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Editors
Prof. Dr. Christoph Wittmann Prof. Dr. Rainer Krull
Technische Universität Braunschweig Technische Universität Braunschweig
Biochemical Engineering Institute Biochemical Engineering Institute
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ISSN 0724-6145 e-ISSN 1616-8542

ISBN 978-3-642-14230-7 e-ISBN 978-3-642-14231-4
DOI 10.1007/978-3-642-14231-4
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Series Editor
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University of Hannover
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Volume Editors
Prof. Dr. Christoph Wittmann Prof. Dr. Rainer Krull
Technische Universität Braunschweig Technische Universität Braunschweig
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Editorial Board
Prof. Dr. S. Belkin Prof. Dr. W.-S. Hu
Interfaculty Biotechnology Program Chemical Engineering
Institute of Life Sciences and Materials Science
The Hebrew University of Jerusalem University of Minnesota
Jerusalem 91904, Israel 421Washington Avenue SE
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Saitama Industrial Technology Center
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Saitama, 333-0844, Japan Chemical Center, Lund University
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and Biotechnology Center for Process Biotechnology
Royal Institute of Technology Technical University of Denmark
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vi Editorial Board

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Technische Universität Hamburg-Harburg
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technik
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Honorary Editors
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Institute of Biotechnology Institute of Technical Chemistry
Eidgenössische Technische Hochschule University of Hannover, Callinstraße 3
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viii Advances in Biochemical Engineering/Biotechnology

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Laudatio

This special volume “Biosystems Engineering” is dedicated to Professor

Dr.-Ing. Dietmar Christian Hempel on the occasion of his 65th birthday, whereby
the different contributions display an excellent reflection of his research during the
past 30 years, bridging engineering and life sciences.
Prof. Hempel, born in Königsberg, studied Construction and Process Engineer-
ing in Dortmund and Berlin (1963–1971). After his PhD work on “Heterogeneous
catalytic fixed bed reactors” he became the head for “Reaction and Biochemical
Engineering” at the Bayer AG in Leverkusen, where he started to apply chemical
engineering principles to biological systems (1975–1980). This was obviously
stimulating his later research as Professor of Technical Chemistry and Chemical
Engineering at the University of Paderborn (1980–1994) and as Professor and
Director of the Institute of Biochemical Engineering at the Technische Universität
Braunschweig (1994–2009) where he kept his research interest on biological
systems in technical applications – covering various aspects process engineering
of biological and biochemical processes. As impressive outcome of his career,
Dietmar Christian Hempel authored and co-authored about 300 publications and
supervised 70 PhD students as a “Doktorvater”. From 2001 to 2008 he was head
and speaker of the still successfully continued interdisciplinary DFG-collaborate
research centre SFB 578 “Development of biotechnological processes by integrat-
ing genetic and engineering methods – From gene to product”. This collaborate
research centre displays a landmark in the German biochemical engineering com-
munity and serves as important link between cellular biology and bioprocess
engineering.
Preface

The special volume “Biosystems Engineering” reflects an emerging field of applied

research that aims at a system-level understanding of biological systems toward
their targeted design and improvement. To obtain system-wide insight, interdisci-
plinary approaches are applied, which integrate expertise from biologists, engi-
neers, and computer scientists, who have developed powerful analytical methods,
modelling concepts, and information technologies for this challenging task. With
the increasing need for sustainable production of fuels and chemicals from renew-
able resources, a major focus in this area is on the optimization of biotechnological
production strains and processes. Of specific importance hereby is the consideration
of the microorganism as a part of the bioprocess in its entirety.
In this regard, the different contributions of this volume provide an outstanding
review of novel tools, methods, and concepts in the field of biosystems engineering,
a still young, but yet powerful direction of interdisciplinary research. Part I “Creat-
ing Superior biocatalysts” of this volume highlights how system-wide analysis,
modelling and understanding of gene regulation, metabolic networks, and fluxes
open a new era for rational design and optimization of superior production strains,
one of the key pre-requisites for bio-based production of pharmaceuticals, chemi-
cals, materials, and fuels. This includes the introduction of systems and synthetic
metabolic engineering approaches as well as industrial application examples. Part II
“Linking Cellular Networks and Bioprocesses” illustrates concepts and tools which
are based on models and experiments to investigate metabolic networks in correlation
with the cell environment. In addition to the introduction of fundamental strategies
introducing thermodynamic principles as well as different modelling approaches
to metabolic network simulation, different examples directly address the link of
cells and bioreactors providing fascinating and valuable insights into the interaction
between cellular metabolism and process environment.

Braunschweig, Summer 2010 Christoph Wittmann

Rainer Krull

xi
.
Contents

Integration of Systems Biology with Bioprocess Engineering:

L-Threonine Production by Systems Metabolic Engineering
of Escherichia Coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sang Yup Lee and Jin Hwan Park

Analysis and Engineering of Metabolic Pathway Fluxes

in Corynebacterium glutamicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Christoph Wittmann

Systems Biology of Industrial Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Marta Papini, Margarita Salazar, and Jens Nielsen

De Novo Metabolic Engineering and the Promise of Synthetic DNA . . . . 101

Daniel Klein-Marcuschamer, Vikramaditya G. Yadav, Adel Ghaderi,
and Gregory N. Stephanopoulos

Systems Biology of Recombinant Protein Production

in Bacillus megaterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Rebekka Biedendieck, Boyke Bunk, Tobias Fürch, Ezequiel Franco-Lara,
Martina Jahn, and Dieter Jahn

Extending Synthetic Routes for Oligosaccharides by Enzyme,

Substrate and Reaction Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Jürgen Seibel, Hans-Joachim Jördening, and Klaus Buchholz

Regeneration of Nicotinamide Coenzymes: Principles and

Applications for the Synthesis of Chiral Compounds . . . . . . . . . . . . . . . . . . . . . 195
Andrea Weckbecker, Harald Gröger, and Werner Hummel

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

xiii
Adv Biochem Engin/Biotechnol (2010) 120: 1–19
DOI: 10.1007/10_2009_57
# Springer‐Verlag Berlin Heidelberg 2010
Published online: 6 February 2010

Integration of Systems Biology with Bioprocess

Engineering: L-Threonine Production by Systems
Metabolic Engineering of Escherichia Coli

Sang Yup Lee and Jin Hwan Park

Abstract Random mutation and selection or targeted metabolic engineering with-

out consideration of its impact on the entire metabolic and regulatory networks can
unintentionally cause genetic alterations in the region, which is not directly related
to the target metabolite. This is one of the reasons why strategies for developing
industrial strains are now shifted towards targeted metabolic engineering based on
systems biology, which is termed systems metabolic engineering. Using systems
metabolic engineering strategies, all the metabolic engineering works are con-
ducted in systems biology framework, whereby entire metabolic and regulatory
networks are thoroughly considered in an integrated manner. The targets for
purposeful engineering are selected after all possible effects on the entire metabolic
and regulatory networks are thoroughly considered. Finally, the strain, which is
capable of producing the target metabolite to a high level close to the theoretical
maximum value, can be constructed. Here we review strategies and applications of
systems biology successfully implemented on bioprocess engineering, with partic-
ular focus on developing L-threonine production strains of Escherichia coli.

Keywords L-threonine, Systems biology, Systems metabolic engineering,

Bioprocess engineering

S.Y. Lee (*), J.H. Park

Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and
Biomolecular Engineering (BK21 program), Center for Systems and Synthetic Biotechnology, Institute
for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
BioProcess Engineering Research Center, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-
701, Republic of Korea
email: [email protected]
S.Y. Lee
Department of Bio and Brain Engineering and Bioinformatics Research Center, KAIST, 335
Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
Department of Biological Sciences, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701,
Republic of Korea
2 S.Y. Lee and J.H. Park

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Use of Omics in Metabolic and Bioprocess Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3 L-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4 Integration of Systems Biology with Bioprocess
Development for L-Threonine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1 Introduction

Systems biology is changing the way biological systems are studied. Genomics,
transcriptomics, proteomics, metabolomics, and fluxomics are generating system-
wide data and information on genomic contents, transcripts, proteins, metabolites,
and metabolic fluxes, respectively. When combined with metabolic, gene regulatory,
and signaling network analysis, these data can be used to decipher the current
metabolic and regulatory status over much wider picture. Also, mathematical model-
ing and simulation became an essential tool for better understanding the biological
system under investigation at the systems-level. The systems-level analysis of micro-
organisms using genome-wide experimental and computational methods has been
proven to be beneficial for elucidating the cell physiology at the whole cell level,
consequently providing us with new strategies for metabolic engineering [1]. Further-
more, midstream and downstream processes are considered together during the
upstream strain development so that the entire bioprocess can be optimized. This
approach has been employed as a general strategy to develop biotechnologically
important microorganisms, which is termed systems metabolic engineering [2, 3].
Microorganisms that are capable of efficiently producing bioproducts, particu-
larly amino acids, have traditionally been constructed by random mutagenesis,
which can cause unwanted alterations in cellular physiology. Metabolic engineer-
ing allows overcoming such problems by specifically altering the metabolic, regu-
latory, and cellular machinery in a desired fashion [4–6]. When combined with
systems biology tools, metabolic engineering becomes an even more powerful
strategy for the enhanced production of target metabolites at a yield near the
theoretical maximum [7, 8]. In this chapter, the general strategy for the integration
of systems biology with bioprocess engineering is reviewed. Also, some recent
successful examples are showcased with particular focus on the development of
L-threonine overproducing strain of Escherichia coli.

2 Use of Omics in Metabolic and Bioprocess Engineering

In this section, the strategies for metabolic and bioprocess engineering based on the
omics technologies are described and their successful examples of applications are
summarized.
Integration of Systems Biology with Bioprocess Engineering 3

2.1 Genome

Genomic information available for industrially important microorganisms can be

usefully employed to develop strains for the enhanced production of desired
bioproducts. In particular, comparative genome analysis allows identification of
genes to be modified for a desirable metabolic phenotype. This approach was
successfully demonstrated by Ohnishi et al. [9] who carried out genome comparison
studies between a wild-type Corynebacterium glutamicum strain and an L-lysine-
producing strain obtained by random mutation and selection. They were able to
identify mutations in the gnd gene that are suspected to contribute to the efficient
production of L-lysine. They introduced the identified mutation into the AHP-3
strain to make an APG-4 strain, which allowed 15% increase in L-lysine production
[9]. Further improvement was performed by introducing mqo mutation selected on
the basis of genome comparison. The final strain allowed production of 95 g L
1
L-lysine by fed-batch culture [10]. In the case of Mannheimia succiniciproducens,
gene knockout studies were conducted to understand its anaerobic fermentative
metabolism and consequently to develop a metabolically engineered strain capable
of producing succinic acid without by-product formation [11]. Likewise, the
biosynthetic pathway of L-methionine in C. glutamicum was elucidated by genome-
wide analysis combined with targeted gene deletion and homologous complementa-
tion [12].
Genome-wide profiling analysis based on the complete genome sequence can
also be used to identify the functions of certain genes encoding enzymes responsi-
ble for the bioproduct to be overproduced. Using this method, McHardy et al. [13]
discovered potential aminotransferase encoding genes and experimentally charac-
terized them, resulting in the discovery of 11 new aminotransferases participating in
the biosynthesis of branched-chain amino acids and phenylalanine in C. glutamicum.
Two genes involved in the biosynthesis of synechoxanthin were identified by
comparative genomics, and their functions were verified by insertional inactivation
[14]. The cruE gene encodes b-carotene desaturase/methyltransferase, which con-
verts b-carotene to renierapurpurin. The cruH gene encodes an enzyme that is
responsible for the hydroxylation/oxidation of the C-18 and C-180 methyl groups
of renierapurpurin. Based on these findings, a complete pathway for synechoxanthin
biosynthesis was proposed.
In another approach, a reduced-genome strain can be constructed by delet-
ing nonessential genes, while preserving those required for good growth profiles
and protein production [15, 16]. As a conclusion, full genome sequence pro-
vides invaluable information for genome-scale engineering, thereby leading to
the development of strains showing significantly improved performance. This
perspective is strongly supported by the fact that recent DNA sequencing tech-
nologies allow sequencing of up to one billion bases in a single day at low
cost [17].
4 S.Y. Lee and J.H. Park

2.2 Transcriptome

Gene expression profiling by DNA microarray captures mRNA transcription level

for thousands of genes of multiple strains under different conditions simulta-
neously, and thus makes it possible to analyze cell physiology and global regulation
at system-wide transcript level. This tool can be used to analyze the cell physiology
and to select target genes to be engineered as well. For example, the target genes for
the enhanced production of recombinant protein have been successfully identified
by DNA microarray experiments by comparing the transcriptome profiles before
and after induction during the high cell density culture [18]. Overexpression of the
prsA (encoding phosphoribosyl pyrophosphate synthetase) and the glpF (glycerol
transporter) genes, which were selected among the downregulated genes, allowed a
significant increase in IFG-If production (from 1.8 to 4.3 g L
1). Transcriptome
analysis can also be used to characterize certain genes playing critical roles in
carbon metabolism. As an example, induction of two genes encoding lactate
permease and lactate dehydrogenase during L-glutamate production using lactate
as a carbon source in C. glutamicum was identified by transcriptome profiling [19].
Recently, enhanced production of L-lysine was achieved by introducing useful
mutations identified by transcriptome analysis into a defined L-lysine producer.
To identify the amino acid biosynthetic genes that were induced, transcriptome
analysis of an L-lysine producer mutant, C. glutamicum B-6, was carried out [20].
Among the genes showing different transcript levels, the leuC gene was selected as
a useful mutation, and subsequently introduced into the defined L-lysine producer,
AHD-2 (hom59 and lysC311), resulting in 14% increase in L-lysine production [21].
In another study, transcriptome analysis was used to identify target genes to be
engineered in L-lysine producing C. glutamicum, in which upregulated genes includ-
ing those encoding a methyltransferase and ammonium uptake system were over-
expressed, resulting in 40% increase in L-lysine production [22]. More recently,
novel factors enhancing heterologous protein secretion in yeast were identified by
transcriptome profiling [23]. Overexpression of the significantly upregulated genes
involved in protein transport, folding, and exocytosis improved both specific pro-
duction rate and volumetric productivity of an antibody fragment by ca. 2.5-fold.
Another recent report indicates that transcriptome analysis is an effective way of
initially screening the genes targets for molecular breeding. As a demonstration of
this approach, a xylitol-producing strain was constructed by inserting the genes
encoding the NADPH-dependent D-xylose reductase and D-xylose permease into
the E. coli chromosome. Comparative transcriptome analysis of xylitol-producing
and nonproducing conditions for the recombinant strain revealed that xylitol pro-
duction caused down-regulation of 56 genes. From this study, the yhbC gene was
selected as a candidate cause for the suppression of NADPH supply and thus
deleted, which resulted in a 2.7-fold increase in xylitol production [24]. Exemplary
results mentioned here demonstrate that novel gene targets for strain improvement
can be identified based on the global gene expression analysis.