ENZYME TECHNOLOGIES

CHEMICAL BIOLOGY OF ENZYMES FORBIOTECHNOLOGY AND PHARMACEUTICAL

APPLICATIONS

(A Series Consisting of Three Volumes)

Volume I. Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, andBiosynthesisEditors: Wu-Kuang Yeh, Hsiu-Chiung Yang, and James R. McCarthy

Volume II. Enzyme Technologies in Drug DiscoveryEditors: Hsiu-Chiung Yang, Wu-Kuang Yeh, and James R. McCarthy

Volume III. Design of Enzyme Inhibitors for TherapeuticsEditors: James R. McCarthy, Hsiu-Chiung Yang, and Wu-Kuang Yeh

ENZYME TECHNOLOGIES

Metagenomics, Evolution, Biocatalysis,and Biosynthesis

Edited by

WU-KUANG YEHPreClinOmics, Inc., Indianapolis, Indiana

HSIU-CHIUNG YANGEli Lilly and Company, Indianapolis, Indiana

JAMES R. MCCARTHYIndiana University–Purdue University, Indianapolis, Indiana

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright 2010 by John Wiley & Sons, Inc. All rights reserved.

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Library of Congress Cataloging-in-Publication Data:

Enzyme technologies: metagenomics, evolution, biocatalysis, and biosynthesis / edited by Wu-Kuang Yeh, Hsiu-Chiung Yang and James R. McCarthy.

p. cm.Includes index.Summary: Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis

highlights how, what, and where enzymes have become critical in biotechnology andpharmaceutical applications. This book provides in-depth reviews of metagenomics, naturalbiodiversity, directed enzyme and pathway evolution, as well as enzyme optimization in thediscovery of novel enzymes and natural products. The book also discusses biocatalysis principleand applications in “green chemistry” for developing and producing active pharmaceuticalingredients with significant economical and environmental benefits. In addition, this volume dealswith applications involving combinatorial biosynthesis and pathway and enzyme engineering toproduce novel bioactive compounds, as well as to improve yields of natural and modified products.– Provided by publisher.

ISBN 978-0-470-28624-1 (hardback)1. Enzymes–Biotechnology. 2. Pharmaceutical biotechnology. I. Yeh, Wu-Kuang, 1942–

II. Yang, Hsiu-Chiung. III. McCarthy, James R., 1943–TP248.65.E59E59144 2010660.6′34–dc22

2010022384Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors vii

Preface ix

PART A NEW APPROACHES TO FINDING ANDMODIFYING ENZYMES 1

1 Functional Metagenomics as a Technique for the Discovery ofNovel Enzymes and Natural Products 3Luke A. Moe, Matthew D. McMahon, and Michael G. Thomas

2 Directed Enzyme and Pathway Evolution 41Jacob Vick and Claudia Schmidt-Dannert

3 Combining Natural Biodiversity and Molecular-DirectedEvolution to Develop New Industrial Biocatalysts and Drugs 77Laurent Fourage, Celine Ayrinhac, Johann Brot, Christophe Ullmann,Denis Wahler, and Jean-Marie Sonet

4 Principles of Enzyme Optimization for the Rapid Creation ofIndustrial Biocatalysts 99Richard J. Fox and Lori Giver

v

vi CONTENTS

PART B BIOCATALYTIC APPLICATIONS 125

5 Enzyme Catalysis in the Synthesis of Active PharmaceuticalIngredients 127Animesh Goswami

6 Enzymatic Processes for the Production of PharmaceuticalIntermediates 185David Rozzell and Jim Lalonde

7 Novel Developments Employing Redox Enzymes: OldEnzymes in New Clothes 199Kurt Faber, Silvia M. Glueck, Birgit Seisser, and Wolfgang Kroutil

PART C BIOSYNTHETIC APPLICATIONS 251

8 Drug Discovery and Development by CombinatorialBiosynthesis 253Matthew A. DeSieno, Carl A. Denard, and Huimin Zhao

9 Reprogramming Daptomycin and A54145 Biosynthesis toProduce Novel Lipopeptide Antibiotics 285Richard H. Baltz, Kien T. Nguyen, and Dylan C. Alexander

10 Pathway and Enzyme Engineering and Applications forGlycodiversification 309Lishan Zhao and Hung-wen Liu

Index 363

CONTRIBUTORS

Dylan C. Alexander, Cubist Pharmaceuticals, Inc., Lexington, Massachusetts

Celine Ayrinhac, Proteus, Nımes, France

Richard H. Baltz, Cubist Pharmaceuticals, Inc., Lexington, Massachusetts

Johann Brot, Proteus, Nımes, France

Carl A. Denard, Department of Chemical and Biomolecular Engineering andInstitute for Genomic Biology, University of Illinois at Urbana-Champaign,Urbana, Illinois

Matthew A. DeSieno, Department of Chemical and Biomolecular Engineer-ing and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois

Kurt Faber, Department of Chemistry, Organic and Bioorganic Chemistry, Uni-versity of Graz, Graz, Austria

Laurent Fourage, Proteus, Nımes, France

Richard J. Fox, Codexis, Inc., Redwood City, California

Lori Giver, Codexis, Inc., Redwood City, California

Silvia M. Glueck, Department of Chemistry, Organic and Bioorganic Chemistry,University of Graz, Graz, Austria

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viii CONTRIBUTORS

Animesh Goswami, Process Research and Development, Bristol-Myers Squibb,New Brunswick, New Jersey

Wolfgang Kroutil, Department of Chemistry, Organic and Bioorganic Chemistry,University of Graz, Graz, Austria

Jim Lalonde, Codexis, Inc., Redwood City, California

Hung-wen Liu, Division of Medicinal Chemistry, College of Pharmacy, andDepartment of Chemistry and Biochemistry, University of Texas at Austin,Austin, Texas

Matthew D. McMahon, Department of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin

Luke A. Moe, Department of Plant and Soil Sciences, University of Kentucky,Lexington, Kentucky

Kien T. Nguyen, Cubist Pharmaceuticals, Inc., Lexington, Massachusctts

David Rozzell, Sustainable Chemistry Solutions, Burbank, California

Claudia Schmidt-Dannert, Department of Biochemistry, Molecular Biology andBiophysics, University of Minnesota, St. Paul, Minnesota

Birgit Seisser, Department of Chemistry, Organic and Bioorganic Chemistry,University of Graz, Graz, Austria

Jean-Marie Sonet, PCAS Biosolution, Longjumeau, France

Michael G. Thomas, Department of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin

Christophe Ullmann, Proteus, Nımes, France

Jacob Vick, Department of Biochemistry, Molecular Biology and Biophysics,University of Minnesota, St. Paul, Minnesota

Denis Wahler, Proteus, Nımes, France

Huimin Zhao, Department of Chemical and Biomolecular Engineering and Insti-tute for Genomic Biology, and Departments of Chemistry, Biochemistry, andBioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois

Lishan Zhao, Amyris Biotechnologies, Emeryville, California

PREFACE

The continuous improvement of human health and quality of life can be linkeddirectly to the discovery, development, manufacture, and applications of pharma-ceutical agents. Although many drugs for various human diseases are available,there are still numerous unmet medical needs, providing plenty of opportunitiesto researchers in the pharmaceutical and biotechnology industries. Drug discov-ery is a high-risk and potentially high-reward endeavor, costing approximately$1 billion in recent years for a new drug to reach the marketplace. Enzymes andtheir multiple applications play a critical role, both in vitro and in vivo, in thediscovery and development process for most new therapeutic agents. To assistresearchers in taking advantage of practical enzyme tools and strategies, one ofthe current editors coordinated the publication of a previous enzyme-based drugdiscovery book: Enzyme Technologies for Pharmaceutical and BiotechnologicalApplications (HA Kirst, WK Yeh, and MJ Zmjewski, eds., Marcel Dekker, NewYork, 2001). With respect to the field of enzymes for drug discovery, there havebeen significant and exciting advances in the first decade of the twenty-first cen-tury, and the current editors are pleased to produce the first in a series of threevolumes on Chemical Biology of Enzymes for Biotechnology and PharmaceuticalApplications.

The three editors have a combined pharmaceutical discovery experience ofover 60 years. Through several years of a highly collaborative effort, the edi-tors anticipate providing the three unique enzyme-focused books soon: Volume I,Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis ;Volume II, Enzyme Technologies in Drug Discovery ; and Volume III, Design ofEnzyme Inhibitors for Therapeutics . The book chapters in each Enzyme Technolo-gies volume, contributed by highly experienced biotechnology and pharmaceuti-cal scientists, present many key enzyme areas that are critical for drug discovery,

ix

x PREFACE

development, and production. Thus, for all potential and practicing researchers,from beginners to experts, the three enzyme-based volumes are unique and infor-mative for both training and improving enzyme skills and strategies for drugdiscovery, development, and manufacture.

The first volume consists of three parts: A, New Approaches to Finding andModifying Enzymes; B, Biocatalytic Applications; and C, Biosynthetic Applica-tions. In Part A, Moe, McMahon, and Thomas describe functional metagenomicsas a technique for the discovery of novel enzymes and natural products inChapter 1. The chapter focuses on the emerging field of metagenomics and appli-cations for identification of novel enzymes and natural products from a full DNAcontent of soil-dwelling microbes. Next, in Chapter 2, Vick and Schmidt-Dannertdescribe directed enzyme and pathway evolution. The authors present the prac-tical applications of directed evolution to enzymes and pathways, discuss thetolerance of enzymes for multiple mutations and the potential benefits of neutraldrift and “adaptive evolution” and describe in vitro evolution of one or moremetabolic functions in assembled pathways, allowing the synthesis of new iso-prenoid or acetate-derived natural products. In Chapter 3, Fourage, Sonet, andcolleagues discuss combining natural biodiversity and molecular-directed evo-lution to develop new industrial biocatalysts and drugs. The authors exploreapproaches to the discovery and design of biocatalysts based on the combineduse of biodiversity screening and molecular-directed evolution, and the impactof these approaches in drug development. Part A is completed with Chapter 4 byFox and Giver on the principles of enzyme optimization for the rapid creation ofindustrial biocatalysts. The authors discuss the critical interplay of three orthog-onal aspects for efficient enzyme optimization: the fitness function, diversitygeneration, and the search algorithm.

In Part B, biocatalytic applications can be considered “green chemistry” forvery significant economical and environmental benefits in developing and produc-ing key pharmaceutical ingredients. Chapter 5, by Goswami, on enzyme catalysisin the synthesis of active pharmaceutical ingredients, based on the high selectivityof enzymatic transformations, provides an extensive review of specific applica-tions of different reactions in producing active pharmaceutical ingredients andpotential benefits with associated issues. Chapter 6, by Rozzell and Lalonde,deals with enzymatic processes for the production of pharmaceutical interme-diates. Examples are given of ketoreductase-based methods for the productionof key precursors of two blockbuster drugs (atorvastatin, trade name Lipitor,Pfizer; and montelukast, trade name Singulair, Merck), providing higher stereo-chemical purity of the final product and dramatic reductions in solvent use andwaste. Chapter 7, by Faber, Glueck, Seisser, and Kroutil, covers novel devel-opments employing redox enzymes. The authors provide an overview of recentdevelopments employing enzymes in organic synthesis and focus on dehydroge-nases for the reduction of sterically demanding ketones. Also covered are clonedenoate reductases from the “old yellow enzyme family” as popular catalysts forasymmetric reduction of activated alkenes.

PREFACE xi

Part C deals with applications involving the modification of enzymes andpathways for producing novel pharmaceutical intermediates and products aswell as for improving yields of natural and modified products. Chapter 8, byDeSieno, Denard, and Zhao, provides an extensive overview of drug discoveryand development by combinatorial biosynthesis. The chapter highlights somepast accomplishments, exemplified by major efforts in polyketide synthases andnonribosomal peptide synthases, as well as recent advances in combinatorialbiosynthesis, including new tools for manipulating biosynthetic pathways andan expanding list of heterologous hosts for the production of improved drugs.Chapter 9, by Baltz, Nguyen, and Alexander describes the reprogramming ofdaptomycin and A54145 biosynthesis to produce novel lipopeptide antibiotics.The chapter is an extensive review on applying combinatorial biosynthesis amongmultiple compatible hosts for generating many new derivatives of daptomycinand A54145, and some that have improved properties relative to daptomycin andA54145. Chapter 10, the final chapter, by Zhao and Liu covers pathway andenzyme engineering and applications for glycodiversification. The authors reportthat numerous promiscuous sugar biosynthetic enzymes and their correspondingglycosyltransferases toward alternative substrates have facilitated efforts to cre-ate novel chemical entities with altered sugar structures via pathway and enzymeengineering and thus highlight the great potential of glycodiversification as aneffective strategy for development of new therapeutic agents in drug discovery.

The editors graciously acknowledge outstanding contributions by two scien-tific consultants to the first volume of the Enzyme Technologies series. HerbKirst and Milt Zmijewski, co-editors of the earlier Enzyme Technologies book,were extremely helpful in identifying and recommending the most appropriateenzyme topics and authors for inclusion in this volume. Also, Dr. Zmijewskiwas involved extensively in reviewing the entire manuscript. Without their sig-nificant assistance, the content of the volume would be much less satisfactoryand timely. The editors are thankful to all the authors for close and in somecases time-consuming collaboration in multiple reviews toward producing thehighest-quality chapter manuscripts possible with consistent formats. The edi-tors are pleased by the agreement, suggestions, and encouragement from thepublishers to produce a truly unique and potentially highly useful Enzyme Tech-nologies series to benefit current and future researchers for drug discovery anddevelopment.

WU-KUANG YEHPreClinOmics, Inc.HSIU-CHIUNG YANGEli Lilly and Company

JAMES R. MCCARTHYIndiana University–Purdue UniversityIndianapolis, Indiana

PART A

NEW APPROACHES TO FINDINGAND MODIFYING ENZYMES

1