ARTICLE IN PRESS

Metabolic Engineering 7 (2005) 291–301

www.elsevier.com/locate/ymben

Lysine and glutamate production by Corynebacterium glutamicum

on glucose, fructose and sucrose: Roles of malic enzyme and
fructose-1,6-bisphosphatase
Tobias Georgi, Doris Rittmann, Volker F. Wendisch
Institute of Biotechnology 1, Research Center Juelich, Juelich D-52428, Germany
Received 30 November 2004; received in revised form 3 May 2005; accepted 10 May 2005
Available online 27 June 2005

Abstract

In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose
or sucrose are typically used. Glutamate production by C. glutamicum ATCC13032 was very similar on glucose, fructose, glucose
plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on
glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and
lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic
enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on
any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine
yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C.
glutamicum with sucrose as the carbon source.
r 2005 Elsevier Inc. All rights reserved.

Keywords: Corynebacterium glutamicum; Lysine production; Glutamate production; Fructose; Sucrose; Glucose; Malic enzyme; malE; Fructose-1,6-
bisphosphatase; fbp

1. Introduction respect to carbon precursor supply (Eggeling, 1994;

Corynebacterium glutamicum is a Gram-positive rod
belonging to the suborder Corynebacterianeae together
with its pathogenic relative C. diphtheria of the family of
Corynebacteriaceae and Mycobacterium tuberculosis of
the family of Mycobacteriaceae (Stackebrandt et al.,
1997). C. glutamicum is widely used for the biotechno-
logical production of 41 200 000 tons of L-glutamate
per year, of 4550 000 tons of L-lysine per year and of
several other amino acids (Hermann, 2003). A number
of molecular targets essential for efficient lysine produc-
tion have been identified in the lysine biosynthesis
pathway, in pathways leading to side products and with
Corresponding author.
E-mail address: v.wendisch@fz-juelich.de (V.F. Wendisch).
Hermann, 2003; Pfefferle et al., 2003; Sahm et al., 2000).
Although media containing glucose, fructose or sucrose
are used for lysine and glutamate production, the
evaluation of the molecular targets for amino acid
production has focused on glucose as carbon source.
Aspartokinase is the key enzyme of the split lysine
biosynthesis pathway (Schrumpf et al., 1991). It is
encoded by lysC and shows cumulative feedback
inhibition by lysine and threonine (Kalinowski et al.,
1991; Shiio and Miyajima, 1969). Overexpression of
lysC (Cremer et al., 1991) and in particular of lysC
alleles coding for versions of aspartokinase that are
not feedback-inhibited by lysine improved lysine
production (Schrumpf et al., 1992; Thierbach et al.,
1990). In addition, overexpression of dapA, which
codes for dihydrodipicolinate synthase, improved lysine

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doi:10.1016/j.ymben.2005.05.001
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292 T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301

Fig. 1. Schematic representation of the central carbon metabolism of C. glutamicum. Enzymatic reactions/pathways are indicated for uptake and
activation of glucose, fructose and sucrose, for glycolysis, pentose phosphate pathway and the tricarboxylic acid cycle and for glutamate and lysine
synthesis. Gene names are indicated besides the reactions of glucose-6-phosphate dehydrogenase (zwf), fructose-1,6-bisphosphatase (fbp), pyruvate
carboxylase (pyc), malic enzyme (malE), aspartokinase (lysC) and homserine dehydrogenase (hom).

production (Eggeling et al., 1998), while identification of
the lysine export system LysE (Vrljic et al., 1996) provided
another target for strain improvement. Side product
formation was decreased by introducing hom alleles
leading to restricted homoserine dehydrogenase enzyme
levels and, in addition, the threonine concentrations in
such hom mutants are too low for feedback inhibition of
aspartokinase and thus lysine production was improved
(Eikmanns et al., 1991; Follettie et al., 1988).
Increasing carbon precursor supply enhanced lysine
production on glucose and was achieved by over-
expression of pyc (Peters-Wendisch et al., 2001), which
encodes pyruvate carboxylase (Peters-Wendisch et al.,
1998) (Fig. 1). Besides the two anaplerotic C3 carbox-
ylating enzymes PEP carboxylase (Eikmanns et al.,
1989) and pyruvate carboxylase (Peters-Wendisch et al.,
1996, 1997), C. glutamicum possesses the C4 decarbox-
ylating enzymes PEP carboxykinase and malic enzyme,
encoded by pck (Riedel et al., 2001) and malE (Gourdon
et al., 2000), respectively (Fig. 1). Deletion of the pck
gene resulted in an altered flux distribution at the PEP/
pyruvate and oxaloacetate/malate nodes (Petersen et al.,
2001), which are rigid metabolic nodes of the C.
glutamicum metabolic biochemical network (Stephano-
poulos and Vallino, 1991), and improved lysine produc-
tion on glucose (Riedel et al., 2001).
Based on the knowledge about molecular targets to
improve lysine production, introduction of point muta-
tions in the aspartokinase gene lysC, in the homoserine
dehydrogenase gene hom and in the pyruvate carbox-
ylase gene pyc, which were discovered in a classically
obtained lysine production strain, into the wild type
resulted in very efficient lysine production on glucose
(Ohnishi et al., 2002).
With respect to the regeneration of the cofactor
NADPH, of which 4 mol are required for the production
 ARTICLE IN PRESS
T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
of 1 mol of lysine (Marx et al., 1997), carbon flux
analysis revealed the critical importance of the pentose
phosphate pathway during growth and lysine produc-
tion on glucose (Marx et al., 1996, 1997, 1999) which
also became evident from a metabolic flux genealogy
study of different lysine-producing strains (Wittmann
and Heinzle, 2002). Deletion of the phosphoglucoisome-
rase gene pgi increased flux through the pentose
phosphate shunt and improved lysine production on
glucose (Marx et al., 2003) (Fig. 1). Similarly, expression
of the glucose-6-phosphate dehydrogenase gene zwf
(Fig. 1) increased pentose phosphate pathway flux and
lysine production on glucose (Ando et al., 2002).
During growth of C. glutamicum on fructose as sole
carbon source or on fructose/glucose mixtures, a low
pentose phosphate flux was demonstrated by carbon
flux analysis (Dominguez et al., 1998; Kiefer et al., 2004;
Pons et al., 1996). It was proposed that to meet the
NADPH requirement for growth and lysine production
on fructose or on fructose/glucose mixtures malic
enzyme activity has to be increased (Dominguez et al.,
1998). Alternatively, as fructose is transported into the
cell and phosphorylated to fructose-1-phosphate by a
PEP-dependent phosphotransferase system and enters
glycolysis after phosphorylation to fructose-1,6-bispho-
sphate (Kotrba et al., 2001; Parche et al., 2001) (Fig. 1),
overexpression of the fructose-1,6-bisphosphatase gene
fbp (Rittmann et al., 2003) was proposed to increase the
pentose phosphate pathway flux as well as lysine
production on fructose (Kiefer et al., 2004; Pons et al.,
1996). In this study, we evaluated the effects of
introducing point mutation alleles of the genes for
pyruvate carboxylase, homoserine dehydrogenase, as-
partokinase and glucose-6-phosphate dehydrogenase
(pycP458S, homV59A lysCT311I and zwfA243T) into the
wild-type C. glutamicum strain ATCC 13032 on lysine
Table 1
Strains and plasmids used in this study
Strain or plasmid Relevant characteristics
E. coli
DH5aMCR endA1 supE44 recA1 gyrA96 relA1 deoR U169 F80dlacZDM15 mcrA D(mrr-hsdRMS-mcrBC)
C. glutamicum
WT wild-type strain, ATCC 13032
DM1727 pycP458S, derived from WT
DM1728 pycP458S, homV59A, derived from DM1727
DM1729 pycP458S, homV59A, lysCT311I, derived from DM1728
DM1730 pycP458S, homV59A, lysCT311I, zwfA243T, derived from DM1729
DM1800 pycP458S, lysCT311I, derived from DM1727
Plasmids
pXMJ19-fbp CmR; C. glutamicum/E. coli shuttle vector for regulated gene expression (Ptac, lacIQ, pBL1
oriVC.g., pK18 oriVE.c.) carrying fbp
pVWEx1 KanR; C. glutamicum/E. coli shuttle vector for regulated gene expression (Ptac, lacIQ, pHM1519
oriVC.g., pACYC177 oriVE.c.)
pVWEx1-fbp pVWEx1 derivative carrying fbp
pVWEx1-malE pVWEx1 derivative carrying malE
293
production when lysine was produced on fructose,
glucose+fructose or sucrose. Additionally, we deter-
mined the roles of malic enzyme and fructose-1,6-
bisphosphatase for lysine production of C. glutamicum
on fructose, fructose/glucose mixtures, on glucose and
on sucrose.
2. Materials and methods
Bacteria, plasmids and growth conditions: Bacterial
strains and plasmids used in this study are listed in Table
1. Escherichia coli was grown on Luria-Bertani (LB)
medium as the standard medium (Sambrook et al.,
1989), while brain–heart infusion medium (BHI; Difco)
was used as complex medium for C. glutamicum. As
minimal medium for C. glutamicum CgXII (Keilhauer et
al., 1993) including 0.03 g protocatechuic acid per liter
was used. When appropriate, E .coli and C. glutamicum
strains were cultured with kanamycin (50 mg/ml). A
reduced concentration of kanamycin (25 mg/ml) was
used to obtain transformants of C. glutamicum. E. coli
was grown at 37 1C in 5 ml medium in test-tubes and
170 rpm agitation while C. glutamicum was grown at
30 1C in 60 ml medium in 500 ml baffled shake-flasks and
120 rpm agitation.
For lysine and glutamate production, a BHI pre-
culture was inoculated from a fresh LB-plate and
cultivated over night. After washing cells with CgXII-
medium without carbon source, the main culture with
minimal medium was inoculated with OD600 of 1 in
60 ml medium in 500 ml baffled shake-flasks. For lysine
production, 100 g/l glucose, 100 g/l fructose, 100 g/l
sucrose or 50 g/l glucose+50 g/l fructose were used
as carbon source, while for glutamate production
40 g/l glucose, 40 g/l fructose, 40 g/l sucrose or 20 g/l
Reference/Source
Grant et al. (1990)
DSMZ
B. Bathe, degussa
B. Bathe, degussa
B. Bathe, degussa
B. Bathe, degussa
B. Bathe, degussa
Rittmann et al. (2003)
Peters-Wendisch et al.
(2001)
This study
This study
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294 T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
glucose+20 g/l fructose were used. To trigger glutamate
production 300 mg/ml ethambutol was added to the
minimal medium (Radmacher et al., 2005). After 72 h
for lysine production and after 24 h for glutamate
production, samples were withdrawn from the cultures
for determination of the concentrations of amino acids
and sugars in the culture medium.
Preparation of crude extracts: For determination of
enzyme activities exponentially growing cells were
harvested by centrifugation (5000g, 5 min, 4 1C) and
stored at
70 1C. For determination of malic enzyme
activity cell pellets were washed in 20 ml 200 mM Tris,
5 mM MgCl2 and 10% (v/v) glycerol, pH 7.0 and
resuspended in 1 ml of the same buffer. For determina-
tion of fructose-1,6-bisphosphatase activity cell pellets
were washed in 20 ml 20 mM tricine, 50 mM KCl, 1 mM
MgCl2, 1 mM DTT, 0,5 mM EDTA, pH 7.7 and
resuspended in 1 ml of the same buffer. Cells were
disrupted by ultrasonication at 4 1C for 6 min
(Dr. Hielscher Ultraschallprozessor, cycle 0.5, ampli-
tude 55%). After centrifugation at 4 1C for 60 min at
14 000g, enzyme activities were determined immediately
in the cell-free supernatant.
Fructose-1,6-bisphosphatase assay: Fructose-1,6-bi-
sphosphatase activity was measured in a coupled
spectrophotometric assay containing 2 mM MnCl2,
100 mM KCl, 20 mM tricine, pH 7.7, 0.25 mM NADP+,
yeast glucose-6-phosphate-dehydrogenase (0.4 U/ml),
yeast phosphoglucoisomerase (0.7 U/ml). The assay
was started with 0.75 mM fructose 1,6-bisphosphate as
described previously (Rittmann et al., 2003). Fructose
6-phosphate formed by the reaction of fructose-1,6-
bisphosphatase was converted to glucose 6-phosphate
and subsequently to 6-phosphogluconate by coupling to
phosphoglucoisomerase and glucose-6-dehydrogenase
and the concomitant formation of NADPH
(e340 nm ¼ 6.3 mM
1 cm
1) was followed at 340 nm.
Malic enzyme assay: Malic enzyme activity was
measured as described (Gourdon et al., 2000; Netzer et
al., 2004). The assay contained 100 mM 3-[4-(2-Hydro-
xyethyl)-1-piperazinyl]propanesulfonic acid, pH 7.8,
5 mM MgCl2, 1 mM NADP and 40 mM D,L-disodium
malate. Malic enzyme was assayed spectrophotometri-
cally at 30 1C by determining the formation of NADPH
(e340 nm ¼ 6.3 mM
1 cm
1) by the change in absorption
at 340 nm.
Homologous overexpression of fbp and malE from C.
glutamicum: Plasmids were constructed in E. coli DH5a
by transformation with the RbCl2 method (Hanahan,
1983). C. glutamicum strains were transformed via
electroporation (van der Rest et al., 1999). In order to
overexpress fbp, the vector pVWEx1–fbp was con-
structed based on the expression vector pVWEx1
(Peters-Wendisch et al., 2001). For cloning fbp into
pVWEx1, a 1075 bp fragment containing the fbp-gene
was isolated by restriction of pXMJ19–fbp (Rittmann
et al., 2003) with SalI and KpnI and ligated in SalI-
restricted and KpnI-restricted pVWEx1, resulting in the
vector pVWEx1–fbp which allows IPTG-inducible
expression of fbp in C. glutamicum. For overexpression
of malE the expression vector pVWEx1–malE was
constructed. Primers with the following sequences: 50 -
GCGTCGACAAGGAGATATAGATATGACCATC-
GACCTGCAGCG-30 (the underlined nt corresponds to
nt 3208280 of NC003450, the SalI restriction site is given
in bold and the sequence highlighted in italics contains a
ribosome binding site) and 50 -GCGTCGAC-
0
TATTGGCGCCTCGACGGG-3 (the nt correspond-
ing to nt 3209500 of NC003450 is underlined and the
SalI restriction site is given in bold) were used to amplify
malE from genomic DNA of C. glutamicum
ATCC13032 by PCR (Expand High Fidelity PCR kit;
Roche Diagnostics). The PCR-generated fragment was
restricted with SalI and cloned into SalI-treated
pVWEx1. Sequence analysis (AGOWA GmbH, Berlin,
Germany) of the resulting vector, pVWEx1–malE,
revealed the absence of undesired mutations that could
have been introduced during the PCR.
Determination of amino acid and sugar concentrations:
Cells were removed from the culture samples by
centrifugation for 10 min at 14 000g and filtration
(Spartan 13/0,2 RC filter unit, Schleicher & Schuell,
Dassel, Germany). L-lysine, L-glutamate, L-alanine and
L-valine concentrations were determined by automatic
precolumn derivatization with ortho-phthaldialdehyde
and reversed-phase high-performance liquid chromato-
graphy (RP-HPLC) (HP1100 series; Hewlett-Packard,
Waldbronn, Germany) with fluorimetric detection (ex-
citation at 230 nm; emission at 450 nm) as described
previously (Schrumpf et al., 1991). Hypersil ODS 5-mm
columns were used (precolumn, 40  4 mm; column,
120  4 mm; Chromatographie Service GmbH, Langer-
wehe, Germany). The buffer gradient consisted of 0.1 M
sodium acetate, pH 7.2 (with 0.03% sodium azide), as
the polar phase and methanol as the nonpolar phase.
Quantification was performed with L-asparagine as an
internal standard and by comparison with external
standards.
D-glucose and D-fructose were quantified by HPLC
with a Biorad HPX-87C 300  7.8 mm column at 70 1C
using isocratic elution with H2O at a flow rate of 0.6 ml/
min. Substances were detected via refraction index
(Merck LaChrom RI detector L-7490). Sucrose was
quantified enzymatically with a Sucrose/D-Glucose kit
(R-Biopharm, Darmstadt, Germany) as described by the
manufacturer. Concentrations were determined by
comparison of the sample probes with external stan-
dards.
Determination of intracellular fructose-6-phosphate
and fructose-1,6-bisphosphate concentrations: Extraction
of intracellular metabolites was performed as described
by (Dominguez et al., 1998). Intracellular metabolites
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T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
were quantified by measuring the increase or decrease of
NAD(P)H fluorescence at a wavelength of 340 nm with
a plate reader (Spectra Max Plus). Fructose-6-phos-
phate and fructose-1,6-bisphosphate were assayed as
described (Lang and Michal, 1974; Michal and Beutler,
1974). For calculations of intracellular metabolite
concentrations a cell volume of 1.8 ml/mg dry cell mass
as determined by (Hoischen and Kramer, 1989) was
used.
3. Results
3.1. Glutamate production of C. glutamicum WT on the
carbon sources glucose, fructose, glucose+fructose and
sucrose
Glucose, fructose and sucrose are carbon sources for
growth and amino acid production of C. glutamicum
and their effect on lysine production by strains obtained
by classical mutagenesis and screening has recently been
described (Kiefer et al., 2002). However, the effects of
these carbon sources on glutamate production with the
wild-type C. glutamicum strain ATCC13032 have to our
knowledge not been tested. Therefore, we compared
glutamate production by C. glutamicum strain
ATCC13032 on glucose, on fructose, on a glucose–
fructose mixture and on sucrose. Glutamate accumu-
lated to similar concentrations in the medium and the
yields, expressed as mol carbon (mol-C) of glutamate
produced per mol-C of carbon source utilized, were
similar as well (Table 2). Thus, glutamate production by
C. glutamicum was not affected significantly by the
tested carbon sources.
3.2. Lysine production of defined C. glutamicum strains
on the carbon sources glucose, fructose, glucose+fructose
and sucrose
Lysine production differs from glutamate production
by a higher NADPH requirement. While 4 mol of
NADPH are used for the production of 1 mol of lysine,
only 1 mol of NADPH per glutamate produced is
needed (Marx et al., 1997). As compared to glucose,
NADPH generation via the pentose phosphate pathway
was lower on fructose and on glucose/fructose mixtures
(Dominguez et al., 1998; Kiefer et al., 2004; Pons et al.,
1996). This correlated to lower lysine product yields
with fructose as carbon source as compared to glucose
for C. glutamicum strain ATCC 21526, which was
obtained by undirected mutagenesis and screening. As
efficient lysine producing strains were recently obtained
by site-directed mutagenesis (Ohnishi et al., 2002), we
determined the effects of these carbon sources on lysine
production by strains derived from C. glutamicum WT
by gene-directed mutagenesis.
295
Table 2
Glutamate production on glucose, fructose, glucose+fructose and
sucrose by C. glutamicum wild type
Carbon sourcea Glutamate yieldb Glutamate
(mol-C/mol-C) concentrationc (mM)
Glucose 0.17 33
Fructose 0.18 38
Fructose+glucose 0.15 40
Sucrose 0.14 36
a
As carbon sources 40 g/l glucose, 40 g/l sucrose, 40 g/l fructose or
20 g/l fructose+20 g/l glucose were used.
b
Molar yields are given as mol-C glutamate produced per mol-C
sugar consumed.
c
Concentrations of amino acids accumulated in the medium at the
end of the cultivations are given. All data are mean values of at least
two independent cultivations with errors o6% for glutamate
concentrations and o11% for glutamate yields.
C. glutamicum DM1730, which was derived from the
sequenced wild-type C. glutamicum ATCC13032
(Kalinowski et al., 2003) by four allelic exchanges
(pycP458S, homV59A, lysCT311I and zwfA243T) showed
large differences with respect to lysine yields on the
carbon sources glucose, fructose, glucose+fructose
and sucrose. Lysine yields were much higher on glucose
as compared to the other carbon sources with fructose
allowing for the lowest lysine yield (Table 3).
An equimolar mixture of glucose and fructose (50 g/l
each) did not result in a yield intermediate between
that on 100 g/l fructose and that on 100 g/l glucose, but
it was nearly as low as with 100 g/l fructose as sole
carbon source (Table 3). Similar results were also
observed for the isogenic strains DM1729 (pycP458S
homV59A lysCT311I), DM1728 (pycP458S homV59A) and
DM1800 (pycP458S lysCT311I). Clearly, lysine production
yields of defined C. glutamicum strains were affected
severely by the kind of PTS-transportable sugar serving
as the carbon source.
Additionally, we observed that the introduction of
homV59A and pycP458S into wild-type C. glutamicum was
sufficient for lysine production on the tested carbon
sources (compare DM1727 and DM1728 in Table 3).
Consistent with earlier observations (Ohnishi et al.,
2002; Schrumpf et al., 1992), the lysine yield on glucose
was increased decisively upon the introduction of
lysCT311I (compare DM1800 to DM1727 and DM1729
to DM1728 in Table 3). To a lesser extent, the
introduction of zwfA243T further improved the lysine
yield on glucose (compare DM1730 to DM1729 in
Table 3). However, comparing lysine yields of C.
glutamicum strains DM1728, DM1800, DM1729 and
DM1730 on fructose, sucrose or glucose+fructose
revealed that on these carbon sources neither the
introduction of lysCT311I nor of zwfA243T improved
lysine yields (Table 3).
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296 T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301

Table 3
Lysine production on glucose, fructose, glucose+fructose and sucrose by different C. glutamicum strains derived from C. glutamicum wild type

Carbon sourcea C. glutamicum strainb Lysine yieldc Concentrationsd (mM)

(mol-C/mol-C)
Lysine Alanine Valine

Glucose DM1727 pyc o0.01 1 27 5

DM1800 pyc lysC 0.13 70 20 o1
DM1728 pyc hom 0.08 46 31 10
DM1729 pyc hom lysC 0.13 66 24 6
DM1730 pyc hom lysC zwf 0.15 75 15 4
Fructose DM1727 pyc o0.01 o1 2 o1
DM1800 pyc lysC 0.04 11 2 o1
DM1728 pyc hom 0.05 12 3 o1
DM1729 pyc hom lysC 0.03 8 3 o1
DM1730 pyc hom lysC zwf 0.05 11 3 o1
Glucose +fructose DM1727 pyc o0.01 o1 2 o1
DM1800 pyc lysC 0.05 13 2 o1
DM1728 pyc hom 0.05 14 3 o1
DM1729 pyc hom lysC 0.05 13 3 o1
DM1730 pyc hom lysC zwf 0.06 15 2 o1
Sucrose DM1727 pyc o0.01 o1 3 o1
DM1800 pyc lysC 0.06 22 4 o1
DM1728 pyc hom 0.05 11 3 o1
DM1729 pyc hom lysC 0.05 16 3 o1
DM1730 pyc hom lysC zwf 0.07 16 3 o1
a
As carbon sources 100 g/l glucose, 100 g/l fructose, 50 g/l fructose+50 g/l glucose or 100 g/l sucrose were used.
b
Strain names and mutated genes are given. The point mutations pycP458S, homV59A lysCT311I and zwfA243T were introduced into the wild-type C.
glutamicum strain ATCC 13032 which did not accumulate lysine (o1 mM) under the tested conditions.
c
Molar yields are given as mol-C lysine produced per mol-C sugar consumed.
d
Concentrations of amino acids accumulated in the medium at the end of the cultivations are given. All data are mean values of at least two
independent cultivations with errors o5% for lysine concentrations, o5% for alanine and o10% for valine concentrations and o10% for lysine
yields.

3.3. Effect of malE overexpression on lysine production
of C. glutamicum DM1730
Based on carbon flux analyses on fructose-grown C.
glutamicum, Dominguez et al. (1998) suggested that
overexpression of the malic enzyme gene malE, which
catalyzes the reductive decarboxylation of malate to
pyruvate with concomitant NADPH generation (Fig. 1)
improves growth and lysine production. Recently,
Ohnishi et al. (2003) proposed malE as a target to
improve lysine production at higher temperatures. To test
whether malE overexpression might improve lysine
production, we constructed C. glutamicum strains carry-
ing a malE overexpression vector or, as a control, an
empty vector and determined the specific activities of
malic enzyme in these strains. The specific activities of
malic enzyme in C. glutamicum DM1730(pVWEx1) were
0.04 mmol min
1 mg
1 on glucose, 0.04 mmol min
1 mg
1
on fructose, 0.03 mmol min
1 mg
1 on glucose+fructose,
and 0.05 mmol min
1 mg
1 on sucrose. C. glutamicum
DM1730(pVWEx1–malE) showed specific activities of
malic enzyme of 0.10 mmol min
1 mg
1 on glucose,
0.12 mmol min
1 mg
1 on fructose, 0.12 mmol min
1 mg
1
on glucose+fructose, and 0.17 mmol min
1 mg
1 on
sucrose. Thus, on the carbon sources tested the specific
activity of malic enzyme was about threefold higher in
C. glutamicum DM1730(pVWEx1–malE) than in the
control strain DM1730(pVWEx1). Similar overexpres-
sion factors were observed for malE in C. glutamicum
wild type (Netzer et al., 2004). A comparative analysis of
C. glutamicum strains DM1730(pVWEx1–malE) and
DM1730(pVWEx1) revealed that malE overexpression
did not lead to significant changes of lysine production
(Fig. 2). Both strains accumulated lysine to similar
concentrations in the medium on the tested carbon
sources and lysine yields were about 0.16, 0.06, 0.07, and
0.06 mol carbon of lysine produced per mol carbon of
substrate utilized, respectively, on the carbon sources
glucose, fructose, glucose+fructose and sucrose, respec-
tively. Therefore, malE overexpression did not improve
lysine production with the tested PTS-sugars as carbon
sources.
3.4. Effect of fbp overexpression on lysine production of
C. glutamicum DM1730
Overexpression of the fructose-1,6-bisphosphatase
gene fbp (Rittmann et al., 2003) was proposed to
increase NADPH generation in the pentose phosphate
pathway during growth and lysine production of
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T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
Fig. 2. Lysine yields of C. glutamicum strains DM1730(pVWEx1)
(white columns), DM1730(pVWEx1-fbp) (black columns) and
DM1730(pVWEx1-malE) (gray columns) on different carbon sources.
Yields are expressed as mol carbon (mol-C) of lysine formed per mol-C
of carbon source utilized and are mean values of two to six
independent cultivations.
C. glutamicum on fructose (Kiefer et al., 2004; Pons
et al., 1996). To test this hypothesis, we constructed fbp
overexpressing strains and analyzed lysine production of
these strains as compared to an empty vector control.
The specific activities of fructose-1,6-bisphosphatase
were low in the C. glutamicum strain carrying the
control vector, DM1730(pVWEx1), regardless of the
carbon source tested (Table 4). In C. glutamicum
DM1730(pVWEx1–fbp) overexpression of fbp led
to seven- to eight-fold higher specific activities of
fructose-1,6-bisphosphatase on the carbon sources
tested (Table 4). Similar factors were described pre-
viously for fbp overexpression in C. glutamicum
ATCC13032 (Rittmann et al., 2003).
Lysine production by C. glutamicum strains
DM1730(pVWEx1–fbp) and DM1730(pVWEx1) were
compared using glucose, fructose, glucose+fructose or
sucrose as carbon sources. While lysine production on
glucose and on fructose was not affected, fbp over-
expression led to slightly increased lysine production on
glucose–fructose mixtures (Fig. 2). A large and sig-
nificant increase of fbp overexpression on lysine
production was observed on sucrose (Fig. 2). While
the lysine yield of C. glutamicum DM1730(pVWEx1)
was 0.0770.01 mol-C lysine per mol-C sucrose, the
lysine yield of DM1730(pVWEx1–fbp) was 0.147
0.01 mol-C lysine per mol-C sucrose (Fig. 2) and thus
297
Table 4
Specific activities of fructose-1,6-bisphosphatase in the C. glutamicum
strains ATCC13032, DM1730(pVWEx1) and C. glutamicum
DM1730(pVWEx1-fbp)
C. glutamicum strain Sp. act. of fructose-1,6-
bisphosphatase (mmol min
1 mg
1)
Glucose Fructose Fructose+ Sucrose
glucose
ATCC13032 0.02 0.01 0.02 0.02
DM1730(pVWEx1) 0.02 0.01 0.02 0.02
DM1730(pVWEx1-fbp) 0.15 0.08 0.15 0.14
All data are mean values of at least two determinations of at least two
independent cultivations with errors o9%.
significantly higher (Po0:001). Therefore, the lysine
yield on sucrose doubled upon fbp overexpression
(Fig. 2). This demonstrated that overexpression of fbp
improves lysine production of C. glutamicum with
sucrose as carbon source.
To understand why fbp overexpression improved
lysine production on sucrose, but not on fructose or
glucose+fructose, the intracellular concentrations of
fructose-1,6-bisphosphate and fructose-6-phosphate
were determined. The concentration of fructose-1,6-
bisphosphate, which inhibits the key enzymes of
the pentose phosphate pathway glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase,
was higher during growth on sucrose, fructose or
glucose+fructose as compared to growth on glucose
(Table 5). Overexpression of fbp decreased the fructose-
1,6-bisphosphate concentration significantly and to
similar concentrations as observed for growth on
glucose only with sucrose, but not with fructose or
glucose+fructose as carbon source (Table 5).
4. Discussion
Glutamate production on different PTS sugars: In
C. glutamicum the requirement for 1 mol of NADPH per
mol of glutamate produced is met by isocitrate
dehydrogenase which provides 2-oxoglutarate as gluta-
mate precursor as well as NADPH by oxidative
decarboxylation of isocitrate (Eikmanns et al., 1995).
Accordingly, as demonstrated by metabolic flux analysis
the total rate of NADPH generation during glutamate
production is lower than during growth and much lower
than during lysine production (Marx et al., 1997).
Moreover, a C. glutamicum strain able to synthesize
glutamate in an NADH-dependent manner, which
was obtained by replacement of the endogenous
NADPH-dependent glutamate dehydrogenase by
a NADH-dependent glutamate dehydrogenase from
Peptostreptococcus asaccharolyticus, showed reduced
 ARTICLE IN PRESS
298 T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
Table 5
Intracellular concentrations of fructose-6-phosphate and fructose-1,6-bisphosphate during growth of C. glutamicum DM1730(pVWEx1) and
DM1730(pVWEx1–fbp) on glucose, fructose, glucose+fructose and sucrose
Carbon sourcea Fructose-6-phosphate concentration (mM)
DM1730 (pVWEx1) DM1730 (pVWEx1–fbp)
Glucose 13 17
Fructose 1 1
Fructose+glucose 5 7
Sucrose 5 7
a
As carbon sources 100 g/l glucose, 100 g/l fructose, 50 g/l fructose+50 g/l glucose or 100 g/l sucrose were used. All data are mean values of at least
two determinations of at least two independent cultivations with errors o15%.
overall NADPH generation as compared to the control
strain (Marx et al., 1999). Although the flux via the
pentose phosphate pathway and thus NADPH genera-
tion on fructose and on fructose–glucose mixtures was
found to be lower than on glucose (Dominguez et al.,
1998; Kiefer et al., 2004; Pons et al., 1996) we showed
here that glutamate production was not affected by the
PTS sugar used as in each case the NADPH requirement
should be met by isocitrate dehydrogenase.
Malic enzyme and lysine production on different PTS
sugars: During the course of C. glutamicum strain
development for lysine production a number of mole-
cular targets have been identified. Their importance for
lysine production has been focused on the use of glucose
as carbon source and has been tested by overexpression
of these target genes (e.g., pyc (Peters-Wendisch et al.,
2001)), by deletion analysis (e.g., pck (Riedel et al.,
2001)) or by allelic exchange (e.g., hom and lysC
(Ohnishi et al., 2002)). Recently, Ohnishi et al. (2003)
proposed that increased malE expression might be
beneficial for lysine production on glucose. This
proposal was based on the observation that the
increased lysine yield of the defined C. glutamicum
strain AHP-3 carrying pycP458S, homV59A and lysCT311I
on glucose at 40 1C as compared to 30 1C correlated to
changed expression of 21 of the 120 genes analyzed
(Ohnishi et al., 2003). Out of these expression changes
the authors highlighted increased expression of the malic
enzyme gene malE and of the PEP carboxykinase gene
pck as well as decreased expression of the citrate
synthase gene gltA (Ohnishi et al., 2003). Although
from the observed increase of pck expression one might
expect positive effects of pck overexpression on lysine
production, it was shown previously that lysine produc-
tion decreased upon overexpression of pck and increased
upon pck deletion (Riedel et al., 2001). Similarly, it was
shown here that malE overexpression did not lead to
higher lysine yields on glucose. Thus, at least at 30 1C
malic enzyme does not limit lysine production of C.
glutamicum. This is commensurate with the view that,
albeit increased pck and malE expression was correlated
to increased lysine production at elevated temperatures
(Ohnishi et al., 2003), these expression changes most
Fructose-1,6-bisphosphate concentration (mM)
DM1730 (pVWEx1) DM1730 (pVWEx1–fbp)
27 25
40 39
33 30
33 24
likely arose as a consequence of changed temperature or
lysine production, but they were not the cause for
increased lysine production.
Besides glucose, overexpression of the malic enzyme
gene malE did also not influence lysine production by C.
glutamicum on fructose, sucrose or glucose+fructose
(Fig. 2). The role of malic enzyme in C. glutamicum still
is not fully understood. Genome comparisons revealed
that the single malic enzyme gene malE of C. glutamicum
(Gourdon et al., 2000) was lost in C. diphtheriae during
evolution (Nishio et al., 2004). Malic enzyme activity
has been detected in C. glutamicum under various
growth conditions, but malic enzyme is not essential
for growth on any of the tested carbon sources
(Gourdon et al., 2000; Netzer et al., 2004). During
growth on acetate or citrate, regulation of malE
expression precluded a role of malic enzyme in the
generation of pyruvate on these gluconeogenic carbon
sources (Netzer et al., 2004). A malE deletion mutant
showed slower growth on L-lactate which might indicate
a role of malic enzyme for generation of NADPH on
this carbon source (Gourdon et al., 2000). This
observation was generalized and it was suggested that
malic enzyme may function in generation of NADPH on
other substrates with low pentose phosphate pathway
flux (Gourdon et al., 2000) and that malE overexpres-
sion may increase lysine production on fructose
(Dominguez et al., 1998). NADPH may be generated
from NADH by a reaction cycle of malic enzyme,
pyruvate carboxylase and the NADH-dependent malate
dehydrogenase with concomitant ATP-hydrolysis. Dur-
ing growth on glucose no flux via malic enzyme was
revealed by carbon flux analysis of wild-type C.
glutamicum ATCC13032 (Petersen et al., 2000) whereas
malic enzyme carried flux in the classically obtained
lysine producing strain ATCC21799 (Klapa et al., 2003).
Besides strain differences, it has to be noted that the flux
estimations were performed in the presence of either
0.45 g L
1 lactate (Petersen et al., 2000) or 1 g L
1
threonine, 0.3 g L
1 methionine and 1 g L
1 leucine
(Klapa et al., 2003). This is important as expression
of the malE gene is regulated by the carbon source
(Gourdon et al., 2000; Netzer et al., 2004). The
 ARTICLE IN PRESS
T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
C. glutamicum strain DM1730(pVWEx1–malE) described
here has elevated levels of two enzymes of
the reaction cycle that forms NADPH from NADH:
pyruvate carboxylase due to the pycP458S allele and malic
enzyme due to malE overexpression. However, lysine
production of C. glutamicum DM1730(pVWEx1–malE)
was not higher than in the control strain DM1730
(pVWEx1) on the tested PTS sugars. It remains to be
shown whether cycling between pyruvate, oxaloacetate
and malate and thus ATP-dependent formation of
NADPH from NADH occurs when malE is over-
expressed in the presence of the pycP458S allele, or
whether in addition increased NAD-dependent malate
dehydrogenase is required and how in turn lysine
production might be influenced.
Fructose-1,6-bisphosphatase and lysine production on
different PTS sugars: Fructose-1,6-bisphosphatase cata-
lyzes the reverse reaction of phosphofructokinase and
their simultaneous action results in an ATP consuming
cycle. Here, we have shown that during growth on
glucose overexpression of fbp did not change lysine
production (Fig. 2). Thus, either overexpression of fbp
did not result in ATP hydrolysis by simultaneous action
of phosphofructokinase and fructose-1,6-bisphospha-
tase or, if it did, this ATP-consuming cycle did not
perturb lysine production of C. glutamicum DM1730.
Fructose enters glycolysis as fructose-1,6-bispho-
sphate and flux from fructose-1,6-bisphosphate towards
glucose-6-phosphate and the oxidative pentose phos-
phate pathway is carried by fructose-1,6-bisphospha-
tase. It was proposed previously that fructose-1,6-
bisphosphatase limits growth and lysine production on
fructose and that overexpression of fbp might improve
lysine production on fructose by way of increasing the
flux into the pentose phosphate pathway (Kiefer et al.,
2004; Pons et al., 1996). Low lysine yields on fructose
were demonstrated for a strain obtained by mutagenesis
and screening procedures (Kiefer et al., 2002) and,
as shown here, for a series of genetically defined
C. glutamicum strains (Table 3). However, lysine
production of the genetically defined C. glutamicum
strain DM1730 on fructose was not significantly
higher when fbp was overexpressed (Fig. 2). Seven-
to eight-fold overexpression of fbp (Table 4) was neither
sufficient to improve lysine production of the defined
C. glutamicum strain DM1730 on fructose nor sufficient
to decrease the intracellular fructose-1,6-bisphosphate
concentration under the tested conditions. At con-
centrations of about 40 mM as observed during
growth on fructose (Table 5) fructose-1,6-bisphosphate
inhibits glucose-6-phosphate dehydrogenase and 6-
phosphogluconate dehydrogenase from C. glutamicum
as these enzymes show half-maximal inhibition by
fructose-1,6-bisphoshate at concentrations of about
14 mM (Moritz et al., 2000; Sugimoto and Shiio,
1987a, b).
299
On sucrose, lysine yields were lower than on glucose
(Table 3 and (Kiefer et al., 2002). The intracellular
fructose-1,6-bisphosphate concentration was higher
during growth on sucrose as compared to growth on
glucose. Seven- to eight-fold overexpression of fbp (Fig.
2 and Table 4) improved lysine yields on sucrose, but
not on fructose. While fbp overexpression did not affect
the intracellular fructose-1,6-bisphosphate concentra-
tion in fructose-grown cells, the concentration of
fructose-1,6-bisphosphate in sucrose-grown cells de-
creased to that observed in glucose-grown cells when
fbp was overexpressed (Table 5). As fructose-1,6-bi-
sphosphate inhibits C. glutamicum glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase
at inhibition constants of about 14 mM (Moritz et al.,
2000; Sugimoto and Shiio, 1987a b) the observed
reduction of the fructose-1,6-bisphosphate concentra-
tion upon fbp overexpression during growth on sucrose
is physiologically relevant. As a consequence of in-
creased NADPH generation by these enzymes lysine
production improves as has been demonstrated for
glucose-grown C. glutamicum (Marx et al., 1996, 1999,
2003; Wittmann and Heinzle, 2002). Recently it was
shown that lysine production and the pentose phosphate
pathway flux increased when an allele for 6-phospho-
gluconate dehydrogenase which is less sensitive to
inhibition by fructose-1,6-bisphophate and other meta-
bolites was introduced into a C. glutamicum lysine
producing strain (Ohnishi et al., 2005). These results
corroborate our findings and identify 6-phosphogluco-
nate dehydrogenase as an important target to improve
lysine production by C. glutamicum.
In B. subtilis, fructose-1,6-bisphosphate does not only
modulate the activity of allosteric enzymes, but also
activates CggR, a transcriptional repressor of glycolytic
genes (Doan and Aymerich, 2003). Moreover, fructose-
1,6-bisphosphate activates the B. subtilis Hpr(ser) kinase
and in turn CcpA becomes active as repressor of
operons for the utilization of secondary carbon sources
(Titgemeyer and Hillen, 2002). In C. glutamicum, all
strains showed near-complete utilization of glucose, but
none of the strains used sucrose, fructose nor a mixture
of fructose and glucose completely. Overexpression of
fbp led to near-complete utilization of sucrose while
substantial concentrations of sucrose remained in the
culture medium of the control strain C. glutamicum
DM1730(pVWEx1) and the plasmid-free strains (Table
3 and data not shown). Transcriptional regulation of the
genes required for uptake of glucose, sucrose and
fructose in C. glutamicum has not been investigated in
detail yet. It is known that during co-utilization of
acetate and glucose the glucose uptake rate is decreased
as compared to growth on glucose alone (Wendisch et
al., 2000) and that the mRNA-level of the glucose-
specific PTS gene ptsG is decreased due to repression
by RamB (Gerstmeir et al., 2003, 2004). Similarly, the
 ARTICLE IN PRESS
300 T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
co-utilization of fructose with glucose and sucrose with
glucose are characterized by a decreased glucose uptake
rate as compared to growth on glucose alone (Pons
et al., 1996) and our unpublished results). Sugar-specific
transcriptional regulation in C. glutamicum differs from
that of low-GC Gram-negative bacteria as it lacks
functional homologs of CcpA and Hpr(ser) kinase
(Kalinowski et al., 2003). Therefore, we are currently
investigating fructose- and sucrose-specific transcrip-
tional regulation in C. glutamicum.
Acknowledgments
We thank Hermann Sahm for continuous support
and Steffen Erkelenz for technical assistance. C.
glutamicum strains DM1727, DM1728, DM1729,
DM1730, and DM1800 were kind gifts of Brigitte
Bathe, Degussa. Part of the described work belongs to
the planned dissertation of Tobias Georgi at the faculty
of Mathematics and Natural Sciences of the Heinrich-
Heine-Universität Düsseldorf.
References
Ando, S., Ochiai, K., Yokoi, H., Hashimoto, S., Yonetani, Y., 2002.
Novel glucose-6-phosphate dehydrogenase. Patent WO0198472
(2002-01-02).
Cremer, J., Eggeling, L., Sahm, H., 1991. Control of the lysine
biosynthetic sequence in Corynebacterium glutamicum as analyzed
by overexpression of the individual corresponding genes. Appl.
Environ. Microbiol. 57, 1746–1752.
Doan, T., Aymerich, S., 2003. Regulation of the central glycolytic
genes in Bacillus subtilis: binding of the repressor CggR to its single
DNA target sequence is modulated by fructose-1,6-bisphosphate.
Mol. Microbiol. 47, 1709–1721.
Dominguez, H., Rollin, C., Guyonvarch, A., Guerquin-Kern, J.L.,
Cocaign-Bousquet, M., Lindley, N.D., 1998. Carbon-flux distribu-
tion in the central metabolic pathways of Corynebacterium
glutamicum during growth on fructose. Eur. J. Biochem. 254,
96–102.
Eggeling, L., 1994. Biology of L-lysine overproduction by Corynebac-
terium glutamicum. Amino Acids 6, 261–272.
Eggeling, L., Oberle, S., Sahm, H., 1998. Improved L-lysine yield with
Corynebacterium glutamicum: use of dapA resulting in increased
flux combined with growth limitation. Appl. Microbiol. Biotech-
nol. 49, 24–30.
Eikmanns, B.J., Follettie, M.T., Griot, M.U., Sinskey, A.J., 1989. The
phosphoenolpyruvate carboxylase gene of Corynebacterium gluta-
micum: molecular cloning, nucleotide sequence, and expression.
Mol. Gen. Genet. 218, 330–339.
Eikmanns, B.J., Metzger, M., Reinscheid, D., Kircher, M., Sahm, H.,
1991. Amplification of three threonine biosynthesis genes in
Corynebacterium glutamicum and its influence on carbon flux in
different strains. Appl. Microbiol. Biotechnol. 34, 617–622.
Eikmanns, B.J., Rittmann, D., Sahm, H., 1995. Cloning, sequence
analysis, expression, and inactivation of the Corynebacterium
glutamicum icd gene encoding isocitrate dehydrogenase and
biochemical characterization of the enzyme. J. Bacteriol. 177,
774–782.
Follettie, M.T., Shin, H.K., Sinskey, A.J., 1988. Organization and
regulation of the Corynebacterium glutamicum hom-thrB and thrC
loci. Mol. Microbiol. 2, 53–62.
Gerstmeir, R., Wendisch, V.F., Schnicke, S., Ruan, H., Farwick, M.,
Reinscheid, D., Eikmanns, B.J., 2003. Acetate metabolism and its
regulation in Corynebacterium glutamicum. J. Biotechnol. 104,
99–122.
Gerstmeir, R., Cramer, A., Dangel, P., Schaffer, S., Eikmanns, B.J.,
2004. RamB, a novel transcriptional regulator of genes involved in
acetate metabolism of Corynebacterium glutamicum. J. Bacteriol.
186, 2798–2809.
Gourdon, P., Baucher, M.F., Lindley, N.D., Guyonvarch, A., 2000.
Cloning of the malic enzyme gene from Corynebacterium glutami-
cum and role of the enzyme in lactate metabolism. Appl. Environ.
Microbiol. 66, 2981–2987.
Grant, S.G., Jessee, J., Bloom, F.R., Hanahan, D., 1990. Differential
plasmid rescue from transgenic mouse DNAs into Escherichia coli
methylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87,
4645–4649.
Hanahan, D., 1983. Studies on transformation of Escherichia coli with
plasmids. J. Mol. Biol. 166, 557–580.
Hermann, T., 2003. Industrial production of amino acids by coryne-
form bacteria. J. Biotechnol. 104, 155–172.
Hoischen, C., Kramer, R., 1989. Evidence for an efflux carrier system
involved in the secretion of glutamate by Corynebacterium
glutamicum. Arch. Microbiol. 151, 342–347.
Kalinowski, J., Cremer, J., Bachmann, B., Eggeling, L., Sahm, H.,
Puhler, A., 1991. Genetic and biochemical analysis of the
aspartokinase from Corynebacterium glutamicum. Mol. Microbiol.
5, 1197–1204.
Kalinowski, J., Bathe, B., Bartels, D., Bischoff, N., Bott, M.,
Burkovski, A., Dusch, N., Eggeling, L., Eikmanns, B.J., Gaigalat,
L., Goesmann, A., Hartmann, M., Huthmacher, K., Kramer, R.,
Linke, B., McHardy, A.C., Meyer, F., Mockel, B., Pfefferle, W.,
Puhler, A., Rey, D.A., Ruckert, C., Rupp, O., Sahm, H.,
Wendisch, V.F., Wiegrabe, I., Tauch, A., 2003. The complete
Corynebacterium glutamicum ATCC 13032 genome sequence and
its impact on the production of L-aspartate-derived amino acids
and vitamins. J. Biotechnol. 104, 5–25.
Keilhauer, C., Eggeling, L., Sahm, H., 1993. Isoleucine synthesis in
Corynebacterium glutamicum: molecular analysis of the ilvB– ilv-
N– ilvC operon. J. Bacteriol. 175, 5595–5603.
Kiefer, P., Heinzle, E., Wittmann, C., 2002. Influence of glucose,
fructose and sucrose as carbon sources on kinetics and stoichio-
metry of lysine production by Corynebacterium glutamicum. J. Ind.
Microbiol. Biotechnol. 28, 338–343.
Kiefer, P., Heinzle, E., Zelder, O., Wittmann, C., 2004. Comparative
metabolic flux analysis of lysine-producing Corynebacterium
glutamicum cultured on glucose or fructose. Appl. Environ.
Microbiol. 70, 229–239.
Klapa, M.I., Aon, J.C., Stephanopoulos, G., 2003. Systematic
quantification of complex metabolic flux networks using stable
isotopes and mass spectrometry. Eur. J. Biochem. 270, 3525–3542.
Kotrba, P., Inui, M., Yukawa, H., 2001. The ptsI gene encoding
enzyme I of the phosphotransferase system of Corynebacterium
glutamicum. Biochem. Biophys. Res. Commun. 289, 1307–1313.
Lang, G., Michal, G., 1974. D-Glucose-6-Phosphate and D-fructose-6-
phosphate. Methods Enzymatic Anal. 3, 1238–1242.
Marx, A., de Graaf, A.A., Wiechert, W., Eggeling, L., Sahm, H., 1996.
Determination of the fluxes in the central metabolism of
Corynebacterium glutamicum by nuclear magnetic resonance
spectroscopy combined with metabolite balancing. Biotechnol.
Bioeng. 49, 111–129.
Marx, A., Striegel, K., de Graaf, A.A., Sahm, H., Eggeling, L., 1997.
Response of the central metabolism of Corynebacterium glutami-
cum to different flux burdens. Biotechnol. Bioeng. 56, 168–180.
 ARTICLE IN PRESS
T. Georgi et al. / Metabolic Engineering 7 (2005) 291–301
Marx, A., Eikmanns, B.J., Sahm, H., de Graaf, A.A., Eggeling, L.,
1999. Response of the central metabolism in Corynebacterium
glutamicum to the use of an NADH-dependent glutamate
dehydrogenase. Metab. Eng. 1, 35–48.
Marx, A., Hans, S., Mockel, B., Bathe, B., de Graaf, A.A., 2003.
Metabolic phenotype of phosphoglucose isomerase mutants of
Corynebacterium glutamicum. J. Biotechnol. 104, 185–197.
Michal, G., Beutler, H.D., 1974. D-Fructose-1,6-diphosphate, dihy-
droxyacetone phosphate and D-glyeraldehyde-3-phosphate. Meth-
ods Enzymatic Anal. 3, 1359–1364.
Moritz, B., Striegel, K., De Graaf, A.A., Sahm, H., 2000. Kinetic
properties of the glucose-6-phosphate and 6-phosphogluconate
dehydrogenases from Corynebacterium glutamicum and their
application for predicting pentose phosphate pathway flux in vivo.
Eur. J. Biochem. 267, 3442–3452.
Netzer, R., Krause, M., Rittmann, D., Peters-Wendisch, P.G.,
Eggeling, L., Wendisch, V.F., Sahm, H., 2004. Roles of pyruvate
kinase and malic enzyme in Corynebacterium glutamicum for
growth on carbon sources requiring gluconeogenesis. Arch.
Microbiol. 182, 354–363.
Nishio, Y., Nakamura, Y., Usuda, Y., Sugimoto, S., Matsui, K.,
Kawarabayasi, Y., Kikuchi, H., Gojobori, T., Ikeo, K., 2004.
Evolutionary process of amino acid biosynthesis in Corynebacter-
ium at the whole genome level. Mol. Biol. Evol. 21, 1683–1691.
Ohnishi, J., Mitsuhashi, S., Hayashi, M., Ando, S., Yokoi, H., Ochiai,
K., Ikeda, M., 2002. A novel methodology employing Corynebac-
terium glutamicum genome information to generate a new L-lysine-
producing mutant. Appl. Microbiol. Biotechnol. 58, 217–223.
Ohnishi, J., Hayashi, M., Mitsuhashi, S., Ikeda, M., 2003. Efficient 40
degrees C fermentation of L-lysine by a new Corynebacterium
glutamicum mutant developed by genome breeding. Appl. Micro-
biol. Biotechnol. 62, 69–75.
Ohnishi, J., Katahira, R., Mitsuhashi, S., Kakita, S., Ikeda, M., 2005.
A novel gnd mutation leading to increased L-lysine production in
Corynebacterium glutamicum. FEMS Microbiol. Lett. 242,
265–274.
Parche, S., Burkovski, A., Sprenger, G.A., Weil, B., Kramer, R.,
Titgemeyer, F., 2001. Corynebacterium glutamicum: a dissection of
the PTS. J. Mol. Microbiol. Biotechnol. 3, 423–428.
Petersen, S., de Graaf, A.A., Eggeling, L., Mollney, M., Wiechert, W.,
Sahm, H., 2000. In Vivo quantification of parallel and bidirectional
fluxes in the anaplerosis of Corynebacterium glutamicum. J. Biol.
Chem. 275, 35932–35941.
Petersen, S., Mack, C., de Graaf, A.A., Riedel, C., Eikmanns, B.J.,
Sahm, H., 2001. Metabolic consequences of altered phosphoenol-
pyruvatecarboxykinase activity in Corynebacterium glutamicum
reveal anaplerotic regulation mechanisms in vivo. Metab. Eng. 3,
344–361.
Peters-Wendisch, P.G., Wendisch, V.F., de Graaf, A.A., Eikmanns,
B.J., Sahm, H., 1996. C3-carboxylation as an anaplerotic reaction
in phosphoenolpyruvate carboxylase-deficient Corynebacterium
glutamicum. Arch. Microbiol. 165, 387–396.
Peters-Wendisch, P.G., Wendisch, V.F., Paul, S., Eikmanns, B.J.,
Sahm, H., 1997. Pyruvate carboxylase as an anaplerotic enzyme in
Corynebacterium glutamicum. Microbiology 143, 1095–1103.
Peters-Wendisch, P.G., Kreutzer, C., Kalinowski, J., Patek, M., Sahm,
H., Eikmanns, B.J., 1998. Pyruvate carboxylase from Corynebac-
terium glutamicum: characterization, expression and inactivation of
the pyc gene. Microbiology 144, 915–927.
Peters-Wendisch, P.G., Schiel, B., Wendisch, V.F., Katsoulidis, E.,
Mockel, B., Sahm, H., Eikmanns, B.J., 2001. Pyruvate carboxylase
is a major bottleneck for glutamate and lysine production by
Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol. 3,
295–300.
Pfefferle, W., Mockel, B., Bathe, B., Marx, A., 2003. Biotechnological
manufacture of lysine. Adv. Biochem. Eng. Biotechnol. 79, 59–112.
301
Pons, A., Dussap, C.G., Pequinot, C., Gros, J.B., 1996. Metabolic flux
distribution in Corynebacterium melassecola ATCC 17965 for
various carbon sources. Biotechnol. Bioeng. 51, 77–189.
Radmacher, E., Stansen, K.C., Besra, G.S., Alderwick, L.J., Maughan,
W.N., Hollweg, G., Sahm, H., Wendisch, V.F., Eggeling, L., 2005.
Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis,
elicits L-glutamate efflux of Corynebacterium glutamicum. Micro-
biology 151, 1359–1368.
Riedel, C., Rittmann, D., Dangel, P., Mockel, B., Petersen, S., Sahm,
H., Eikmanns, B.J., 2001. Characterization of the phosphoenol-
pyruvate carboxykinase gene from Corynebacterium glutamicum
and significance of the enzyme for growth and amino acid
production. J. Mol. Microbiol. Biotechnol. 3, 573–583.
Rittmann, D., Schaffer, S., Wendisch, V.F., Sahm, H., 2003. Fructose-
1,6-bisphosphatase from Corynebacterium glutamicum: expression
and deletion of the fbp gene and biochemical characterization of
the enzyme. Arch. Microbiol. 180, 285–292.
Sahm, H., Eggeling, L., de Graaf, A.A., 2000. Pathway analysis and
metabolic engineering in Corynebacterium glutamicum. Biol. Chem.
381, 899–910.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
Schrumpf, B., Schwarzer, A., Kalinowski, J., Puhler, A., Eggeling, L.,
Sahm, H., 1991. A functionally split pathway for lysine synthesis in
Corynebacterium glutamicium. J. Bacteriol. 173, 4510–4516.
Schrumpf, B., Eggeling, L., Sahm, H., 1992. Isolation and prominent
characteristics of an L-lysine hyperproducing strain of Corynebac-
terium glutamicum. Appl. Microbiol. Biotechnol. 37, 566–571.
Shiio, I., Miyajima, R., 1969. Concerted inhibition and its reversal by
end products of aspartate kinase in Brevibacterium flavum.
J. Biochem. (Tokyo) 65, 849–859.
Stackebrandt, E., Rainey, F.A., Ward-Rainey, N.L., 1997. Proposal
for a new classification system, Actinobacteria classis nov. Int.
J. Syst. Bacteriol. 47, 479–491.
Stephanopoulos, G., Vallino, J.J., 1991. Network rigidity and
metabolic engineering in metabolite overproduction. Science 252,
1675–1681.
Sugimoto, S.I., Shiio, I., 1987a. Regulation of 6-phosphogluconate
dehydrogenase in Brevibacterium flavum. Agric. Biol. Chem. 51,
1257–1263.
Sugimoto, S.I., Shiio, I., 1987b. Regulation of glucose-6-phosphate
dehydrogenase in Brevibacterium flavum. Agric. Biol. Chem. 51,
101–108.
Thierbach, G., Kalinowski, J., Bachmann, B., Puhler, A., 1990.
Cloning of a DNA fragment from Corynebacterium glutamicum
conferring aminoethyl cysteine resistance and feedback resistance
to aspartokinase. Appl. Microbiol. Biotechnol. 32, 443–448.
Titgemeyer, F., Hillen, W., 2002. Global control of sugar metabolism:
a Gram-positive solution. Antonie Van Leeuwenhoek 82, 59–71.
van der Rest, M.E., Lange, C., Molenaar, D., 1999. A heat shock
following electroporation induces highly efficient transformation of
Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl.
Microbiol. Biotechnol. 52, 541–545.
Vrljic, M., Sahm, H., Eggeling, L., 1996. A new type of transporter
with a new type of cellular function: L-lysine export from
Corynebacterium glutamicum. Mol. Microbiol. 22, 815–826.
Wendisch, V.F., de Graaf, A.A., Sahm, H., Eikmanns, B.J., 2000.
Quantitative determination of metabolic fluxes during coutilization
of two carbon sources: comparative analyses with Corynebacterium
glutamicum during growth on acetate and/or glucose. J. Bacteriol.
182, 3088–3096.
Wittmann, C., Heinzle, E., 2002. Genealogy profiling through strain
improvement by using metabolic network analysis: metabolic flux
genealogy of several generations of lysine-producing corynebacter-
ia. Appl. Environ. Microbiol. 68, 5843–5859.