Table Of ContentTHE METABOLIC
PATHWAY
ENGINEERING
HANDBOOK
Tools and Applications
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The Metabolic Pathway Engineering Handbook, 1st Edition
The Metabolic Pathway Engineering Handbook: Fundamentals
The Metabolic Pathway Engineering Handbook: Tools and Applications
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THE METABOLIC
PATHWAY
ENGINEERING
HANDBOOK
Tools and Applications
Edited by
Christina D. Smolke
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CRC Press
Taylor & Francis Group
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Contents
Introduction ..................................................................................................ix
Editor .............................................................................................................xv
Contributors ...............................................................................................xvii
SECTION I Evolutionary Tools in Metabolic Engineering
Claudia Schmidt-Dannert
1 Evolutionary Engineering of Industrially Important
Microbial Phenotypes .....................................................................................1-1
Stephen S. Fong
2 Improving Protein Functions by Directed Evolution ................................... 2-1
Nikhil U. Nair and Huimin Zhao
3 Engineering DNA and RNA Regulatory Regions through Random
Mutagenesis and Screening ............................................................................3-1
Ichiro Matsumura, Sean A. Lynch, and Justin P. Gallivan
4 Evolving Pathways and Genomes for the Production of Natural and
Novel Compounds ..........................................................................................4-1
Ethan T. Johnson and Claudia Schmidt-Dannert
5 Models Predicting Optimized Strategies for Protein Evolution ...................5-1
Jonathan J. Silberg and Peter Q. Nguyen
SECTION II Gene Expression Tools for Metabolic Pathway
Engineering
Christina D. Smolke
6 Low-Copy Number Plasmids as Artificial Chromosomes ............................6-1
Kristala L. Jones Prather
7 Chromosomal Engineering Strategies ............................................................7-1
Kenan C. Murphy
v
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vi Contents
8 Regulating Gene Expression through Engineered RNA
Technologies ...................................................................................................8-1
Maung Nyan Win and Christina D. Smolke
9 Tools Designed to Regulate Translational Efficiency ................................... 9-1
Claes Gustafsson
10 Metabolic Engineering of the Secretory Processing Pathway
in Eukaryotes.................................................................................................10-1
Mohak Mhatre, Maira P. Pellegrini, and Michael J. Betenbaugh
11 Engineering Multifunctional Enzyme Systems for Optimized
Metabolite Transfer between Sequential Conversion Steps .........................11-1
Robert J. Conrado, Thomas J. Mansell, and Matthew P. DeLisa
12 Practical Pathway Engineering—Demonstration in
Integrating Tools ...........................................................................................12-1
Sung Kuk Lee and Jay D. Keasling
SECTION III Application of Emerging Technologies
to Metabolic Engineering
Jay D. Keasling
13 Genome-Wide Technologies: DNA Microarrays, Phenotypic
Microarrays, and Proteomics .......................................................................13-1
Seh Hee Jang, Mee-Jung Han, Sang Yup Lee, Jong Hyun Choi, and Xiao Xia Xia
14 Monitoring and Measuring the Metabolome ...............................................14-1
Maria Rowena N. Monton and Tomoyoshi Soga
SECTION IV Future Prospects in Metabolic Engineering
Bernhard Ø. Palsson and Sang Yup Lee
15 Systems Biology, Genome-Scale Models, and
Metabolic Engineering ..................................................................................15-1
Sang Yup Lee, Hyun Uk Kim, Hongseok Yun, Seung Bum Sohn, Jin Sik Kim,
Bernhard Ø. Palsson, Markus J. Herrgård, and Vasiliy A. Portnoy
16 Cell-Free Systems for Metabolic Engineering ..............................................16-1
Kara A. Calhoun and James R. Swartz
17 In Silico Models for Metabolic Systems Engineering ...................................17-1
Kumar Selvarajoo, Satya Nanda Vel Arjunan, and Masaru Tomita
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Contents vii
SECTION V Tools for Experimentally Determining Flux
through Pathways
Ralf Takors
18 GC–MS for Metabolic Flux Analysis ............................................................18-1
Christoph Wittmann
19 Tools for Measuring Intermediate and Product Formation ........................19-1
Marco Oldiges
20 Use of AEX-HPLC-ESI-MS for 13C-Labeling Based Metabolic Flux
Analysis in Saccharomyces cerevisiae and Penicillium chrysogenum .........20-1
Wouter A. van Winden, Roelco J. Kleijn, Walter M. van Gulik, and Joseph J. Heijnen
SECTION VI Future Applications of Metabolic Engineering
Brian F. Pfleger
21 Energy and Cofactor Issues in Fermentation and
Oxyfunctionalization Processes ...................................................................21-1
Bruno Bühler, Lars M. Blank, Birgitta E. Ebert, Katja Bühler, and Andreas Schmid
22 Microbial Biosynthesis of Fine Chemicals:
An Emerging Technology .............................................................................22-1
Zachary L. Fowler and Mattheos Koffas
23 Applications of Metabolic Engineering for Natural
Drug Discovery .............................................................................................23-1
Yi Tang, Suzanne Ma, and Wladyslaw A. Wojcicki
24 Metabolic Engineering for Alternative Fuels ...............................................24-1
Yandi Dharmadi and Ramon Gonzalez
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Introduction
Progression of Biological Synthesis Methods
toward Commercial Relevance
The advent of recombinant DNA in the 1970s brought transformative technologies for the synthesis and
manipulation of artificial genetic material. The ability to amplify, cut, and piece together fragments of
DNA outside of a cell and to get (or transform) that DNA into a cell of interest resulted in a set of molec-
ular cloning tools that enabled the field of genetic engineering. In genetic engineering, foreign DNA
that encodes for new or altered functions or traits is inserted into an organism of interest. Many early
applications of recombinant DNA technology focused on heterologous protein production in microbial
hosts. The first medicine made through recombinant DNA technology that was approved by the United
States Federal Drug Administration was the synthesis of synthetic “human” insulin in Escherichia coli.
This was an important early application of recombinant DNA technology, as the success of producing
a safe and effective synthetic hormone in a bacteria led to the widespread acceptance of the technology
and significant resources and funding to be directed to its support and advancement.
As the technologies in support of synthesizing and manipulating artificial DNA matured and
advanced, so did the applications to which they were applied. The early successful applications of recom-
binant DNA technology resulted in alternative routes to the synthesis of medicines, such as insulin,
human growth factor, and erythropoietin, vaccines, and even genetically modified organisms, includ-
ing crops that exhibit more desirable traits. Technologies were developed for the manipulation of artifi-
cial DNA in both prokaryotic and eukaryotic host organisms, including mammalian and plant cells. In
addition, inspired by the diversity of natural products, chemicals, and materials synthesized by biologi-
cal systems that are observed in the natural world, researchers began to look beyond applications that
were limited to the synthesis of a single heterologous protein product in a cellular host to more com-
plicated engineering feats. In particular, these new applications focused on the manipulation of sets or
combinations of proteins, or enzymes, that acted in conjunction in a cell, within metabolic pathways, to
convert energy and precursor chemicals into desired natural and non-natural products.
The production of chemicals, materials, and energy through biology presents an alternative to tra-
ditional chemical synthesis routes. While the development of chemical synthesis methods for the
production of valuable chemicals and small molecule pharmaceuticals is a more mature field and has
demonstrated significant successes, many chemicals remain difficult to be synthesized through such
strategies, particularly those with many chiral centers. Biological catalysts, or enzymes, have dem-
onstrated remarkable adeptness at the synthesis of very complex molecules. In addition, cellular bio-
synthesis strategies offer several advantages over traditional chemical synthesis strategies in that the
former is often conducted under less harsh conditions, thereby enabling “green” synthesis strategies
that are associated with the production of fewer toxic by-products. In addition, cellular biosynthesis
ix
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