Strain improvement by metabolic engineering: lysine production
as a case study for systems biology
Mattheos Koffas1 and Gregory Stephanopoulos2
A central goal of systems biology is the elucidation of cell
function and physiology through the integrated use of broad
based genomic and physiological data. Such systemic
approaches have been employed extensively in the past, as
they are a central element of metabolic flux analysis, the
distribution of kinetic control in pathways, and the key
differentiating characteristic of metabolic engineering. In one
case study, these tools have been applied to the improvement
of lysine-producing strains of Corynebacterium glutamicum.
The systematic study of the physiology of this organism
allowed the identification of specific metabolic targets and
subsequently led to significant improvements in product yield
and productivity. This case study can serve as a guide for the
development of systems biology tools for the utilization of large
volumes of cell- and genome-wide transcriptional and
physiological data.
Addresses
1
Department of Chemical and Biological Engineering, University at
Buffalo, The State University of New York, 904 Furnas Hall, Buffalo,
New York 14260, USA
2
Department of Chemical Engineering, Massachusetts Institute of
Technology, Room 56-469, Cambridge, Massachussetts 02139, USA
Corresponding author: Koffas, Mattheos (mkoffas@eng.buffalo.edu)
Current Opinion in Biotechnology 2005, 16:361–366
This review comes from a themed issue on
Systems biology
Edited by Hans V Westerhoff
Available online 16th May 2005
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2005.04.010
Introduction
L-Lysine is an essential amino acid that has to be available in sufficient amounts in feed-stuffs to meet the
nutritional requirements of animals and humans. This
supplementation is realized by the direct addition of
lysine and, as a result, a tremendous growth in the market
has taken place in the past ten years. It is estimated that
more than 600 000 metric tons of lysine are produced
annually and, owing to the exploitation of new uses in
pharmaceuticals, cosmetics and polymer materials, the
market shows a growth potential of 7–10% per year.
In 1956, a remarkable soil bacterium, Corynebacterium
glutamicum, capable of producing large amounts of gluwww.sciencedirect.com
tamic acid was isolated by a researcher at Kyowa Hakko
Kogyo Co. in Japan. As both glutamic acid and lysine are
derived from tricarboxylic acid (TCA) cycle metabolites,
such a glutamic acid overproducing strain soon allowed
the development of C. glutamicum lysine overproducers.
Since then, a growing number of companies and academic
researchers have carried out research aimed at the development of more efficient L-lysine production platforms.
In this review we shall summarize some of the major
accomplishments involving the use of systems biology
approaches to optimize L-lysine biosynthesis with an
emphasis on research carried out since 2002.
Early studies on Corynebacterium glutamicum
The soil bacterium that is currently used for the biosynthesis of L-lysine was initially classified as Micrococcus
glutamicus, but is known today as Corynebacterium glutamicum [1]. As early as 1958, mutant auxotrophs (such as
homoserine auxotrophs) and later regulatory mutants of
this strain were developed that are capable of high rates of
amino acid production. Since then, C. glutamicum mutants
have become the sole producers of L-lysine manufactured today.
The initial auxotrophs were developed through empirical,
iterative procedures involving mutagenesis and selection
[2]. Strain optimization was performed by random
approaches (chemical mutagenesis or UV irradiation) to
release key enzymes from strict metabolite regulation
(feedback inhibition) [3]. The resulting strains were
grown in small-scale fermentations to select optimal
producers and this recursive procedure would be repeated
for the newly optimized strains. Strains developed by
such approaches could reach relatively low conversion
yields of approximately 0.2–0.3 g of L-lysine (g sugar) 1
[4,5]. Later on, the disadvantage of supplementing the
defined medium with large amounts of the auxotrophic
amino acids would be overcome by developing ‘leaky
strains’. Such strains would synthesize a limited amount
of the required metabolite and, as a result, its intracellular
level would be low, thus avoiding feedback inhibition and
repression of key enzymes in the lysine biosynthetic
pathway. Finally, cell fusion using the method of protoplast fusion has also been applied for the successful
development of industrial lysine producers [6].
Metabolite balancing
Though successful, random mutational approaches were
uncertain and tedious. A more rational design of lysine
overproducers was initiated in the 1980s and was based on
biochemical and physiological measurements, usually
Current Opinion in Biotechnology 2005, 16:361–366
362 Systems biology
obtained from continuous culture experiments. Using
these data, a mathematical formulation based on mass
balances of extracellular substrate consumption and product formation rates was developed to analyze the complex metabolic network of lysine biosynthesis [7]. This
approach offered a more rational methodology for strain
design and allowed the consolidation and validation of
metabolic networks, the identification of key branch
points, and the determination of L-lysine maximum
theoretical yields.
In the first such attempt to analyze the metabolic network, stoichiometrically based mass balances were
applied by Vallino and Stephanopoulos in C. glutamicum
fermentation experiments to determine metabolic fluxes
and to identify potential metabolic bottlenecks [8,9].
Some of the main contributions of this work were the
solidification of the C. glutamicum biochemistry (guided
mainly by enzyme assays), the determination of basal
metabolic flux distributions during growth and lysine
biosynthesis, the characterization of the principal nodes
of the lysine biosynthetic pathway, and the determination of the maximum possible lysine yield [8–12].
Carbon flux distributions resulting from metabolic perturbations demonstrated that lysine yield appeared to
be constrained by the flexibility of the phosphoenol
pyruvate (PEP)/pyruvate node. Moreover, it was shown
that the rate of lysine biosynthesis was always less than
or equal to the rate of oxaloacetate synthesis via the
anaplerotic pathways.
The value of the intracellular (or metabolic) flux analysis
that was initiated by this research is evident in the
diversity of applications that have since made use of this
approach. The main weakness of the extracellular metabolite balancing method was its inability to provide flux
estimates in cases of metabolic network structural singularities (i.e. where the metabolic network is structured
such that simple metabolite balances cannot provide flux
estimation for separate reactions that lead to the same
product from different substrates). For example, it was
not possible to estimate carbon fluxes through the PEP
carboxylase and pyruvate carboxylase anaplerotic reactions. In addition, the rates of extracellular metabolite
excretion and consumption could only provide net fluxes,
while no information about the extent of reversibility of a
reaction could be obtained. Finally, assumptions about
the cellular energy balance had to be included, thus
raising further uncertainties. To overcome these problems, additional experimental data were required to
establish a reliable stationary flux analysis: isotopic tracer
experiments have emerged as a prominent tool for this
purpose.
Isotopic tracer methods
The advantage of introducing stable isotopic labeling
methods is that the label can be traced from substrate
Current Opinion in Biotechnology 2005, 16:361–366
to product with a specific pattern that is completely
dependent upon the structure and fluxes of the biochemical pathway reactions. Thus, labeling methods can be
used to determine flux distribution in structurally ‘singular’ groups as well as to elucidate the reversibility of
intracellular reactions. These more refined methods can
also validate the flux estimates and accompanying assumptions used in the overall material balance technique.
In one of the first efforts to use isotopomer labeling
to elucidate the C. glutamicum physiology, the nuclear
magnetic resonance (NMR) technique was applied by
Yamaguchi et al. [13] to study actual flux distributions in
two routes of lysine biosynthesis: the one-step diaminopimelate dehydrogenase route and the four-step succinylase route (Figure 1). The authors found that the one-step
route accounted for 30–40% of the flux towards lysine.
Later, by using knockout mutants and recombinant
strains, Shaw-Reid et al. [14] established that the fourstep route is dispensable for lysine production. In a similar
analysis, Sonntag et al. [15] fed cultures with (6-13C)glucose and showed that flux contribution to the one-step
route (diaminopimelate dehydrogenase) decreased from
72% to zero over the course of shake flask culture time.
This was traced to the availability of ammonium in the
culture medium. Together, these studies demonstrate the
powerful use of the NMR technique to determine accurate split ratios of pathways.
Moving to the more challenging problem of analysis of
the central carbon metabolism of C. glutamicum, Park and
colleagues [16] developed and used a mathematical
model to study isotopomer distributions of TCA cycle
intermediates following the administration of 13C-labeled
substrates. Previous studies had utilized isotopomer
labeling to quantify fluxes at the PEP/pyruvate node
and to quantify exchange fluxes. However, these studies
either investigated cases in which only a single flux was
considered in each direction, or cases in which parallel
reactions were lumped together. Applying this approach
to select genetic backgrounds, Park and colleagues were
able not only to identify a novel pyruvate carboxylating
anaplerotic pathway in C. glutamicum, but also demonstrated that 90% of total anaplerotic activity resulted from
pyruvate carboxylation. This result was later confirmed in
wild-type C. glutamicum lysine-producing strains [17] by
utilizing a thorough and universal model for carbon isotopomer labeling experiments, which takes into account
the bidirectionality of biochemical reactions [18–20]. The
modeling work complemented earlier physiological data
suggesting that the only other anaplerotic reaction in C.
glutamicum, catalyzed by phosphoenolpyruvate carboxylase, is dispensable for lysine biosynthesis [21]. Separate
studies have confirmed the presence of pyruvate carboxylase in C. glutamicum [22,23] and its overexpression
confirmed that this anaplerotic reaction plays a key role
in lysine biosynthesis [24,25].
www.sciencedirect.com
Strain improvement by metabolic engineering Koffas and Stephanopoulos 363
Figure 1
O
OH
HO
Aspartate
O
NH2
ATP
Aspartate
kinase
ADP
O
O
OH
O
L-4-Aspartylphosphate
NH2
HO P OH
O
NADPH
Aspartate
semialdehyde
dehydrogenase
NADP
O
H
HO
L-Aspartate-4-semialdehyde
NH2
Dihydrodipicolinate
synthase
O
Pyruvate
L-2,3,-Dihydrodipicolinate
HO
N
COOH
O
NADPH
Dihydrodipicolinate
reductase
NADP
L-2,3,4,5-Tetrahydrodipicolinate
HO
N
COOH
Succinyl-CoA
O
CoA
Tetrahydrodipicolinate
succinylase
O
O
HN
HO
COOH
N-Succinyl-2-L-amino-6oxopimelate
COOH
O
NH4+
Glutamate
Succinyl-amino
ketopimelate
transaminase
NADPH
Oxoglutarate
O
NADP
N-Succinyl-LL-2,6diaminopimelate
COOH
HO
Diaminopimelate
dehydrogenase
COOH
HN
NH2
O
Succinyl-amino
pimelate
succinylase
Succinate
O
COOH
HO
NH2
LL-2,6-Diaminopimelate
H2N
Diaminopimelate
epimerase
O
COOH
HO
NH2
D,L-2,6-Diaminopimelate
H2N
Diaminopimelate
decarboxylase
CO2
O
NH2
HO
L-Lysine
NH2
Current Opinion in Biotechnology
The L-lysine biosynthetic pathway in Corynebacterium glutamicum.
www.sciencedirect.com
Current Opinion in Biotechnology 2005, 16:361–366
364 Systems biology
Despite the success stories, NMR-based approaches are
limited by their low sensitivity: labeling patterns are
mostly analyzed from amino acids obtained from hydrolyzed cellular proteins [26]. In this respect, labeling
patterns of intermediary metabolites, usually occurring
at low concentrations, are difficult to measure directly
[27]. To address this limitation, mass spectrometry (MS)
methods have emerged as a powerful alternative for
analyzing labeling patterns: these methods require small
amounts of sample and are faster and are more sensitive
than NMR [28]. These features make MS suitable for the
measurement of intracellular metabolite labeling patterns, and thus for the investigation of dynamic responses
of the metabolism to defined changes of cultivation conditions.
The application of MS for identifying the topology of
metabolic networks has been successfully demonstrated
in many different organisms, including C. glutamicum
during lysine biosynthesis. Wittmann and Heinzle [29]
first determined metabolite fluxes in the lysine biosynthetic network using MALDI-TOF (matrix-assisted laser
desorption ionization time-of-flight) MS, although they
were unable to resolve all the metabolic network singularities. Later on, gas chromatography MS was introduced
to perform a comparative metabolic network analysis of a
genealogy of five successive generations of lysine-producing C. glutamicum strains [30] and to make comparative
studies between growth on glucose and fructose [31]. It
appears, however, that the major potential of MS analysis
lies in its applicability to high-throughput metabolic flux
analysis that will allow broad quantitative screening of
lysine-producing C. glutamicum mutants [32].
Genome sequencing and functional genomics
The completion of the genome sequence of C. glutamicum
provides a leap forward both for understanding the biology of the organism and for enabling further metabolic
engineering for the production of lysine and other biochemical products [33,34,35]. Annotation of the genome
sequence provided valuable hints for missing metabolic
steps in the lysine biosynthetic pathway, while comparative genomics allowed the identification of beneficial
mutations for the improvement of lysine production
[36,37]. In an excellent example of inverse metabolic
engineering, a comparative genomics analysis between
an engineered lysine-producing strain and its parental
strain identified beneficial point mutations that were then
used to guide the construction of a high lysine-producing
strain [38,39]. When the fermentation temperature was
raised to 40 8C, lysine production increased even further
and reached 85 g/L within 28 h [40].
The availability of the whole genome sequence provided
the necessary tools for performing genome-wide expression analyses (functional genomics). The use of DNA
microarrays in the elucidation of C. glutamicum transcripCurrent Opinion in Biotechnology 2005, 16:361–366
tion regulation has been reviewed extensively by Wendisch and colleagues and has already been utilized in
efforts to develop valine-producing strains [41,42].
Furthermore, global transcription profiling has been
employed for elucidating C. glutamicum physiology; for
example, 92 genes that are up- or downregulated during
phosphate starvation were identified [43] and the effect of
the deletion of acnR, a TetR-type repressor of the aconitase gene, was assessed [44]. Other related studies have
revealed the effect of deletions of signal transduction
systems [45] and the effect of deletion of the pyruvate
kinase ( pyk) gene [46] on the C. glutamicum transcriptome.
The expanded view of C. glutamicum physiology, made
possible by genome sequencing and parallel, highthroughput technologies, has facilitated the application
of a more holistic approach. Such an approach will allow
links between the different components of cell physiology, such as the transcriptome and fluxome, to be elucidated [47]. It is expected that such a systems approach
will prove a very useful adjunct in designing optimized C.
glutamicum strains for maximal lysine production, in a
similar way to that already demonstrated for other important industrial strains and applications [48].
Conclusions
It might be noted that the topic of this issue, systems
biology, was hardly mentioned in this article. Yet, a
systemic approach to the analysis of C. glutamicum physiology and the improvement of lysine-producing strains
is a recurrent theme of the presented work that spans
approximately two decades [49]. The analysis and
determination of fluxes through an integrated reconstruction of the biosynthetic and central carbon metabolic
pathways of the organism was one of the first demonstrations of a systems approach to the analysis of microbial
physiology. Furthermore, the calculation of theoretical
maximum yields and the identification of key branch
points harboring control of the entire network flux could
not have been accomplished without a systems mindframe. Finally, perhaps the most outstanding illustration
of the application of systems concepts that led to the
improvement of lysine-production strains is the coordinated overexpression of two genes (encoding pyruvate
carboxylase and aspartokinase), in realization of the fact
that pathway flux control is shared and not localized to any
particular enzyme [25]. This resulted in a marked
improvement in specific lysine productivity, where many
similar efforts had failed in the past.
We offer this case study as an example of what can be
accomplished by systems-oriented approaches to strain
improvement. The need for such approaches will be
intensified in the near future in light of the large volumes
of data that will be generated from cell-wide and genomewide measurements. On the one hand, we do not yet have
good methods that will allow us to make judicious use of
www.sciencedirect.com
Strain improvement by metabolic engineering Koffas and Stephanopoulos 365
this avalanche of information whereas, on the other hand,
there is a sufficient arsenal of tools that can be applied to
smaller scale, more focused investigations. We believe
that significant progress can be accomplished in strain
improvement with these methods, while more powerful
approaches are being developed that will allow a more
complete view of the metabolic machinery of microorganisms and will facilitate their modification for industrial
applications by metabolic engineering.
15. Sonntag K, Eggeling L, De Graaf AA, Sahm H: Flux partitioning in
the split pathway of lysine synthesis in Corynebacterium
glutamicum. Quantification by 13C- and 1H-NMR
spectroscopy. Eur J Biochem 1993, 213:1325-1331.
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