BIOCATALYSIS FOR THE PHARMACEUTICAL INDUSTRY Discovery, Development, and Manufacturing

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1 BIOCATALYSIS FOR THE PHARMACEUTICAL INDUSTRY Discovery, Development, and Manufacturing Junhua (Alex) Tao Elevance Renewable Sciences, USA Guo-Qiang Lin Shanghai Institute of Organic Chemistry, CAS, China Andreas Liese Hamburg University of Technology, Germany

3 BIOCATALYSIS FOR THE PHARMACEUTICAL INDUSTRY

5 BIOCATALYSIS FOR THE PHARMACEUTICAL INDUSTRY Discovery, Development, and Manufacturing Junhua (Alex) Tao Elevance Renewable Sciences, USA Guo-Qiang Lin Shanghai Institute of Organic Chemistry, CAS, China Andreas Liese Hamburg University of Technology, Germany

6 Copyright # 2009 John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop, # 02-01, Singapore Visit our Home Page on All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as expressly permitted by law, without either the prior written permission of the Publisher, or authorization through payment of the appropriate photocopy fee to the Copyright Clearance Center. Requests for permission should be addressed to the Publisher, John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop, # 02-01, Singapore , tel: , fax: , enquiry@wiley.com. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book. All trademarks referred to in the text of this publication are the property of their respective owners. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Other Wiley Editorial Offices John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA , USA Wiley-VCH Verlag GmbH, Boschstrasse 12, D Weinheim, Germany John Wiley & Sons Australia Ltd, 42 McDougall Street, Milton, Queensland 4064, Australia John Wiley & Sons Canada Ltd, 5353 Dundas Street West, Suite 400, Toronto, ONT, M9B 6H8, Canada Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Library of Congress Cataloging-in-Publication Data Tao, Junhua. Biocatalysis for the pharmaceutical industry: discovery, development, and manufacturing/junhua Tao, Guo-Qiang Lin, Andreas Liese. p. cm. Includes bibliographical references and index. ISBN (cloth) 1. Pharmaceutical biotechnology. 2. Enzymes Biotechnology. I. Lin, Guo-Qiang. II. Liese, A. (Andreas) III. Title. RS380.T dc ISBN (HB) Typeset in 10/12pt Times by Thomson Digital, Noida, India. Printed and bound in Singapore by Markono Print Media Pte Ltd, Singapore. This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production.

7 Contents Preface xi 1 Enzymes and Their Synthetic Applications: An Overview Introduction Enzyme Families Enzyme Discovery and Optimization Enzyme Production Enzymes and Synthetic Applications Ketoreductases (EC ) Enoate Reductases or Ene Reductases (EC ) Oxygenases (EC. xxxx) Alcohol Oxidases (EC X) Peroxidases (EC X) Halogenases (EC. xxxx) Nitrilases (EC ) Nitrile Hydratases (EC ) Epoxide Hydrolases (EC X) !-Transaminases (EC X) Hydroxynitrile Lyases (EC X) Aldolases (EC. xxxx) Glycosidases (EC. xxxx) Glycosyltransferase (EC. xxxx) Conclusions 15 2 Expression Hosts for Enzyme Discovery and Production Introduction How to Choose an Expression System Prokaryotic Expression Systems Posttranslational Modification in Prokaryotes Escherichia coli Bacilli Pseudomonas fluorescens Other Prokaryotic Expression Systems 28

8 vi Contents 2.4 Eukaryotic Expression Systems Yeasts Filamentous Fungi Insect/Baculovirus System Mammalian Cell Cultures Other Expression Systems Cell-Free Expression Systems Conclusions 37 3 Directed Enzyme Evolution and High-Throughput Screening Introduction Directed Evolution Library Creation Strategies Random and Semi-Rational Mutagenesis Gene Shuffling Directed Evolution Library Screening/Selection Methods In Vivo Methods: Genetic Complementation In Vivo Methods: Chemical Complementation In Vivo Methods: Surface Display In Vitro Methods: Lysate Assay In Vitro Methods: Ribosome Display In Vitro Methods: In Vitro Compartmentalization Equipment/Automation Selected Industrial Examples Activity Thermostability Substrate Specificity Product Specificity Enantioselectivity Conclusions and Future Directions 60 4 Applications of Reaction Engineering to Industrial Biotransformations Introduction Metabolic Bioconversion Enzymatic Biotransformations Cofactor Regeneration Racemic Mixtures Equilibrium Conversion By-Product Formation Substrate Inhibition Low Solubility Conclusions 83 5 Chiral Synthesis of Pharmaceutical Intermediates Using Oxynitrilases Introduction HNL The Natural Function and Distribution of HNLs 89

9 Contents vii Classification of HNLs New HNLs and High-Throughput Screening Reaction of HNLs Reaction System Immobilization of Enzyme Continuous Reactors Henry Reaction Transformation of Cyanohydrins Transformation of Hydroxyl Group Transformation of Nitrile Group Intramolecular Reaction Summary Expanding the Scope of Aldolases as Tools for Organic Synthesis Directed Evolution and Rational Mutagenesis Reaction Engineering Broad Substrate Tolerance of Wild-Type Aldolases Conclusions Synthetic Applications of Ketoreductases and Alcohol Oxidases Ketoreductases Wild-Type Whole-Cell Biocatalysts Recombinant Whole-Cell Biocatalysts Overexpressing Catalytic Enzymes Isolated Enzyme Alcohol Oxidases Primary Alcohol Oxidases Secondary Alcohol Oxidases Applications of Nitrile Hydratases and Nitrilases Introduction NHase New NHases Applications Nitrilase New Nitrilases Applications Conclusions Biosynthesis of Drug Metabolites Introduction Metabolite Synthesis Using Mammalian Bioreactors Selection of In Vitro Systems Reaction Condition Optimization Large Scale Incubations 190

10 viii Contents Examples with Mammalian Bioreactors In Vivo Samples Metabolite Synthesis Using Microbial Bioreactors Microbial Bioreactors Used in Metabolite Structure Elucidation Microbial Bioreactors Used in Synthesis of Key Metabolites Strain Selection Microbial Glycoside Conjugation Large Scale Reactions Examples for Metabolite Synthesis with a Microbial Bioreactor Recombinant Enzyme Bioreactors Advantages of Using CYP Enzymes for Producing Drug Metabolites Human Cytochrome Biocatalysts Microbial CYP Enzymes Summary Application of Whole-Cell Biotransformation in the Pharmaceutical Industry Introduction Whole-Cell Biotransformation Processes Used in Commercial Production of Pharmaceuticals Application of Whole-Cell Biotransformation Process in the Synthesis of Chiral Pharmaceutical Intermediates Disadvantages of Whole-Cell Process Compared with the Isolated Enzyme Process Substrate Availability and Recovery of Products in Low Concentrations Undesirable Side Reactions Toxicity of Substrate and Product Advantages of Whole-Cell Process Compared with the Isolated Enzyme Process More Stable Sources than Isolated Enzymes Regeneration of Cofactors and Multi-Enzymes Reactions Diversity and Availability Reactions with Non-Commercially Available Isolated Enzymes for Preparative Scale Synthesis Cost Effectiveness and Ease of Operation Approaches to Address the Disadvantages of Whole-Cell Biotransformation Control of Substrate and Product Concentration by Absorbing Resins Immobilized-Cell Technology Aqueous Organic Two-Phase System Genetic Engineering Approaches Conclusions 224

11 Contents ix 11 Combinatorial Biosynthesis of Pharmaceutical Natural Products Introduction Combinatorial Biosynthesis: The Natural Way for Structural Diversity Examples of Combinatorial Biosynthesis of Pharmaceutical Natural Products Erythromycin (Polyketide Biosynthesis) Daptomycin (Nonribosomal Peptide Biosynthesis) Patellamide (Ribosomal Peptide Biosynthesis) Summary and Perspectives Metabolic Engineering for the Development and Manufacturing of Pharmaceuticals Introduction Metabolic Engineering Tools Tools for the Cellular Metabolic Network Analysis Tools for Rational Genetic Modification Metabolic Engineering for the Development and Production of Polyketide Pharmaceuticals Biosynthesis of Polyketides Metabolic Engineering for Improved Erythromycin Production Metabolic Engineering for Overproduction of 6dEB in Heterologous Hosts Metabolic Engineering of Other Polyketides Development of Novel Polyketides for Drug Discovery Metabolic Engineering for the Production of -Lactam Metabolic Engineering for Isoprenoid Production Biosynthesis Pathway of Isoprenoids Metabolic Engineering for Enhancing Precursor Supply for Isoprenoid Production Metabolic Engineering for Artemisinine Development and Production Metabolic Engineering for Carotenoid Production Metabolic Engineering for Taxol Development and Production Conclusions Multimodular Synthases and Supporting Enzymes for Chemical Production Introduction Background Multimodular Synthase Architecture Natural Product Biosynthetic Cycle Metabolic Engineering of Megasynthases Daptomycin: Metabolic Engineering by Domain Swap Avermectin: Metabolic Engineering by Directed Fermentation 281

12 x Contents 13.4 Excised Domains for Chemical Transformations Function of Individual Domains, Domain Autonomy Cyclization Halogenation Heterocyclization/Aromatization Methylation Oxygenation Glycosylation Conclusions Green Chemistry with Biocatalysis for Production of Pharmaceuticals Introduction Enzymatic Resolutions: Higher Yields, Less Waste Bioreductions: Greener Ligands, Renewable Hydride Donors, No Metals Enzymatic Oxidations: Clean, Highly Selective and Catalytic C C Bond Formations: Atom Efficiency at Its Best Summary and Outlook 318 Index 323

13 Preface Biocatalysis is evolving to be a transformational technology as a result of a confluence of factors, which include (1) large scale and ever increasingly cost-efficient DNA sequencing technologies; (2) exponential growth in GenBank; (3) powerful directed enzyme evolution and high-throughput screening technologies; (4) robust expression systems for enzyme production; (5) deep understanding of the logic of natural product biosynthesis; (6) industrial successes of metabolic engineering and pathway engineering. Consequently, many successful stories and a number of reviews have been reported recently in developing biocatalysis for the pharmaceutical industry, across drug discovery, development, and manufacturing. The book is dedicated to these advances, and divided into four parts:. Chapters 1 4 serve as an introduction to emerging biocatalysts, modern expression hosts, state of the art of directed evolution, high-throughput screening, and bioprocess engineering for industrial applications.. Chapters 5 8 are directed to emerging enzymes, which include oxynitrilases, aldolases, ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications especially in synthesis of chiral drugs and intermediates.. Chapters 9 and 10 focus on synthesis of drug metabolites and intermediates catalyzed by P450s or whole cells.. Chapters are devoted to combinatorial biosynthesis, metabolic engineering, and autonomous enzymes for the synthesis and development of complex medicinal molecules.. Chapter 14 discusses the recent impact of biocatalysis in green chemistry and chemical development. Our main goal is to come up with a concise but comprehensive, practical but insightful book covering the topics discussed above. We hope you enjoy reading this book. Any suggestions and comments are welcome. Junhua (Alex) Tao Elevance Renewable Sciences, USA Junhua_tao@yahoo.com Guo-Qiang Lin Guo-Qiang Lin Shanghai Institute of Organic Chemistry, China lingq@mail.sioc.ac.cn Andreas Liese Hamburg University of Technology, Germany liese@tuhh.de

15 1 Enzymes and Their Synthetic Applications: An Overview Junhua (Alex) Tao 1 and Jian-He Xu 2 1 Elevance Renewable Sciences, 175 E. Crossroads Parkway, Bolingbrook, IL 60440, USA 2 East China University of Science and Technology (ECUST) 130 Meilong Road, Shanghai, , PR China 1.1 Introduction Whole-cell biocatalysis has been exploited for thousands of years; for example, in preparing barley for beer brewing. While the chemical, economic and social advantages of biocatalysis over traditional chemical approaches were recognized a long time ago, their applications for the drug industry have been largely underexplored until the recent technological breakthroughs in large-scale DNA sequencing, robust protein expression systems, metabolic engineering and directed evolution. In this chapter, emphasis will be directed to the discussion of those isolated enzymes which are uniquely suited for the synthesis of small-molecule pharmaceutical ingredients. 1.2 Enzyme Families Based on reactions they catalyze, enzymes can be broadly classified into six major categories (Table 1.1) [1]. It was estimated that about 60% of biotransformations currently rely on the use of hydrolases, followed by 20% of oxidoreductases [2]. On the other hand, some of the C C bond-forming and oxygenation enzymes catalyze reactions with very high reaction efficiency and very low waste generation, underlining the potential of emerging enzymes. Biocatalysis for the Pharmaceutical Industry Ó 2009 John Wiley & Sons Asia (Pte) Ltd edited by Junhua Tao, Guo-Qiang Lin, and Andreas Liese

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