Strain Engineering: Methods and Protocols

476 Pages·2011·6.484 MB·English

by  Jindan Zhou, Kenneth E. Rudd (auth.), James A. Williams (eds.)

M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
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Strain Engineering
Methods and Protocols
Edited by
James A. Williams
Nature Technology Corporation, Lincoln, NE, USA
Editor
James A. Williams, Ph.D
Nature Technology Corporation
Lincoln, NE
USA
[e-mail]
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-196-3 e-ISBN 978-1-61779-197-0
DOI 10.1007/978-1-61779-197-0
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Preface
Microbial strain engineering is used to improve production of bioproducts. Classical strain
engineering is performed by repeated cycles of random mutation and selection. These
methods have greatly contributed to strain improvement, but have serious drawbacks.
Uncharacterized “non-specific” secondary deleterious mutations will be introduced into
the genome during each mutagenesis cycle, and accumulate in the selected strain. Classical
methods also do not allow the introduction of new genetic material and are not suitable
for complex strain development applications such as metabolic engineering of organisms
to enable cell-based conversion of biomass into biofuels.
For complex strain engineering projects such as metabolic engineering for biofuels
production, a starting point “chassis” organism must be selected. This may be a commonly
used industrial organism such as Escherichia coli or Sacharromyces cerevisiae. While these
industrial organisms are not inherently adapted for production of biofuels, new genes and
functions can be rapidly imported using existing comprehensive strain engineering toolkits.
Many of these methods draw upon the fully annotated genome sequences of E. coli and
S. cerevisiae that ushered in a new age of rationale design-based strain engineering.
Alternatively, a native organism with existing biochemical pathways and production
potential for biofuels is selected as the chassis. However, native strains often are not
adapted for industrial fermentation and lack existing molecular biology tools necessary for
efficient strain engineering.
Recently, fully annotated genome sequences of many important native microbial
organisms have become publically available as a resource for researchers. The availability
of these genomic resources will enable adaptation of E. coli or S. cerevisiae-based rationale
design strain engineering methods to native organisms.
In this book, powerful new genetic engineering-based strain engineering methods are
presented for rational modification of a variety of model organisms. These methods are
particularly powerful when utilized to manipulate microbes for which sequenced and
annotated genomes are available. Collectively, these methods systematically introduce
genome alterations in a precise manner, allowing creation of novel strains carrying only
desired genome alterations.
In Section 1, E. coli-based bacterial strain engineering strategies are reviewed. State-of-
the-art methods for targeted gene knockout are presented, as well as their sequential
application for scarless genome modification. Methods for random gene knockout by
transposon mutagenesis are also described.
Cutting edge methods for identification of adaptation-selected genes are presented in
chapters describing genome engineering using oligonucleotide-mediated targeted gene
replacement and microarray-based genetic footprinting of random transposon libraries.
Methods to optimize synthetic operons for metabolic engineering applications are
described. Methods for introduction of genes and operons into the bacterial chromosome
are presented in a chapter on integration plasmid-based chromosomal expression of native
and foreign genes.
Strategies to assemble combinations of tagged integration plasmids, gene knockouts,
or knockout collections (e.g., Keio collection) are discussed in a chapter on high-through-
v
vi Preface
put double mutant assembly via conjugation. Protocols to assemble multiply modified
strains are provided in a chapter on P1 transduction.
In Section 2, analogous microbial engineering strategies for eukaryotic cells are pre-
sented, using the yeast S. cerevisiae as a model. This section also includes chapters describ-
ing creation and phenotypic trait selection with signature-tagged barcoded mutant
collections and libraries of mutant transcription factors; these methodologies have applica-
tion in a wide range of microorganisms.
In Section 3, examples of the proliferative adaptations of these base technologies to
strain engineer industrially important prokaryotic or eukaryotic microbial systems are pre-
sented. Introductory chapters on transformation and broad host range plasmid vectors
provide design guidance to develop robust methods for the critical first step of efficiently
introducing functional DNA into new microbes. This effort is guided by identification in
the annotated genome of genes whose products are detrimental to efficient transforma-
tion, for example, restriction endonucleases and secreted nonspecific nucleases. Targeted
elimination or neutralization of these genes improves broad host range plasmid transfor-
mation. In the case of fungi, nonhomologous recombination genes are also identified and
eliminated, to facilitate development of targeted homologous recombination-based methods.
This subsection then describes methods for applied strain engineering of microbial
organisms (prokaryotic and eukaryotic) with bioenergy potential for which sequenced and
annotated genomes are available. Once basic DNA transformation, replicating plasmids,
and homologous recombination-based chromosome integration methods in new organ-
isms are available, other techniques described in Sections 1 and 2 can be adapted. For
example, to facilitate application of the E. coli integration plasmid technology described in
Chapter 8, phage integration sites can be integrated into the genome at a permissive site
by homologous recombination, and the corresponding phage integrase supplied on a
broad host range plasmid.
Written for: Molecular and cellular biologists, molecular geneticists, bioengineers, and
microbiologists working in academia, pharmaceutical and biotechnology that perform
microbial strain engineering.
Lincoln, NE, USA James A. Williams
Contents
Preface ............................................................ v
Contributors......................................................... ix
PART I E. COLI
1 Bacterial Genome Reengineering ..................................... 3
Jindan Zhou and Kenneth E. Rudd
2 Targeted Chromosomal Gene Knockout Using PCR Fragments.............. 27
Kenan C. Murphy
3 Scarless Chromosomal Gene Knockout Methods ......................... 43
Bong Hyun Sung, Jun Hyoung Lee, and Sun Chang Kim
4 Random Chromosomal Gene Disruption In Vivo Using Transposomes ........ 55
Les M. Hoffman
5 Genome Engineering Using Targeted Oligonucleotide Libraries
and Functional Selection ........................................... 71
Elie J. Diner, Fernando Garza-Sánchez, and Christopher S. Hayes
6 Microarray-Based Genetic Footprinting Strategy to Identify Strain
Improvement Genes after Competitive Selection of Transposon Libraries ....... 83
Alison K. Hottes and Saeed Tavazoie
7 Optimization of Synthetic Operons Using Libraries
of Post-Transcriptional Regulatory Elements ............................ 99
Daniel E. Agnew and Brian F. Pfleger
8 Marker-Free Chromosomal Expression of Foreign and Native Genes
in Escherichia coli ................................................. 113
Chung-Jen Chiang, Po Ting Chen, Shan-Yu Chen, and Yun-Peng Chao
9 Array-Based Synthetic Genetic Screens to Map Bacterial
Pathways and Functional Networks in Escherichia coli...................... 125
Mohan Babu, Alla Gagarinova, Jack Greenblatt, and Andrew Emili
10 Assembling New Escherichia coli Strains by Transduction Using Phage P1....... 155
Sean D. Moore
PART II SACCHAROMYCES CEREVISIAE
11 Yeast Bioinformatics and Strain Engineering Resources..................... 173
Audrey L. Atkin
12 Delete and Repeat: A Comprehensive Toolkit for Sequential Gene Knockout
in the Budding Yeast Saccharomyces cerevisiae ............................ 189
Johannes H. Hegemann and Sven Boris Heick
13 Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae
and Candida albicans.............................................. 207
Tao Xu, Nikë Bharucha, and Anuj Kumar
vii
viii Contents
14 Signature-tagged Mutagenesis to Characterize Genes Through Competitive
Selection of Bar-coded Genome Libraries............................... 225
Julia Oh and Corey Nislow
15 Global Strain Engineering by Mutant Transcription Factors ................. 253
Amanda M. Lanza and Hal S. Alper
16 Genomic Promoter Replacement Cassettes to Alter Gene Expression
in the Yeast Saccharomyces cerevisiae ................................... 275
Andreas Kaufmann and Michael Knop
P III S E O I
ART TRAIN NGINEERING THER NDUSTRIALLY
I M
MPORTANT ICROBES
17 Microbial Genome Analysis and Comparisons: Web-Based
Protocols and Resources............................................ 297
Medha Bhagwat and Arvind A. Bhagwat
18 Plasmid Artificial Modification: A Novel Method for Efficient
DNA Transfer into Bacteria ......................................... 309
Tohru Suzuki and Kazumasa Yasui
19 Broad-Host-Range Plasmid Vectors for Gene Expression in Bacteria........... 327
Rahmi Lale, Trygve Brautaset, and Svein Valla
20 A Simple Method for Introducing Marker-Free Deletions
in the Bacillus subtilis Genome....................................... 345
Takuya Morimoto, Katsutoshi Ara, Katsuya Ozaki,
and Naotake Ogasawara
21 Transposon-Mediated Random Mutagenesis of Bacillus subtilis............... 359
Adam C. Wilson and Hendrik Szurmant
22 Integrative Food Grade Expression System for Lactic Acid Bacteria............ 373
Grace L. Douglas, Yong Jun Goh, and Todd R. Klaenhammer
23 ClosTron-Mediated Engineering of Clostridium.......................... 389
Sarah A. Kuehne, John T. Heap, Clare M. Cooksley,
Stephen T. Cartman, and Nigel P. Minton
24 High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum.... 409
Nobuaki Suzuki, Masayuki Inui, and Hideaki Yukawa
25 Mini-Mu Transposon Mutagenesis of Ethanologenic Zymomonas mobilis........ 419
Katherine M. Pappas
26 Engineering Thermoacidophilic Archaea using Linear DNA Recombination..... 435
Yukari Maezato, Karl Dana, and Paul Blum
27 Targeted Gene Disruption in Koji Mold Aspergillus oryzae .................. 447
Jun-ichi Maruyama and Katsuhiko Kitamoto
28 Selectable and Inheritable Gene Silencing through RNA Interference
in the Unicellular Alga Chlamydomonas reinhardtii........................ 457
Karin van Dijk and Nandita Sarkar
Erratum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E1
Index ................................................................... 477
Contributors
DANIEL E. AGNEW (cid:115) Department of Chemical and Biological Engineering,
University of Wisconsin-Madison, Madison, WI, USA
HAL S. ALPER (cid:115) Department of Chemical Engineering, The University of Texas
at Austin, Austin, TX, USA
KATSUTOSHI ARA (cid:115) Biological Science Laboratories, Kao Corporation, Tochigi, Japan
AUDREY L. ATKIN (cid:115) School of Biological Sciences, University of Nebraska – Lincoln,
Lincoln, NE, USA
MOHAN BABU (cid:115) Banting and Best Department of Medical Research,
University of Toronto, Toronto, ON, Canada
ARVIND A. BHAGWAT (cid:115) Environmental Microbial and Food Safety Laboratory,
U.S. Department of Agriculture, Beltsville, MD, USA; Division Environmental
Microbial & Food Safety Laboratory, Organization USDA-ARS, Beltsville, MD, USA
MEDHA BHAGWAT (cid:115) NIH Library, Office of Research Services, National Institutes
of Health, Bethesda, MD, USA
NIKË BHARUCHA (cid:115) Department of Molecular, Cellular, and Developmental Biology,
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
PAUL BLUM (cid:115) School of Biological Sciences, University of Nebraska, Lincoln, NE, USA
TRYGVE BRAUTASET (cid:115) Department of Biotechnology, SINTEF Materials and Chemistry,
Trondheim, Norway
STEPHEN T. CARTMAN (cid:115) Clostridia Research Group, BBSRC Sustainable Bioenergy
Centre, School of Molecular Medical Sciences, Centre for Biomolecular Sciences,
The University of Nottingham, Nottingham, UK
YUN-PENG CHAO (cid:115) Department of Chemical Engineering, Feng Chia University,
Taichung, Taiwan
PO TING CHEN (cid:115) Department of Biotechnology, Southern Taiwan University, Tainan,
Taiwan
SHAN-YU CHEN (cid:115) Graduate School of Biotechnology and Bioengineering,
Yuan Ze University, Taoyuan, Taiwan
CHUNG-JEN CHIANG (cid:115) Department of Medical Laboratory Science and Biotechnology,
China Medical University, Taichung, Taiwan
CLARE M. COOKSLEY (cid:115) Clostridia Research Group, BBSRC Sustainable Bioenergy
Centre, School of Molecular Medical Sciences, Centre for Biomolecular Sciences,
The University of Nottingham, Nottingham, UK
KARL DANA (cid:115) School of Biological Sciences, University of Nebraska, Lincoln, NE, USA
ELIE J. DINER (cid:115) Biomolecular Science and Engineering Program, University of
California, Santa Barbara, Santa Barbara, CA, USA
GRACE L. DOUGLAS (cid:115) Department of Food, Bioprocessing & Nutrition Sciences,
North Carolina State University, Raleigh, NC, USA
ANDREW EMILI (cid:115) Department of Molecular Genetics, Donelly Centre for Cellular and
Biomolecular Research (CCBR),University of Toronto, Toronto, ON, Canada
ix
x Contributors
ALLA GAGARINOVA (cid:115) Department of Molecular Genetics, University of Toronto,
Toronto, ON, Canada
FERNANDO GARZA-SÁNCHEZ (cid:115) Department of Molecular, Cellular and Developmental
Biology, University of California, Santa Barbara, Santa Barbara, CA, USA
YONG JUN GOH (cid:115) Department of Food, Bioprocessing & Nutrition Sciences,
North Carolina State University, Raleigh, NC, USA
JACK GREENBLATT (cid:115) Banting and Best Department of Medical Research,
University of Toronto, Terrence Donnelly Center for Cellular and Biomolecular
Research, 160 College Street Toronto, ON, Canada; Department of Molecular
Genetics, University of Toronto, 1 King’s College Circle, Toronto, ON, Canada
CHRISTOPHER S. HAYES (cid:115) Biomolecular Science and Engineering Program,
Department of Molecular, Cellular and Developmental Biology,
University of California, Santa Barbara, Santa Barbara, CA, USA
JOHN T. HEAP (cid:115) Clostridia Research Group, BBSRC Sustainable Bioenergy Centre,
School of Molecular Medical Sciences, Centre for Biomolecular Sciences,
The University of Nottingham, Nottingham, UK
JOHANNES H. HEGEMANN (cid:115) Heinrich-Heine-Universität, Lehrstuhl für Funktionelle
Genomforschung der Mikroorganismen, Düsseldorf, Germany
SVEN BORIS HEICK (cid:115) Heinrich-Heine-Universität, Lehrstuhl für Funktionelle
Genomforschung der Mikroorganismen, Düsseldorf, Germany
LES M. HOFFMAN (cid:115) Epicentre Biotechnologies, an Illumina company,
Madison, WI, USA
ALISON K. HOTTES (cid:115) Department of Molecular Biology, Lewis-Sigler Institute
for Integrative Genomics, Princeton University, Princeton, NJ, USA
MASAYUKI INUI (cid:115) Research Institute of Innovative Technology for the Earth (RITE),
Kizugawa-Shi, Kyoto, Japan
ANDREAS KAUFMANN (cid:115) LMC RISC, ETH Zürich, HPM F16, Zürich, Switzerland
SUN CHANG KIM (cid:115) Department of Biological Sciences, Korea Advanced Institute
of Science and Technology (KAIST), Daejeon, South Korea
KATSUHIKO KITAMOTO (cid:115) Department of Biotechnology, The University of Tokyo, Tokyo,
Japan
TODD R. KLAENHAMMER (cid:115) Department of Food, Bioprocessing & Nutrition Sciences,
North Carolina State University, Raleigh, NC, USA
MICHAEL KNOP (cid:115) Cell Biology and Biophysics, ZMBH, Univeristät Heidelberg,
Heidelberg, Germany
SARAH A. KUEHNE (cid:115) Clostridia Research Group, BBSRC Sustainable Bioenergy
Centre, School of Molecular Medical Sciences, Centre for Biomolecular Sciences,
The University of Nottingham, Nottingham, UK
ANUJ KUMAR (cid:115) Department of Molecular, Cellular, and Developmental Biology,
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
RAHMI LALE (cid:115) Department of Biotechnology, Norwegian University of Science
and Technology (NTNU), Trondheim, Norway
AMANDA M. LANZA (cid:115) Department of Chemical Engineering,
The University of Texas at Austin, Austin, TX, USA
JUN HYOUNG LEE (cid:115) Department of Biological Sciences, Korea Advanced Institute of
Science and Technology (KAIST), Daedeok Science Town, Daejeon, South Korea