

96, 240

2, 105

26.9

85

110c

n. a.

2.9

0.8

0.76, 1.72

0.07

1.66

0.15d

n. a.

1.4

510–960, 20–200 74–800

Product(s) Cofactor(s) Effectors KM [µM]a kcat [s–1]b

Table 1.2 Published kinetic properties of enzymes involved in vitamin B6 metabolism

Cane et al., 2001; Kuzuyama et al., 2000

Zhao and Winkler, 1995

Cane et al., 1999

Cane et al., 1998, 1999; Laber et al., 1999

Drewke et al., 1996

Tazoe et al., 2006

Rudolph et al., 2010

Zhao et al., 1995; Boschi-Muller et al., 1997

References

14 Fermentative Production of Vitamin B6

Mg2+, Mn2+

n. a. n. a.

n. a.

385

n. a.

n. a.

n. a.

Tazoe et al., 2005

Saito et al., 2006; Kuznetsova et al., 2006; Sugimoto et al., 2017

for the substrates/cofactors. b Assay conditions differ for the enzymes. creverse reaction was analysed. n. a., not analyzed.

PN, PL

PL

aAffinities

PdxY

Yang et al., 1998; di Salvo et al., 2004

Park et al., 2004

PNP, PLP

n. a.

0.032

White and Dempsey, 1970; di Salvo et al., 2004; Ghatge et al., 2012; di Salvo et al., 2015

Sinorhizobium meliloti

n. a.

46.6

2.34 (0.34, 0.67)

PdxP

n. a.

n. a.

100, (25, 30)

PLP

Mg2+

Mg2+

PL, PLP

0.33 × 10–3 Raschle et al., 2005

Escherichia coli

PLP

PLP

Mg2+

68, 77, 990

YbhA

PL (PN, PM), ATP

Bacillus subtilis

PdxK

PLP, (PNP, PMP)

n. a.

Salvage pathway

n. a.

PL

PL (PN, PM), ATP

Escherichia coli

PdxK

PLP

Escherichia coli

R5P, G3P, Glutamine

Bacillus subtilis

PdxST

DXP-independent pathway

Engineering Microorganisms for the Production of B6 Vitamers 15

16

Fermentative Production of Vitamin B6

Inhibitory interactions have also been observed between novel vitamin B6 metabolic routes and the native metabolic network of E. coli (Kim and Copley, 2012). Thus, the expression levels of the enzymes of a heterologous or a novel vitamin B6 pathway need to be fine-tuned to prevent the accumulation of intermediates to toxic levels. An alternative approach could be to adapt the host organisms to the heterologous pathway through continuous growth under selection for PL production. B. subtilis has also been engineered for the conversion of 4-hydroxy-L-threonine (4HT) to the B6 vitamer PN (Commichau et al., 2015). The conversion of 4HT to PN by B. subtilis required the genomic adaptation of the bacteria because like 4HTP also 4HT interferes with threonine and isoleucine biosynthesis (Westley et al., 1971; Katz et al., 1974; Drewke et al., 1993; Kim et al., 2010; Commichau et al., 2014; Rosenberg et al., 2016). The evolved B. subtilis strains had acquired mutations in (1) branched chain amino acid transporters that probably reduce uptake rate of 4HT from the medium and (2) the Phom promoter, resulting in the de-regulation of threonine biosynthesis, thus endogenous resistance to 4HT (Commichau et al., 2015; Rosenberg et al., 2016). The evolved bacteria expressing the native thrB gene, which encodes the promiscuous homoserine kinase ThrB that converts 4HT to 4HTP (Fig. 1.2), and the E. coli pdxA and S. meliloti pdxJ genes produced 65 mg/L PN from 120 mg/L 4HT (Commichau et al., 2015). Surprisingly, although 4HT was completely consumed by the engineered bacteria they did not fully convert it to PN. This observation suggests that unknown host metabolic pathways compete with the heterologous pathway for 4HT. Given the fact that the various attempts to produce industrially significant levels of B6 vitamers were unsuccessful, it is evident that we know too little about vitamin B6 function, toxicity and the regulation of its metabolism. However, there is potential for strain optimization because the improvement of a S. meliloti wild type strain producing 103 milligrams per liter within 168 h to 1.3 grams per liter could be achieved by only overproducing two genes of the B6 synthesis pathway. To overcome the hurdles and to identify the rate-limiting bottlenecks, we will need the information about the flow of metabolites into and out of the relevant synthetic pathways. This is because the production of

Novel Routes for Vitamin B6 Biosynthesis and Production

B6 in large amounts will deplete significant amounts of metabolites from glycolysis and the pentose phosphate pathway, and also cause the accumulation of toxic intermediates. Therefore, metabolic flux analysis needs to be performed as has been done for an N-acetylglucosamine producing B. subtilis strain (Liu et al., 2016).

1.5 Novel Routes for Vitamin B6 Biosynthesis and Production

Reactions in the cellular metabolic networks may occur spon­ taneously and independent of metabolic enzymes (Piedrafita et al., 2015). Moreover, many enzymes have promiscuous activities that are not required for the primary function of the protein and generally serve no purpose (Khersonsky and Tawfik, 2010). The promiscuous enzymes can cause a so-called “underground metabolism” and they may either act on substrates for which they evolved or on structurally related compounds (D’Ari and Casadeus, 1998; Notebaart et al., 2018). The non-enzymatic reactions as well as promiscuous enzymes are important for the evolution of metabolic pathways (Jensen, 1976; Copley, 2012; Schulenburg and Miller, 2014; Rosenberg and Commichau, 2019). About 37% of the E. coli enzymes were estimated to act on a variety of substrates and catalyze 65% of the known metabolic reactions (Nam et al., 2012). Thus, enzyme promiscuity might be an excellent starting point for the evolution of novel catalysts (Khersonsky and Tawfik, 2010; Copley, 2012). Indeed, the overexpression of native enzymes with promiscuous activities generated so-called serendipitous vitamin B6 pathways satisfy­ ing the PLP auxotrophy of an E. coli pdxB mutant strain (Cooper, 2010; Kim et al., 2010). Interestingly, although the serendipitous pathways divert intermediates from other metabolic pathways, they were shown to feed in into the native DXP-dependent PLP biosynthetic route (Fig. 1.2). A detailed characterization of one of the three pathways, which were identified in E. coli, revealed that the four enzymes SerA, YeaB, LtaE and ThrB connect serine to vitamin B6 biosynthesis by conversion of 3-phosphoglycerate to 4HTP through 3-phosphohydroxypyruvate, 3-hydroxypyruvate, glycolaldehyde, and 4HT. (Fig. 1.2) (Kim et al., 2010; Kim and

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18

Fermentative Production of Vitamin B6

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