ENGINEERING COMPLEX PHENOTYPES IN INDUSTRIAL STRAINS

ENGINEERING COMPLEX PHENOTYPES IN INDUSTRIAL STRAINS

Edited by

RANJAN PATNAIK

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Engineering complex phenotypes in industrial strains / edited by Ranjan Patnaik. p. cm. Includes bibliographical references and index. ISBN 978-0-470-61075-6 (hardback) 1. Industrial microorganisms. 2. Genetic engineering. I. Patnaik, Ranjan, 1969– QR53.E54 2012 579'.163–dc23 2012015254

Printed in the United States of America

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CONTENTS

Foreword viiJohn Pierce

Preface ix

Contributors xi

1 Classical Strain Improvement 1Nathan Crook and Hal S. Alper

2 Tracer-Based Analysis of Metabolic Flux Networks 35Michael Dauner

3 Integration of “Omics” Data with Genome-Scale Metabolic Models 77Stephen Van Dien, Priti Pharkya, and Robin Osterhout

4 Strain Improvement via Evolutionary Engineering 111Byoungjin Kim, Jing Du, and Huimin Zhao

5 Rapid Fermentation Process Development and Optimization 133Jun Sun and Lawrence Chew

6 The Clavulanic Acid Strain Improvement Program at DSM Anti-Infectives 169Bert Koekman and Marcus Hans

v

vi CONTENTS

7 Metabolic Engineering of Recombinant E. coli for the Production of 3-Hydroxypropionate 185Tanya Warnecke Lipscomb, Matthew L. Lipscomb, Ryan T. Gill, and Michael D. Lynch

8 Complex System Engineering: A Case Study for an Unsequenced Microalga 201Michael T. Guarnieri, Lieve M.L. Laurens, Eric P. Knoshaug, Yat-Chen Chou, Bryon S. Donohoe, and Philip T. Pienkos

9 Meiotic Recombination-Based Genome Shuffling of Saccharomyces Cerevisiae and Schefferomyces Stiptis for Increased Inhibitor Tolerance to Lignocellulosic Substrate Toxicity 233Dominic Pinel and Vincent J.J. Martin

Index 251

vii

FOREWORD

The increasing demands for renewable chemicals, materials, and fuels, as well as the continuing evolution of capabilities in biology, chemistry, and engineer-ing, are giving rise to significant efforts in using biotechnological approaches in new process configurations. These approaches are particularly well suited to conversions of carbohydrate and other biological starting compounds into useful materials, as enzymes and microbes naturally transform these sub-stances. Building on a fair history of industrial use of microbes in the produc-tion of high-value, low-volume materials, such as pharmacologically active compounds, vitamins, and amino acids, we are now extending these approaches to the production of higher volume/lower value chemicals, such as monomers for making polymers, lubricants, and fuels. As we progress up this volume curve, the demands on the bioprocess become more and more stringent, and highly integrative approaches among disciplines are required to produce the biocatalysts and associated processes necessary for commercially viable outcomes.

Coincident with this evolution, a number of books and monographs have appeared on the subject of metabolic engineering and systems biology, and the primary literature is becoming more and more detailed. With this back-drop, this book does not attempt to be an authoritative reference on tools and techniques, but rather focuses on the strategies and approaches that enable commercial biocatalyst design. It should be of use to graduate students and early career professionals in the field, or to other generalists and professionals from related disciplines who are eager to grasp the basic tenets of engineering biocatalysts. In addition, it may well be of value in providing corporate manag-ers and government officials with insights into the requirements for successful program outcomes.

viii ForeWord

This book gives an overview of current approaches, with examples drawn from academia and industry and covering biocatalysts ranging from Esche-richia coli and Steptomyces to yeast and microalgae. The vitality of the field is exemplified by the relatively young ages of the contributors, who are shaping the field with their novel approaches, and the inclusion of case studies adds a realistic dimension to the exposition.

John PierceLondon

ix

PREFACE

This book highlights current trends and developments in the area of engineer-ing industrial strains for the production of bulk chemicals and biofuels from renewable biomass. The commercialization of bioprocesses derived from the use of superior engineered strains often requires the balance between unknowns and trade-off between multiple complex traits of the biocatalyst. Complex phenotypes are traits in a microbe that require more than one genetic change (multigenic) to be modulated simultaneously in the microor-ganism’s genome for full expression. Knowing what those genetic changes are for a given trait and how to manipulate those targets in the most efficient way, forms the motivation for writing this book. The chapters address tools and methodologies developed for engineering such complex traits or phenotypes in industrial strains. Emphasis is on the multidisciplinary (metabolic engineer-ing, screening, fermentation, downstream) nature of the approach or strategy that is used during the course of developing such a commercial biocatalyst. Keeping in perspective the multidisciplinary nature of activity and the inter-ests of a broader range of readers, the topics included in the chapters are not meant to be fully exhaustive in their respective areas; rather, the emphasis is on comparison and integration of different tools and objectives. Chapters 1–5 summarize broadly the current tools and technologies available for engineer-ing a complex phenotype in an industrial strain with brief reference to exam-ples, while Chapters 6–9 highlight in detail the application of such tools and methodologies in the form of case studies.

Chapter 1 summarizes the age-old proven approach for engineering in -dustrial strains using mutagenesis, followed by screening or selection, often termed classical strain engineering (CSI). Discussions of the applicability of CSI for engineering complex traits provide information on its suitability and

x PrEfaCE

limitations. Chapter 2 describes the current state of the art in the use of 13C tracer-based analysis and metabolic flux analysis for engineering complex pathways. Chapter 3 describes the utility of genome-scale models by integra-tion of “omics” technology and physiological data to address engineering of complex traits.

The probability of commercial success of a bioprocess that uses microbial catalysts and renewable feedstocks, as compared with platforms that use chem-ical catalysts and fossil fuel-derived feedstocks, greatly depends on the time it takes to engineer these microbes to perform the desired reaction under harsh manufactur ing conditions at rates, titers, and yields that meet the criteria for economic feasibility. Chapter 4 addresses new evolutionary strain engi-neering approaches that are superior to CSI in developing complex traits rapidly. Transitioning from laboratory-scale demonstration to commercial-scale operation is not only time-consuming but also expensive, especially with the uncertainties associated with scalability of complex traits. Chapter 5 describes an integrative platform for rapid fermentation process development and strain evaluation that not only minimizes the number of false positives from a strain engineering program but also provides a cost-effective approach to optimize fermentation conditions.

Chapter 6 is a case study on the use of CSI (Chapter 1) and improved strain screening strategies (Chapter 5) at Dutch State Mines for engineering Streptomyces clavuligerus for commercial production of anti-infectives. Chapter 7 is a case study on the use of evolutionary approaches (Chapter 4) at Opx Biotechnologies for improving tolerance of Escherichia coli to 3-hydroxypropionoic acid. Chapter 8 is a complete strain engineering case study from the National renewable Energy Laboratory in an unsequenced micro-alga, Chlorella vulgaris, for production of biofuels. The authors have high-lighted integration of improved analytics and strain screening approaches (Chapters 1 and 5) with “omics” technology (Chapter 3) for addressing needed improvements in multiple complex traits. Chapter 9 demonstrates the feasibil-ity of using genome-shuffling approaches (Chapter 4) in Saccharomyces cere-visiae and Schef feromyces stiptis for improving tolerance to inhibitors in lignocellulosic substrates.

Scientists, engineers, and project managers who are leaders in their respec-tive areas of research and drawn from diverse fields of science and engineering have contributed to the above chapters. The book has attempted to capture the thought processes on which they so often rely during the initiation and development of a commercial biocatalyst project. I hope the readers find the content of the book to be intellectually satisfying.

I would like to thank the editors at John Wiley & Sons for being patient and for their cooperation during the course of this project.

ranjan Patnaik

xi

CONTRIBUTORS

Hal S. Alper,  Ph.D.,  Department  of  Chemical  Engineering,  University  of Texas, Austin, TX

Lawrence Chew,  Ph.D., Pfenex Inc., San Diego, CA

Yat-Chen Chou,  National Renewable Energy Laboratory, Golden, CO

Nathan Crook,  Department  of  Chemical  Engineering,  University  of  Texas, Austin, TX

Michael Dauner,  Ph.D., E. I. du Pont de Nemours and Company, Wilmington, DE

Bryon S. Donohoe,  Ph.D., National Renewable Energy Laboratory, Golden, CO

Jing Du,  Ph.D., Department of Chemical and Biomolecular Engineering, Uni-versity of Illinois at Urbana-Champaign, Urbana, IL

Ryan T. Gill,  Ph.D.,  Department  of  Chemical  and  Biological  Engineering, University of Colorado-Boulder, Boulder, CO

Michael T. Guarnieri,  Ph.D., National Renewable Energy Laboratory, Golden, CO

Marcus Hans,  Ph.D., DSM Biotechnology Center, Delft, The Netherlands

Byoungjin Kim,  Ph.D., Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL

Eric P. Knoshaug,  National Renewable Energy Laboratory, Golden, CO

Bert Koekman,  Ph.D., DSM Biotechnology Center, Delft, The Netherlands

xii  CONTRIBUTORS

Lieve M.L. Laurens,  Ph.D., National Renewable Energy Laboratory, Golden, CO

Matthew L. Lipscomb,  Ph.D., OPX Biotechnologies, Inc., Boulder, CO

Tanya Warnecke Lipscomb,  Ph.D., OPX Biotechnologies, Inc., Boulder, CO

Michael D. Lynch,  Ph.D., OPX Biotechnologies, Inc., Boulder, CO

Vincent J.J. Martin,  Ph.D., Department of Biology, Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada

Robin Osterhout,  Ph.D., Genomatica, Inc., San Diego, CA

Ranjan Patnaik,  Ph.D.,  Head  Biofuels  R&D,  DuPont  India  Private  Ltd., Hyderabad, India

Priti Pharkya,  Ph.D., Genomatica, Inc., San Diego, CA

Philip T. Pienkos,  Ph.D.,  National  Renewable  Energy  Laboratory,  Golden, CO

John Pierce,  Ph.D., Chief Bioscientist, BP, London, UK

Dominic Pinel,  Department of Biology, Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada

Jun Sun,  Ph.D., E. I. du Pont de Nemours and Company, Newark, DE

Stephen Van Dien,  Ph.D., Genomatica, Inc., San Diego, CA

Huimin Zhao,  Ph.D., Department of Chemical and Biomolecular Engineer-ing, University of Illinois at Urbana-Champaign, Urbana, IL

1

1CLASSICAL STRAIN IMPROVEMENT

Nathan Crook and Hal S. Alper

1.0 INTRODUCTION

Improving complex phenotypes, which are typically multigenic in nature, has been a long-standing goal of the food and biotechnology industry well before the advent of recombinant DNA technology and the genomics revolution. For thousands of years, humans have (whether intentionally or not) placed selec-tive pressure on plants, animals, and microorganisms, resulting in improve-ments to desired phenotypes. Clear evidence of these efforts can be seen from the dramatic morphological changes to food crops since domestication (1). These improvements have been predominantly achieved through a “classical” approach to strain engineering, whereby phenotypic improvements are made by screening and mutagenesis of strains that use methods naive of genome sequences or the resulting genetic changes. This approach is well suited for strain optimization in industrial microbiology, which commonly exploits complex phenotypes in organisms with poorly defined or monitored genetics. As a recognition of importance, Arnold Demain and Julian Davies begin their Handbook of Industrial Microbiology and Biotechnology with “Almost all industrial microbiology processes require the initial isolation of cultures from nature, followed by small-scale cultivations and optimization, before large-scale production can become a reality” (2). The classical approach is concerned

Engineering Complex Phenotypes in Industrial Strains, First Edition. Edited by Ranjan Patnaik.© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.