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.

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

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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

vii

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

1FUNCTIONAL METAGENOMICSAS A TECHNIQUE FOR THEDISCOVERY OF NOVEL ENZYMESAND NATURAL PRODUCTS

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

Matthew D. McMahon, and Michael G. ThomasDepartment of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin

I. INTRODUCTION

The industrial use of biocatalysts and the recent reemphasis on the isolationof natural products with desired biological activities are driving the search fornew mechanisms for accessing the metabolic potential of microorganisms. Thisemphasis on microorganisms comes from an appreciation of the enormous biodi-versity found within them [1–4] and the understanding that traditional culturingtechniques to isolate these organisms have only enabled us to access approx-imately 1% of the microbial population present in a soil environment [5–7].Further, it is reasonable to assume that this low level of culturability will befound in many other ecological niches. Due to this limited access to the fullmetabolic potential in a targeted environment, there is a clear interest in devel-oping techniques allowing us access to the metabolic potential of the remaining99% of microorganisms.

One approach that aims to circumvent the limitations of culturability is the useof metagenomics. The term metagenome was introduced to define the combined

Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis,Edited by Wu-Kuang Yeh, Hsiu-Chiung Yang, and James R. McCarthyCopyright 2010 John Wiley & Sons, Inc.

3

4 NOVEL ENZYMES AND NATURAL PRODUCTS

FIGURE 1 Construction and functional screening of a metagenomic DNA library fromsoil. (See insert for color representation of the figure.)

genomes of all the organisms in a particular environment, with metagenomicsreferring to a collection of techniques allowing access to this genomic informa-tion [8]. The shared features of metagenomic techniques are that the total DNAof the microbial population is extracted from the environment and cloned intoappropriate bacterial cloning vectors in a culture-independent manner (Fig. 1);thus, this technique has the potential to access the 99% of the metabolic potentialthat has previously been inaccessible.

Once metagenomic DNA has been extracted from an environment ofinterest and the associated DNA library has been constructed, there are twomechanisms for analyzing the metabolic potential present. One approach is touse sequence-based methods whereby the library is sequenced and analyzedfor genes of interest. Although this approach has been successful in identifyingnew homologs of enzymes of interest (e.g., bacteriorhodopsin homologs [9])and in gaining an understanding of total microbial community present [10], itdoes have limitations regarding the identification of new and novel biocatalysts.Because sequence-based identification of enzymes relies on relatively highlevels of sequence identity, only those enzymes displaying significant homologyto enzymes known to catalyze a specific reaction will be recognized. Thus, thoseenzymes displaying low sequence identity to known functional classes, andnovel enzymes that exhibit the desired activity but show no homology to knownfunctional classes, will not be identified using sequence-based techniques alone.

Alternatively, functional metagenomics is based on detecting a desired bio-logical activity that has been introduced into the host organism by virtue of therecombinant metagenomic clone. Using this function-based approach, metage-nomic clones encoding a variety of desired activities have been discovered; these

CONSTRUCTION OF METAGENOMIC DNA LIBRARIES 5

activities include esterases [11,12] and nitrile hydratases [13], enzymes respon-sible for the production of natural products (e.g., antibiotics [11] and pigments[14]), and antibiotic resistance genes [15,16]. The power of this approach isderived from the ability to identify clones encoding the desired biological activityin a large metagenomic library (often more than 106 recombinant clones) withoutprior sequence knowledge and without requiring a large-scale sequencing project.

Although successful, this approach to metagenomic DNA analysis also hasits limitations. The most obvious limitation is the ability to devise a phenotypicscreen to identify clones of interest. The second limitation deals with the heterol-ogous host itself. Although Escherichia coli has proven to be an extraordinarilycompliant host, it is not always able to express genes and produce functionalproteins from distantly related microorganisms. This limitation can be due toE. coli lacking the necessary regulatory elements to turn on expression of thegene clusters or the failure of E. coli to recognize the gene promoters due toa lack of the appropriate sigma factors. Additional issues arise when screen-ing for the production of natural products, since E. coli may lack the necessarycofactors or precursors required for production of the metabolite. The promise offunctional metagenomics is emphasized in this chapter and we provide the readerwith information on the steps that various researchers have taken to circumventthe limitations discussed above.

The field of metagenomics has grown enormously recently and it is nearlyimpossible to adequately summarize all the advances in sequence-based and func-tional genomics. Instead, what we have chosen to do here is to provide the readerwith information on, and references for construction of, a metagenomic DNAlibrary from a specific environment (e.g., soils/sediments) and to provide optionsfor how such a library can be screened using functional genomics approaches.

In discussing the construction and functional screening of a soil metagenome,we have divided this chapter into several sections. First, we discuss briefly thechoice of source material, which can be important in enrichment for a desired bio-logical activity. Second, we provide details on how to isolate metagenomic DNAfrom soils/sediments and prepare it for cloning. Third, we discuss the construc-tion of a metagenomic DNA library. Fourth, we provide examples of functionalmetagenomic screens used for identifying clones with various biological activi-ties. Finally, we provide a brief summary and some thoughts on what challengeslay ahead for metagenomics.

II. CONSTRUCTION OF METAGENOMIC DNA LIBRARIES

A. Choice of Source Material

Our research has focused on functional metagenomics from soil-derived DNAlibraries. Our focus on this environment is driven by two factors. First, it is nowquite evident that the soil environment has enormous biological and metabolicdiversity. One gram of soil has between 4 × 107 and 2 × 109 prokaryotic cells[17,18]. Furthermore, Torsvik and colleagues have estimated that this vast numberof prokaryotic cells consists of 3000 to 11,000 different genomes [5,6]. This

6 NOVEL ENZYMES AND NATURAL PRODUCTS

estimate is likely to be an underestimate based on the likelihood that there willbe a number of rare and underrepresented members of this population. Thus,there is enormous metabolic potential from a single gram of soil.

Although we have focused on mesophilic soils/sediments, there are a numberof examples of other soil environments and other ecological niches that havebeen investigated. For soils/sediments these include extreme environments suchas high temperatures [19], high and low pH [20], and high salt [21], in addition tocultivated and uncultivated fields [11,15,22], and different types of soils (e.g., clay[23], sandy [24], loam [15]). There is also a trans-European project that focuseson metagenomic analysis of disease-suppressive soils to identify componentsproduced by soil inhabitants that can be exploited for agricultural purposes [25].

Although there has been a significant focus on soils/sediments, this has notbeen the only environment of focus. Other environments include such diverseareas as animal gastrointestinal tracts for the identity of β-glucanases [26], marinesponges to identify natural products [27], and the resident microbiota of the gypsymoth for resistance determinants [16]. In the end, the choice of starting materialcan influence the success of a functional metagenomic screen.

B. Isolating DNA from Soils and Sediments

There are two general methods by which DNA is obtained from soil samples:the direct lysis and cell separation (or cell extraction) methods. As their namesimply, they differ according to whether bacterial cells are separated from theenvironmental matrix prior to lysis. In the following section we provide overviewsof both methods, including advantages and disadvantages of each. However, forthe purposes of this chapter, we believe the reader may be more interested in thedirect lysis method, and we give a more extensive outline of a protocol in use inour labs. Although we do not offer a comprehensive review of methods for theisolation of DNA from environmental samples, we refer the reader to a numberof references that address these techniques in detail [28–33].

Direct Lysis Method The direct lysis method for obtaining DNA reportedlyresults in larger quantities of DNA isolated from samples [30] and thus a morerepresentative picture of the genetic material present in a sample. However,because lysis is done in the presence of the soil matrix, isolated DNA extractstypically include contaminants that may hinder downstream DNA manipulationsteps. Further, the harsh chemical and mechanical steps involved in processingsamples typically result in smaller DNA fragments available for cloning [29].Nevertheless, direct lysis is the more popular method of the two for researchersinterested in functional screening, due primarily to its relative simplicity (com-mercial kits are available for small-insert DNA library cloning), ease in sampleprocessing, limited time input, and demonstrated success in producing librariesfrom which enzymes of interest can be identified (Table 1). A number of papersdescribe individual steps meant to enhance the viability of the DNA during theprocessing steps; these papers are noted during the description of the protocol.

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15

16 NOVEL ENZYMES AND NATURAL PRODUCTS

The protocol described below has been used in our laboratories to obtainDNA sufficient for small- and large-insert libraries; it was developed by LynnWilliamson (University of Wisconsin–Madison, Department of Bacteriology),drawing from collaborative research and experimentation with a number of pub-lished protocols.

1. Sieve soil through a fine (ca. 2 mm, sterile) mesh to eliminate roots andparticulates.

2. Split 100 g of the sieved soil into two 50-g samples, and add each sampleto a sterile 250-mL centrifuge bottle. To each bottle add 75 mL of Zbuffer [at pH 8.0: 100 mM Tris-HCl; 100 mM sodium phosphate; 100 mMethylenediaminetetraacetic acid (EDTA); 1.5M NaCl; 1% w/v cetrimoniumbromide (CTAB)].

3. Lyse the soil samples using two freeze–thaw cycles; freezing must becomplete and can be accomplished using liquid nitrogen or a dry ice/ethanolbath. The samples can be thawed by incubation in a 65◦C water bath.Freezing in a dry ice bath will take about 40 min; complete thawing willtake about the same amount of time.

4. To the final thaw at 65◦C, add 9 mL of 20% sodium dodecyl sulfate and4.5 mL of 5 M guanidinium isothiocyanate. Mix by gentle inversion.

5. Incubate at 65◦C for 2 h with occasional mixing.6. Centrifuge at 10◦C, 20 min at 15,000 × g.7. Pipette off the supernatant, containing DNA, into two clean, sterile 250-mL

centrifuge bottles. Be sure to use wide-bore pipette tips when handling theDNA in this step, and in the remaining steps, to avoid shearing.

8. To the DNA-containing solution, add 25 mL of chloloform/isoamyl alcohol(24 : 1) and mix gently for 10 min at room temperature.

9. Centrifuge at 10◦C, 20 min at 15,000 × g.10. Pipette off the supernatant, containing DNA, into two clean, sterile 250-mL

centrifuge bottles.11. Precipitate DNA by adding isopropanol to 70% (about 40 mL per bottle)

and mixing gently for 5 min. Let this sample incubate without mixing atroom temperature for an additional 20 min.

12. Centrifuge at 10◦C, 40 min at 15,000 × g.13. A visible brownish pellet should form—this is the DNA. Carefully pour

off the supernatant, and remove all of the remaining liquid from the bottle.14. Resuspend the DNA pellet gently in a minimal amount of T10E10 (at pH 8.0:

10 mM Tris-HCl; 10 mM EDTA) using wide-bore pipette tips. 1 to 2 mLof T10E10 per tube should suffice. Using wide-bore pipette tips, aliquot theresuspended DNA solution equally into four 1.5-mL Eppendorf tubes.

15. Extract the DNA with an equal volume of Tris-buffered phenol/chloroform(commercially available, pH 8.0), invert to mix, and centrifuge at16,000 × g for 10 min at room temperature.