Metagenomics: Methods and Protocols

311 Pages·2017·7.682 MB·English

by  Wolfgang R. Streit, Rolf Daniel (eds.)

Methods in
Molecular Biology 1539
Wolfgang R. Streit
Rolf Daniel Editors
Metagenomics
Methods and Protocols
Second Edition
M M B
ethods in olecular iology
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John M. Walker
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Metagenomics
Methods and Protocols
Second Edition
Edited by
Wolfgang R. Streit
Mikrobiologie & Biotechnologie, Biocenter Klein Flottbek, University of Hamburg,
Hamburg, Germany
Rolf Daniel
Göttingen Genomics Laboratory, Department of Genomic and Applied Microbiology,
Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen,
Göttingen, Germany
Editors
Wolfgang R. Streit Rolf Daniel
Mikrobiologie & Biotechnologie Göttingen Genomics Laboratory
Biocenter Klein Flottbek Department of Genomic and Applied
University of Hamburg Microbiology
Hamburg, Germany Institut für Mikrobiologie und Genetik
Georg-August-Universität Göttingen
Göttingen, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6689-9 ISBN 978-1-4939-6691-2 (eBook)
DOI 10.1007/978-1-4939-6691-2
Library of Congress Control Number: 2016956093
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Preface
Metagenomics is a key technology to the DNA-based exploration of the genomic potential
from not-yet-cultivated microbes for ecology and biotechnology. Since the term “metage-
nome” was coined almost two decades ago, metagenomics has dramatically changed our
view on many research areas such as microbial ecology, community biology, and microbi-
ome research, and it has resulted in the rapid identification of many novel biomolecules
with potential value to bio-based industrial processes. Function-driven metagenomics has
been the focus of many laboratories around the world to quickly encounter novel func-
tional genes encoding enzymes with new and/or improved traits. In this way, the diversity
of biocatalysts and other valuable biomolecules useful for downstream applications increased
significantly. Industries demand enzymes that can be directly applied in biotechnological
processes and catalyze a wide variety of different reactions. Ideally, these biocatalysts/bio-
active molecules should be highly active with a broad range of substrates under harsh reac-
tion conditions and, at the same time, should possess a predictable substrate specificity and
enantioselectivity. Today, only a limited number of truly well-suited enzymes fulfill these
requirements. To identify novel biomolecules different strategies are employed: While the
sequence-based detection of novel enzymes and other biomolecules certainly provides rapid
access to novel genes and enzymes, it suffers from the fact that only sequences with putative
function and similarities to already known genes are recovered. Function-based screens
overcome this bottleneck but are limited by low hit rate due to the often poor capabilities
of the employed host to express foreign genes and to produce active recombinant proteins.
Thus, function-driven detection of novel biocatalysts or other valuable biomolecules is still
a very time-consuming process that slows down development times for novel products.
However, it has the huge advantage that functional biocatalysts and bioactive compounds
are recovered.
In recent years, various novel technologies have been developed to access the metage-
nomes of microbial communities using high-throughput technologies often in combina-
tion with next-generation sequencing approaches. Within the second edition of this book,
we provide up-to-date technologies on various function-based technologies currently used
in metagenomics. Our goal is that this book serves as a manual for researchers who are
interested in establishing metagenomics in their laboratories. All working steps involved are
presented in the chapters: Starting from the DNA isolation from soils and marine samples
followed by the construction and screening of the libraries for diverse enzymes and biomol-
ecules. The book provides a comprehensive overview of current methods used to isolate
DNA and construct large-insert and small-insert libraries from terrestrial and marine habi-
tats, including plant and fungal microbiomes. It further summarizes methods for establish-
ing metagenome libraries in non-E. coli hosts such as Streptomyces, and it highlights novel
molecular tools ready to use for function-driven mining of metagenomic DNA. Lastly,
several chapters provide detailed insights into screening protocols for a wide array of differ-
ent genes encoding enzymes with relevance to biotechnology and ecology. Protocols are
offered for the screening of lipases/esterases, cellulases, hydrogenases, ligninolytic enzymes,
v
vi Preface
glycosyl transferases, and quorum-quenching enzymes involved in the destruction of N-acyl
homoserine lactone-based cell–cell communication signals. Furthermore, the book pro-
vides detailed screening protocols for phosphatases, poly-hydroxyalkanoate metabolism-
related enzymes, stereoselective hydrolases, and microbial signals for the discovery of
secondary metabolites. Finally, detailed insights into the pipeline necessary for the recon-
struction of metabolic pathways are given.
In our view, this book provides a comprehensive collection of up-to-date protocols for
metagenomics and tools for the recovery of many major types of biocatalysts and allows an
easy setup of these screens in any microbiology laboratory.
Hamburg, Germany Wolfgang R. Streit
Göttingen, Germany Rolf Daniel
Contents
Preface.......................................................... v
Contributors.......................................................... ix
1 Construction of Small-Insert and Large-Insert Metagenomic Libraries ...... 1
Carola Simon and Rolf Daniel
2 Extraction of Total DNA and RNA from Marine Filter
Samples and Generation of a cDNA as Universal Template
for Marker Gene Studies......................................... 13
Dominik Schneider, Franziska Wemheuer, Birgit Pfeiffer,
and Bernd Wemheuer
3 Construction and Screening of Marine Metagenomic Large Insert Libraries.... 23
Nancy Weiland-Bräuer, Daniela Langfeldt, and Ruth A. Schmitz
4 Constructing and Screening a Metagenomic Library
of a Cold and Alkaline Extreme Environment ......................... 43
Mikkel A. Glaring, Jan K. Vester, and Peter Stougaard
5 DNA-, RNA-, and Protein-Based Stable-Isotope Probing
for High-Throughput Biomarker Analysis of Active Microorganisms........ 57
Eleanor Jameson, Martin Taubert, Sara Coyotzi, Yin Chen, Özge Eyice,
Hendrik Schäfer, J. Colin Murrell, Josh D. Neufeld, and Marc G. Dumont
6 Assessing Bacterial and Fungal Diversity in the Plant Endosphere .......... 75
Bernd Wemheuer and Franziska Wemheuer
7 Shotgun Metagenomic Sequencing Analysis of Soft-Rot
Enterobacteriaceae in Polymicrobial Communities...................... 85
James Doonan, Sandra Denman, James E. McDonald,
and Peter N. Golyshin
8 Cloning and Expression of Metagenomic DNA in Streptomyces lividans
and Subsequent Fermentation for Optimized Production ................ 99
Yuriy Rebets, Jan Kormanec, Andriy Luzhetskyy, Kristel Bernaerts,
and Jozef Anné
9 Degradation Network Reconstruction Guided by Metagenomic Data ....... 145
Rafael Bargiela and Manuel Ferrer
10 Novel Tools for the Functional Expression of Metagenomic DNA.......... 159
Nadine Katzke, Andreas Knapp, Anita Loeschcke, Thomas Drepper,
and Karl-Erich Jaeger
11 A Microtiter Plate-Based Assay to Screen for Active and Stereoselective
Hydrolytic Enzymes in Enzyme Libraries ............................ 197
Dominique Böttcher, Patrick Zägel, Marlen Schmidt,
and Uwe T. Bornscheuer
vii
viii Contents
12 Screening for Cellulase Encoding Clones in Metagenomic Libraries......... 205
Nele Ilmberger and Wolfgang R. Streit
13 Liquid Phase Multiplex High-Throughput Screening of Metagenomic
Libraries Using p-Nitrophenyl-Linked Substrates for Accessory
Lignocellulosic Enzymes......................................... 219
Mariette Smart, Robert J. Huddy, Don A. Cowan, and Marla Trindade
14 Screening Glycosyltransferases for Polyphenol Modifications.............. 229
Nele Ilmberger and Ulrich Rabausch
15 Methods for the Isolation of Genes Encoding Novel PHA Metabolism
Enzymes from Complex Microbial Communities....................... 237
Jiujun Cheng, Ricardo Nordeste, Maria A. Trainer, and Trevor C. Charles
16 Function-Based Metagenomic Library Screening and Heterologous
Expression Strategy for Genes Encoding Phosphatase Activity............. 249
Genis A. Castillo Villamizar, Heiko Nacke, and Rolf Daniel
17 Activity-Based Screening of Metagenomic Libraries for Hydrogenase
Enzymes..................................................... 261
Nicole Adam and Mirjam Perner
18 Screening for N-AHSL-Based-Signaling Interfering Enzymes ............. 271
Stéphane Uroz and Phil M. Oger
19 Mining Microbial Signals for Enhanced Biodiscovery
of Secondary Metabolites ........................................ 287
F. Jerry Reen, Jose A. Gutiérrez-Barranquero, and Fergal O’Gara
Erratum to: ...................................................... E1
Index ............................................................... 301
Contributors
Nicole AdAm • Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek,
University of Hamburg, Hamburg, Germany
Jozef ANNé • Lab. Molecular Bacteriology, Department Microbiology and Immunology,
Rega Institute, KU Leuven (University of Leuven), Leuven, Belgium
RAfAel BARgielA • Institute of Catalysis, Consejo Superior de Investigaciones Científicas
(CSIC), Madrid, Spain
KRistel BeRNAeRts • Department of Chemical Engineering, KU Leuven (University
of Leuven), Leuven, Belgium
Uwe t. BoRNscheUeR • Department of Biotechnology and Enzyme Catalysis, Institute of
Biochemistry, Greifswald University, Greifswald, Germany
domiNiqUe BöttcheR • Department of Biotechnology and Enzyme Catalysis, Institute of
Biochemistry, Greifswald University, Greifswald, Germany
tRevoR c. chARles • Department of Biology, University of Waterloo, Waterloo, ON, Canada
YiN cheN • School of Life Sciences, University of Warwick, Coventry, UK
JiUJUN cheNg • Department of Biology, University of Waterloo, Waterloo, ON, Canada
doN A. cowAN • Institute for Microbial Biotechnology & Metagenomics, University
of the Western Cape, South Africa; Centre for Bioprocess Engineering Research, University
of Cape Town, South Africa
sARA coYotzi • Department of Biology, University of Waterloo, Waterloo, ON, Canada
Rolf dANiel • Göttingen Genomics Laboratory, Department of Genomic and Applied
Microbiology, Institut für Mikrobiologie und Genetik, Georg-August-Universität
Göttingen, Göttingen, Germany
sANdRA deNmAN • Centre for Ecosystems Society and Biosecurity, Forest Research, Surrey, UK
JAmes dooNAN • School of Biological Sciences, Bangor University, Bangor, Gwynedd, UK
thomAs dReppeR • Institute of Molecular Enzyme Technology, Heinrich-Heine- University
Düsseldorf, Jülich, Germany
mARc g. dUmoNt • Centre for Biological Sciences, University of Southampton,
Southampton, UK
özge eYice • School of Biological and Chemical Sciences, Queen Mary University
of London, London, UK
mANUel feRReR • Institute of Catalysis, Consejo Superior de Investigaciones Científicas
(CSIC), Madrid, Spain
miKKel A. glARiNg • Department of Plant and Environmental Sciences, University
of Copenhagen, Frederiksberg, Denmark
peteR N. golYshiN • School of Biological Sciences, Bangor University, Bangor, Gwynedd,
UK
Jose A. gUtiéRRez-BARRANqUeRo • BIOMERIT Research Centre, University College
Cork–National University of Ireland, Cork, Ireland
RoBeRt J. hUddY • Institute for Microbial Biotechnology & Metagenomics, University
of the Western Cape, South Africa; Centre for Bioprocess Engineering Research, University
of Cape Town, South Africa
ix
x Contributors
Nele ilmBeRgeR • Microbiology & Biotechnology, Biocenter Klein Flottbek, University of
Hamburg, Hamburg, Germany
KARl-eRich JAegeR • Institute of Molecular Enzyme Technology, Heinrich-Heine- University
Düsseldorf, Jülich, Germany
eleANoR JAmesoN • School of Life Sciences, University of Warwick, Coventry, UK
NAdiNe KAtzKe • Institute of Molecular Enzyme Technology, Heinrich-Heine- University
Düsseldorf, Jülich, Germany
ANdReAs KNApp • Institute of Molecular Enzyme Technology, Heinrich-Heine- University
Düsseldorf, Jülich, Germany
JAN KoRmANec • Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava,
Slovak Republic
dANielA lANgfeldt • Institut für Allgemeine Mikrobiologie, Christian-Albrechts-
Universität zu Kiel, Kiel, Germany
ANitA loeschcKe • Institute of Molecular Enzyme Technology, Heinrich-Heine- University
Düsseldorf, Jülich, Germany
ANdRiY lUzhetsKYY • Actinobacteria Metabolic Engineering Group, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), University of Saarland, Saarbrücken,
Germany; Department of Pharmaceutical Biotechnology, University of Saarland,
Saarbrücken, Germany
JAmes e. mcdoNAld • School of Biological Sciences, Bangor University, Bangor, Gwynedd, UK
J. coliN mURRell • School of Environmental Sciences, University of East Anglia, Norwich, UK
heiKo NAcKe • Institut für Mikrobiologie und Genetik, Georg-August-Universität
Göttingen, Göttingen, Germany
Josh d. NeUfeld • Department of Biology, University of Waterloo, Waterloo, ON, Canada
RicARdo NoRdeste • Department of Biology, University of Waterloo, Waterloo, ON, Canada
feRgAl o’gARA • BIOMERIT Research Centre, University College Cork–National
University of Ireland, Cork, Ireland; School of Biomedical Sciences, Curtin Health
Innovation Research Institute, Curtin University, Perth, Australia
phil m. ogeR • Univ Lyon, INSA-Lyon, UCBL, Villeurbanne Cedex, France; Univ Lyon,
ENS-Lyon, Lyon, France
miRJAm peRNeR • Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek,
University of Hamburg, Hamburg, Germany
BiRgit pfeiffeR • Institut für Mikrobiologie und Genetik, Georg-August-Universität
Göttingen, Göttingen, Germany
UlRich RABAUsch • Microbiology and Biotechnology, Biocenter Klein Flottbek, University of
Hamburg, Hamburg, Germany
YURiY ReBets • Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
University of Saarland, Saarbrücken, Germany
f. JeRRY ReeN • BIOMERIT Research Centre, University College Cork–National
University of Ireland, Cork, Ireland
heNdRiK schäfeR • School of Life Sciences, University of Warwick, Coventry, UK
mARleN schmidt • Department of Biotechnology and Enzyme Catalysis, Institute of
Biochemistry, Greifswald University, Greifswald, Germany
RUth A. schmitz • Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität
zu Kiel, Kiel, Germany
domiNiK schNeideR • Institut für Mikrobiologie und Genetik, Georg-August-Universität
Göttingen, Göttingen, Germany
cARolA simoN • Pohl-Boskamp GmbH, Hamburg, Germany