Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122, available online at http:www.idealibrary.com on
Metabolic Analysis of Glutamate Production by
Corynebacterium glutamicum
Pierre Gourdon and Nicholas D. Lindley 1
Centre de Bioingenierie Gilbert Durand, Centre National de la Recherche ScientifiqueUnite Mixte de Recherche 5504, UR 792 INRA,
Institut National des Sciences Appliquees, Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex 4, France
Received March 22, 1999; accepted June 22, 1999
The dynamic behavior of the metabolism of Corynebacterium
glutamicum during l-glutamic acid fermentation, was evaluated by
quantitative analysis of the evolution of intracellular metabolites and
key enzyme concentrations. Glutamate production was induced
by an increase of the temperature and a final concentration of
80 g l was attained. During the production phase, various other
compounds, notably lactate, trehalose, and DHA were secreted to
the medium. Intracellular metabolites analysis showed important
variations of glycolytic intermediates and NADH, NAD coenzymes
levels throughout the production phase. Two phenomena occur
during the production phase which potentially provoke a decrease in
the glutamate yield: Both the intracellular concentrations of
glycolytic intermediates and the NADH NAD ratio increase signi-
ficantly during the period in which the overall metabolic rates
decline. This correlates with the decrease in glutamate yield due
in part to the production of lactate and also to the period of the
fermentation in which growth no longer occurred.  1999 Academic Press
INTRODUCTION
Corynebacterium glutamicum is widely used for the produc-
tion of amino acids, such as l-glutamic acid and l-lysine.
Because of the economic importance of these fermentations,
a great deal of effort has been made, in the last decade, to
improve our working knowledge of the physiology of this
bacterium. Investigations have focused on the regulation of
amino acid biosynthetic pathways and the carbon flux
distribution within the central metabolism. Various
groups have examined the fate of glucose in the central
pathways, using NMR (Rollin et al., 1995; Marx et al., 1996;
Dominguez et al., 1998), enzymatic (Cocaign-Bousquet
et al., 1996) and mathematical modelling (Vallino and
Stephanopoulos, 1993; Pons et al., 1996; Park et al., 1997a)
1
To whom correspondence should be addressed at centre de
Bioingenierie Gilbert Durand, INSA, Complexe Scientifique de Rangueil,
31077 Toulouse cedex 4, France. Fax: (33) 5 61 55 94 00. E-mail:
lindleyinsa-tlse.fr.
1096-717699 30.00 224
Copyright  1999 by Academic Press
All rights of reproduction in any form reserved.
approaches. Particular attention have been paid to the
pyruvatePEP branching point in an attempt to define the
relative contribution of each anaplerotic enzyme for TCA
cycle during growth and amino acid synthesis (Park et al.,
1997b; Peters-Wendisch et al., 1996). Much of this work has
involved either growth of the organism on various sub-
strates or l-lysine production. The metabolic constraints
associated with l-glutamic acid production have not been
as thoroughly characterized. However, NMR experiments
(Ishino et al., 1991; Sonntag et al., 1995; Marx et al., 1997),
have reported significant modifications of metabolic flux
between exponential growth and l-glutamate production.
Most important are a diminished flux through the pentose
pathway and an increased flux through isocitrate dehydro-
genase during the phase of l-glutamate production. The
smaller contribution of the pentose pathway was assumed
to occur because of the diminished requirement of NADPH
in glutamic acid formation, postulating a correlation
between the carbon flux distribution and the energetic
demand though this has proved difficult to exploit (Marx et
al., 1999). However, these experiments though elegant in
their laboratory conception often used either poor produc-
tion strains or fermentation strategies somewhat different
from those suitable for industrial use. Little attempt was
made to see how flux distribution varied throughout the
production phase, notably at glutamate concentrations of
industrial interest. In this study, a temperature-triggered
process of glutamate overproduction has been used and
dynamic variations in intracellular metabolite pools have
been correlated to whole cell kinetic behavior. Important
variations of metabolites levels were measured during the
fermentation indicating that the production phase cannot
be assumed to be a period of quasi-steady-state behavior.
This dynamic analysis suggests that strain improvement
strategies based upon data obtained during the early phase
of production may fail to perform up to expectations due to
the modified metabolic constraints intervening later in the
fermentation.
Metabolic Constraints during Glutamate Fermentation
MATERIALS AND METHODS
Organism and Cultivation
The strain used in this work was Corynebacterium
glutamicum 2262. Cultivation was done in a 3.5-liter reactor
(Chemap). The pH was maintained at 7.6 by automatic
addition of NH 4 (12 N). During the production phase, the
culture was pulsed with a concentrated glucose solution
(500 gl) to avoid periods of glucose limitation. The
medium was based on MCGC medium (Von der Osten et
al., 1989) although citrate was replaced by deferoxamine.
The medium contained glucose (60 gl), Na 2HPO 4 (3 gl),
KH 2PO 4 (6 gl), NaCl (2 gl), (NH 4 ) 2SO 4 (8 gl), MgSO 4,
7H 2O (0.4 gl), FeSO 4, 7H 2O (40 mgl), FeCl 3 (4 mgl),
ZnSO4, 7H 2O (1 mgl), CuCl 2, 2H 2O (0.4 mgl), MnSO 4,
H 2O (2 mgl), (NH 4 ) 6Mo 7O 24, 4 H 2O (0.2 mgl), Na 2B 4O 7 ,
10 H 2O (0.4 mgl), CaCl 2 (84 mgl), biotin (2 mgl),
thiamine (20 mgl), desferoxamine (3 mgl), and betaine
(2 gl). The onset of glutamate production was induced by
increasing the growth temperature from 33 to 39.5%C over a
5 min period when the biomass concentration reached
5.6 gl.
Analytical Methods
Biomass was estimated by absorbance at 650 nm and by
a direct gravimetric method following drying of washed cells
to constant weight under partial vacuum at 60%C for at least
24 h. In this manner, potential errors in biomass estimation
linked to morphological changes were avoided. Sugars and
organic acids were determined on the culture supernatant
by HPLC using a HPX87H column (Biorad) maintained at
48%C with H 2SO 4 (5 mM) as eluant. Detection was per-
formed by UV and refractometer detectors. Gas phase
composition was determined by gas chromatography with a
Porapak Q column maintained at 40%C with helium as the
carrier gas and catharometer detection. Glutamate and
other amino acid concentrations was determined with an
AminoQuant 1090 high-pressure liquid chromatography
(HewlettPackard) after derivatization by orthophthalade-
hyde in the presence of 3-mercaptopropionic acid, separation
with a C 18 column, and spectrophotometric detection at
338 nm.
Enzymatic Assays
Most enzyme activities were assayed by spectrophoto-
metric measurement of variation in NAD(P)H concentration
at 340 nm (==6.223 M &1 cm &1 ). One unit of activity was
the amount of enzyme required to produce 1 nmol of
product per min. All enzyme activity were measured in
crude cell-free extracts obtained by sonication. Crude
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Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
extracts were prepared as follows: cell were harvested,
washed twice with 0.20 KCl, and resuspended in Tris-
tricarballylate buffer (250 mM, pH 7.8) containing glycerol
(300), and MgCl 2 (5 mM). Cells were disrupted by sonica-
tion and cell debris was removed by centrifugation at
10,000g, for 10 min at 4%C. The supernatant was used for
enzyme assay and the protein concentration of the extract
was determined by the Lowry method with bovine serum
albumin as standard.
Glucose 6-phosphate dehydrogenase was assayed by a
method based on that of Sugimoto and Shiio (1987a) in
a reaction mixture containing Tris-HCl buffer (100 mM,
pH 7.8), MgCl 2 (10 mM), NADP + (0.5 mM), and glucose
6-phosphate (2 mM) as the substrate. 6-phosphogluconate
dehydrogenase was assayed by a method based on that of
Sugimoto and Shiio (1987b) using the same reaction
mixture as above, except that 6-Phosphogluconate (1 mM)
was added as the substrate instead of glucose 6-phosphate.
Phosphoenolpyruvate carboxylase was assayed by a method
based on that of Mori and Shiio (1984) in a reaction
mixture containing Tris-HCl buffer (100 mM, pH 7.8),
MnSO 4 (5 mM), KHCO 3 (10 mM), NADH 2 (0.15 mM),
acetyl coenzyme-A (0.1 mM), 10 +g } ml &1 malate dehy-
drogenase, and PEP (2 mM). The reaction was started
by the addition of PEP. Glyceraldehyde-3-phosphate
dehydrogenase was assayed according to the method of
Crow and Wittenberger (1979) with the following reaction
mixture: NAD (1 mM), sodium arsenate (5 mM), cysteine
HCl (5 mM), triethanolamine buffer (125 mM, pH 7.8),
dl-glyceraldehyde-3-P (4 mM), and diluted enzyme extract.
Malic enzyme was assayed by the method described by
Mori and Shiio (1987) with an optimized reaction mixture
consisting of phosphate buffer (100 mM, pH 7.8),
MgCl 2(5 mM), NADP + (0.6 mM), and malate (40 mM) as
the substrate. Assays were initiated by the addition of
malate. Isocitrate dehydrogenase was assayed with the
following reaction mixture : Tris-HCl buffer (100 mM,
pH 7.8), MnSO 4 (5 mM), NADP + (0,6 mM), and
isocitrate (10 mM). Assays were initiated by the addition of
isocitrate. Glutamate dehydrogenase was assayed using a
reaction mixture containing Tris-HCl buffer (100 mM,
pH 7.8), NH 4Cl (40 mM), NADPH 2 (0.6 mM), and
:-ketoglutarate (10 mM). Nonspecific NADH oxidation
was assayed using a reaction mixture containing Tris-HCl
buffer (100 mM, pH 7.8), MnSO 4 (5 mM), and NADH 2
(0.3 mM).
Extraction and Estimation of Intracellular Metabolite
Concentrations
The extraction procedure optimized by Dominguez et al.
(1998) was used in which cell samples (10 ml) of known cell
Metabolic Engineering 1, 224231 (1999) Gourdon and Lindley
Article ID mben.1999.0122

dry weight, were removed directly from the culture, frozen

immediately in liquid nitrogen, and stored at &80%C. After
rapid thawing, either HCl (3N) or KOH (5N) were added
to give a final pH of 1.5 or 12.5, respectively. The acid
extraction procedure was used for all metabolites except
NADH and was achieved by incubating the HCl-treated
culture (pH 1.5) at 25%C for 10 min before neutralizing
to pH 6.8 with KOH (10 N) while agitating vigorously to
avoid transiently alkaline pH conditions. After centrifuga-
tion at 12,000g (8 min) to remove cell debris (no visible
chemical precipitation occurred) the supernatant was used
for assays. Extraction procedures were tested with aqueous
solutions of all metabolites and loss during extraction was
shown to be less than 50 in all cases, though this loss
increased if more acidic or prolonged extraction procedures
were used. Acid-labile coenzyme NADH were extracted by
incubating the KOH-treated culture at pH 12.5 at 25%C for
10 min. After centrifugation at 4%C for 5 min at 12,000g the
supernatant was immediately tested for NADH without
neutralizing to avoid destruction of NADH by locally high
concentrations of acid. Intracellular metabolites were assayed
by measuring the increase (or decrease) of NAD(P)H
fluorescence, using a spectrofluorometer (Hitachi F 2000).
The excitation wavelength was 350 nm and the emission
wavelength was 460 nm. The enzyme-coupled assay proce- FIG. 1. Fermentation time course for glutamate overproduction by C.
dures used were those described by Le Bloas et al. (1993). glutamicum. Data points represent biomass (Q), glucose consumed (M),
All metabolite concentrations were given relative to dry cell production of glutamate (g), trehalose (G), lactate (m), and DHA (q).
weight since the significant changes in culture medium
composition made estimations of cell volume via the silicon
oil centrifugation technique prone to error due to poor rate of glutamate production decreased progressively in
pellet formation. parallel with the specific rate of glucose consumption, coin-
ciding with the stopping of biomass proliferation and an
acceleration of lactate production which reached a value of
RESULTS 0.032 gg } h &1. In the final hours of the fermentation, the
specific rate of lactate production further increased to 0.06
Fermentation Kinetics gg } h &1. Trehalose was produced continuously throughout
After a growth phase of approximately 5 h, glutamate the production phase and constituted the main coproduct
production is induced by a rapid increase of the fermenta- throughout the fermentation. The specific production rate
tion temperature (Fig. 1). Throughout the production increase regularly from 0.04 gg } h &1 to reach 0.06 gg } h &1
phase, the temperature was maintained at 39.5\0.5%C. In after 14 h and remained constant thereafter. Production of
less than 2 h, the growth rate decreased from 0.58 h &1 to 0.2 dihydroxyacetone was considerably less important and was
h &1 and the specific rate of glutamate production reached a maintained throughout the fermentation.
maximum value of 0.55 g } g &1 } h &1 (Fig. 2). Glutamate
production and glucose consumption remained maximal for Stoichiometric Analysis of the Fermentation
6 h while the growth rate continued to decrease slowly.
During this phase, trehalose, lactate, and dihydroxyacetone Stoichiometric analysis based upon product yields,
were detected at low levels in the medium. The presence of calculated from data given above, illustrates that metabolic
these metabolites indicates metabolic leakage at three levels changes occurred throughout the fermentation (Fig. 3).
within glycolysis (glucose 6-phosphate, pyruvate, and triose Throughout the fermentation carbon balances were good,
phosphates). Such overflow metabolism was not due to i.e., more than 950 of the sugar consumed was accounted
oxygen limitation since dissolved oxygen was maintained for in the major products (biomass, CO 2 , glutamate,
above 200 saturation. After 12 h of fermentation, the specific trehalose, lactate and dihydroxyacetone). The remainder

226
Metabolic Constraints during Glutamate Fermentation
FIG. 2. Evolution of specific rates throughout the fermentation. (A)
Glucose consumption ( - - ), glutamate production (   ), and growth
(www). (B) trehalose production ( - ), lactate production (  ), and
DHA production (-------).
was in part associated with a number of minor products
present at trace amounts (succinate, :-ketoglutarate,
acetate, alanine, glutamine, and N-acetyl-glutamine). The
post-temperature shift part of the fermentation can be
divided into three distinct phases. The first phase which
lasted approximately 2 h is characterized by a rapid shift
from growth to glutamate production. A second phase is
then initiated until growth stops after 15 h of fermentation.
This phase is marked by a reduction of the biomass yield
associated with co-metabolite production. It can be specu-
lated that the decrease of the anabolic carbon flux leaving
glycolysis for biomass synthesis provokes an increase of the
glycolytic flux and probably leads to overflow metabolism.
The glutamate yield remains stable during this phase.
A final phase begins after the growth arrest with an increase
in the yields of lactate and trehalose at the expense of
glutamate production. This tendency accelerates in the final
3 h of the fermentation in which a marked decrease in
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Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
FIG. 3. Instantaneous yields relative to glucose consumption
throughout the fermentation. The vertical division lines indicate distinct
phases during the glutamate production phase as mentioned in the text.
(A) Glutamate (   ), biomass (www), and CO 2 ( - - ). (B) trehalose
( -), lactate (  ), and DHA (-------).
carbon conversion efficiency is seen for glutamate with con-
comitant increases in the molar yields for lactate, trehalose,
and CO 2 .
Enzyme Activity Profiles
The specific activities of various enzymes, representative
of different pathways of central metabolism, were assayed
throughout the culture (Fig. 4). A transient decrease of all
activities was observed immediately after the temperature
shift, corresponding, to an increased cellular protein content,
probably due to the induced synthesis of stress proteins.
Glutamate dehydrogenase activity remained constant
throughout the fermentation. The enzyme concentration
seems not to be affected by the shift to glutamate pro-
duction. A similar profile was obtained when glutamate
production was induced by biotin limitation, addition of
penicillin G or surfactant (Kawahara et al., 1997). The GAP
Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
FIG. 4. Evolution of specific enzyme activities of C. glutamicum
throughout the fermentation. (A) Glyceraldehyde 3-phosphate dehydro-
genase (s), isocitrate dehydrogenase (S), and glutamate dehydrogenase
(Q). (B) 6-phosphoguconate dehydrogenase (m), glucose 6-phosphate
dehydrogenase (M), phosphoenolpyruvate carboxylase (G), malic enzyme
(g), and NADH oxidizing activity (q). Each value represents the average
of three independent determinations with error bars.
dehydrogenase activity increased rapidly during the first
half of the production phase prior to stabilising at a value
approximately twofold higher than during exponential
growth when growth no longer occurred. There is an
apparent contradiction between the increase of GAP
dehydrogenase specific activity and DHA production
indicative of a possible flux limitation through this enzyme
though clearly this will depend upon the in vivo regulation
of this enzyme activity. The specific activity of isocitrate
dehydrogenase increased progressively throughout the
production phase.
The two dehydrogenation reactions (G6P dehydrogenase
and 6PG dehydrogenase) constituting the initial entry into
the pentose pathway showed different variations in specific
activity profiles: 6PG dehydrogenase behaved similarly to
GAP dehydrogenase while G6P dehydrogenase activity
decreased markedly once growth ceased.
228
Gourdon and Lindley
The two anaplerotic enzymes assayed display a different
evolution. The specific activity of malic enzyme decreased
rapidly after the temperature shift whereas the PEP car-
boxylase activity increased to a maximal value between 10
and 15 h of fermentation, when the growth rate is very low.
This finding corroborates previous observations concerning
the growth rate dependent activity of each anaplerotic
enzyme in glucose-grown cells (Cocaign-Bousquet et al.,
1996). In view of the rapid disappearance of malic enzyme
activity it can probably be concluded that this enzyme
plays no major role in TCA cycle replenishment during
the greater part of the glutamate production phase. The
PEP carboxylase activity is significantly lower than the
anaplerotic flux required for glutamate except during the
terminal phase of the fermentation once growth had
stopped. As concluded elsewhere (Park et al., 1997b; Peters-
Wendisch et al., 1996) a pyruvate carboxylating enzyme is
probably ensuring the majority of the anaplerotic flux dur-
ing the production phase though we were unable to measure
reliably this activity in the strain used in the present study.
Finally an NADH oxidizing activity, initially believed to be
due to NADH oxidase activity (Cocaign-Bousquet et al.,
1996) but now believed to be attributable to NADH
dehydrogenase components of the respiratory chain (Hertz,
1998) decreased rapidly in parallel to the growth rate.
Metabolite Concentrations
The intracellular concentrations of key phosphorylated
glycolytic intermediates, pyruvate and NAD(H) were
followed during the fermentation. The concentration of all
glycolytic metabolites increased during the second half of
the production phase indicating a progressive limitation
of the flux through glycolysis (Fig. 5). Both glucose
6-phosphate and fructose 6-phosphate concentrations
remained constant for about 6 h after the temperature
shift at levels comparable with these measured during
exponential growth phase, but increased sharply thereafter
throughout the following 12 h until the end of the fermenta-
tion to concentrations more than fourfold higher than seen
in exponential growth. Fructose 1,6-diphosphate concentra-
tion increases sharply during the initial phase of glutamate
production and then more slowly throughout the remainder
of the production phase. Triose-Ps (GAP and DHAP) levels
increase linearly from the onset of the production phase to
stabilize transiently at a value of 20 +molg coinciding with
the peak of DHA production. Thereafter, the pool concen-
tration increased sharply to reach a maximal value of
80 +molg when growth ceased: value maintained through-
out the remainder of the fermentation. Phosphorylated
intermediates situated downstream of GAP but upstream of
PEP were at all times lower than the analytical precision
Metabolic Constraints during Glutamate Fermentation
FIG. 5. Concentrations of intracellular metabolites in cell samples taken throughout the fermentation. Each data point represents the average of four
independent determinations with error bars.
threshold (<0.2 +mol). The manner in which the pool of
PEP evolved throughout the production phase was similar
to that observed for sugar phosphates (glucose 6-phosphate
and fructose 6-phosphate) possibly involving the type of
coordinated regulation of phosphofructokinase and
pyruvate kinase activities described for Lactobacillus
bulgericus (Le Bras et al., 1998). The pyruvate pool shows
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Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
a progressive but small increase in concentration
throughout the production phase until the final hours of the
fermentation during which the pyruvate concentration
increases rapidly, concomitant with the increased rate of
lactate production.
Although the intracellular concentration of NAD remained
approximately constant throughout the fermentation
Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
the NADH concentration increased in a pattern similar to
that observed for many of the glycolytic intermediates, i.e.,
a slight increase throughout the initial period of the pro-
duction phase with a more marked increase in the later
period of glutamate production. This leads to a significant
modification of the NADHNAD ratio, known to play a
major role in the in vivo regulation of glycolysis and notably
as an inhibitor of pyruvate and GAP dehydrogenases
(Snoep et al., 1992; Garrigues et al., 1997; Dominguez et al.,
1998) while such an increase in intracellular NADH concen-
tration is also known to activate lactate dehydrogenase
activity (Garrigues et al., 1997) which is constitutively
expressed in C. glutamicum but increases progressively
throughout the production phase to reach activities
threefold higher than those measured in exponentially
growing cells (results not shown). It would appear likely
that this increase in NADHNAD ratio, probably due to a
decreased respiratory capacity since NADH oxidation
decreases rapidly after the temperature shift, provokes
many of the intracellular modifications of metabolite pools
and the consequences this has on whole cell behavior.
DISCUSSION
Metabolic engineering strategies attempt to obtain a
realistic view of the metabolic constraints associated with
the specific operating conditions used in industrial fermen-
tations to pragmatically identify targets for rational
improvement of either the process or the biological compo-
nent of the process. However, this approach frequently
assumes that metabolite overproduction obeys a rigid
steady-state type behavior and that rate-controlling reac-
tions remain so over the entire production phase despite the
frequently observed changes in kinetic behavior. Fermenta-
tion processes are intrinsically changing systems, or at least
in the more commonly used batch systems, in which the
physicochemical environment seen by the cells is being con-
stantly modified, not least by the accumulation of desired
metabolites. During the fermentation, the temperature-
induced overflow of glutamate leads to an important
accumulation of products (notably glutamic acid) with
associated osmotic stress. This progressive increase in
osmolarity is associated with trehalose production whose
intracellular accumulation is known to contribute to
osmoprotective mechanisms (Guillouet and Engasser,
1995) but is frequently reported to be excreted into the
medium during amino acid overproduction using C.
glutamicum (Ishino et al., 1991; Park et al., 1997a).
In the temperature induced production phase it appears
that the capacity to maintain cellular division is important
for process performance. The progressive loss of cell division
and modified catabolic potential is seen in the slowing down
230
Gourdon and Lindley
of the specific metabolic rates and even more clearly in the
shift from glutamate production to alternative products in
the final phase of the fermentation. This is particularly
noticeable for lactate production which accompanies the
increased NADHNAD ratio. Whole cell kinetics and
intracellular analysis of metabolites and enzyme activities
are similar to what would be expected under oxygen limit-
ing conditions though only trace amounts of succinate,
indicative of anoxic conditions (Dominguez et al., 1993),
were measured during the production phase. Indeed the
situation appears to be more closely akin to the behavior of
C. glutamicum during growth on fructose in which both
DHA and lactate accumulate due to the modified
intracellular flux partition and the resulting increase in
glycolysis (Dominguez et al., 1998). This was attributed to
an increased NADHNAD ratio and the control exerted
over dehydrogenation reactions such that glycolysis
becomes saturated. In this case, the overflow of lactate and
DHA is again correlated to an increased NADHNAD ratio
though in this case this would appear to be associated to the
diminished capacity to oxidize NADH. Future work needs
to establish to what extent respiratory activity is affected
under such conditions.
Clearly increasing product yields in such a fermentation
will probably depend upon our capacity to apply correctly
the metabolic knowledge that can be gained in physiological
studies. Analysis of the initial period of glutamate produc-
tion would no doubt lead to credible targets for improving
carbon conversion efficiency, but the loss of metabolic
activity observed throughout the second half of the fermen-
tation would most probably attenuate the anticipated
gains. An alternative approach, derived directly from the
metabolic analysis used in this study would be to modify
the process constraints so as to maintain a higher level of
metabolic activity and thereby prolong the period of high
rate and high yield of glutamate production, retarding the
increased NADHNAD ratio and associated consequences
for product formation and sugar consumption rate.
ACKNOWLEDGEMENTS
The authors thank Mrs. Monique Suderie for valuable technical
assistance. This work was made possible by financial assistance from
ORSAN-Amylum France, the CNRS, and as part of the work package
supported by the European Union Grant BIO4-CT96-01145.
REFERENCES
Cocaign-Bousquet, M., Guyonvarch, A., and Lindley, N. D. (1996).
Growth rate dependent modulation of carbon flux through central
metabolism and the kinetic consequences for glucose-limited chemostat
cultures of Corynebacterium glutamicum. Appl. Env. Microbiol. 62,
429436.
Metabolic Constraints during Glutamate Fermentation
Crow, W. L., and Wittenberg, C. L. (1979). Separation and properties
of NAD +- and NADP +-dependent glyceraldehyde-3-phosphate
dehydrogenase from Streptococcus mutans. J. Biol. Chem. 254,
11341142.
Dominguez, H., Nezondet, C., Lindley, N. D., and Cocaign, M. (1993).
Modified carbon flux during oxygen-limited growth of Corynebacterium
glutamicum and the consequences for amino acid production.
Biotechnol. Lett. 15, 449454.
Dominguez, H., Rollin, C., Guyonvarch, A., Guerquin-Kern, J. L.,
Cocaign-Bousquet, M., and Lindley, N. D. (1998). Carbon-flux
distribution in the central metabolic pathways of Corynebacterium
glutamicum during growth on fructose. Eur. J. Biochem. 254, 96102.
Garrigues, C., Loubiere, P., Lindley, N. D., and Cocaign-Bousquet,
M. (1997). Control of the shift from homolactic acid to mixed-acid
fermentation in Lactococcus lactis : Predominant role of the
NADHNAD + ratio. J. Bacteriol. 179, 52825287.
Guillouet, S., and Engasser, J. M. (1995). Growth of Corynebacterium
glutamicum in glucose-limited continuous cultures under high osmotic
pressure. Influence of growth rate on the intracellular accumulation of
proline, glutamate and trehalose. Appl. Microbiol. Biotechnol. 44,
496500.
Hertz, P. F. (1998). ``Cha@^ ne respiratoire et deshydrogenases flaviniques
chez Corynebacterium melassecola ATCC 17965: Contribution a l'etude
des voies d'utilisation de coenzymes intervenant dans les reactions
d'oxydoreduction.'' Ph.D. thesis. Universite Paris Sud, France.
Ishino, S., Shimomura-Nishimuta, J., Yamaguchi, K., Shirahata, K., and
Araki, K. (1991). 13C Nuclear magnetic resonance studies of glucose
metabolism in L-glutamic acid and L-lysine fermentation by Corynebac-
terium glutamicum. J. Gen. Appl. Microbiol. 37, 157165.
Kawahara, Y., Takahashi-Fuke, K., Shimizu, E., Nakamatsu, T., and
Nakamori, S. (1997). Relationship between the glutamate production
and the activity of 2-oxoglutarate dehydrogenase in Brevibacterium
lactofermentum. Biosci. Biotech. Biochem. 61, 11091112.
Lebloas, P., Guilbert, N., Loubiere, P., and Lindley, N. D. (1993). Growth
inhibition and pyruvate overflow during glucose metabolism of
Eubacterium limosum are related to a limited capacity to reassimilate
CO 2 by the acetyl-CoA pathway. J. Gen. Microbiol. 139, 18611868.
Le Bras, G., de la Torre, F., Branny, P., and Garel, J. R. (1998). Enzymatic
and genetic regulation of glycolysis in Lactobacillus delbrueckii subsp.
bulgaricus. Lait 78, 8590.
Marx, A., de Graaf, A. A., Wiechert, W., Eggeling, L., and Sahm, H. (1996).
Determination of the fluxes in the central metabolism of Corynebac-
terium glutamicum by nuclear magnetic resonance spectroscopy
combined with metabolic balancing. Biotechnol. Bioeng. 49, 111129.
Marx, A., Striegel, K., de Graaf, A. A., Sahm, H., and Eggeling,
L. (1997). Response of the central metabolism of Corynebacterium
glutamicum to different flux burdens. Biotechnol. Bioeng. 56, 168180.
231
Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122
Marx, A., Eikmanns, B. J., Sahm, H., de Graaf, A. A., and Eggeling, L.
(1999). Response of the central metabolism in Corynebacterium gluta-
micum to the use of an NADH-dependent glutamate dehydrogenase.
Metabolic Engineering 1, 3548.
Mori, M., and Shiio, I. (1984). Production of aspartic acid and enzymatic
alteration in pyruvate kinase mutants of Brevibacterium flavum. Agric.
Biol. Chem. 48, 11891197.
Mori, M., and Shiio, I. (1987). Pyruvate formation and sugar metabolism
in an amino acid-producing bacterium, Brevibacterium flavum. Agric.
Biol. Chem. 51, 129138.
Park, S. M., Sinskey, A. J., and Stephanopoulos, G. (1997a). Metabolic
and physiological studies of Corynebacterium glutamicum mutants.
Biotechnol. Bioeng. 55, 864879.
Park, S. M., Shaw-Reid, C., Sinskey, A. J., and Stephanopoulos, G.
(1997b). Elucidation of anaplerotic pathways in Corynebacterium
glutamicum via 13C-NMR spectroscopy and GC-MS. Appl. Microbiol.
Biotechnol. 47, 430440.
Peters-Wendisch, P. G., Wendisch, V. F., de Graaf, A. A., Eikmanns, B. J.,
and Sahm, H. (1996). C3-carboxylation as an anaplerotic reaction in
phosphoenolpyruvate-deficient Corynebacterium glutamicum. Arch.
Microbiol. 165, 387396.
Pons, A., Dussap, C. G., Pequignot, C., and Gros, J. B. (1996). Metabolic
flux distribution in Corynebacterium glutamicum ATCC 17965 for
various carbon sources. Biotechnol. Bioeng. 51, 177189.
Rollin, C., Morgant, V., Guyonvarch, A., and Guerquin-Kern, J. L. (1995).
13
C-NMR studies of Corynebacterium melassecola metabolic pathways.
Eur. J. Biochem. 227, 488493.
Snoep, J. L., Teixeira de Mattos, M. J., Starrenburg, M., and Hugenholtz,
J. (1992). Isolation, characterisation and physiological role of the
pyruvate dehydrogenase complex and :-acetolactate synthase of
Lactococcus lactis subsp. diacetylactis. J. Bacteriol. 174, 48314841.
Sonntag, K., Schwinde, J., de Graaf, A. A., Marx, A., Eikmanns, B. J.,
Wiechert, W., and Sahm, H. (1995). 13C NMR studies of the fluxes in
the central metabolism of Corynebacterium glutamicum during growth
and overproduction of amino acids in batch cultures. Appl. Env.
Microbiol. 44, 489495.
Sugimoto, S., and Shiio, I. (1987a). Regulation of glucose 6-phosphate
dehydrogenase in brevibacterium flavum. Agric. Biol. Chem. 51, 101108.
Sugimoto, S., and Shiio, I. (1987b). Regulation of 6-phosphogluconate
dehydrogenase in brevibacterium flavum. Agric. Biol. Chem. 51,
12571263.
Vallino, J., and Stephanopoulos, G. (1993). Metabolic flux distributions in
Corynebacterium glutamicum during growth and lysine overproduction.
Biotechnol. Bioeng. 41, 633646.
Von der Osten, C. H., Gioannetti, C., and Sinskey, A. J. (1989). Design of
a defined medium for growth of Corynebacterium glutamicum in which
citrate facilitates iron uptake. Biotechnol. Lett. 11, 1116.