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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

approaches. Particular attention have been paid to the


The dynamic behavior of the metabolism of Corynebacterium pyruvatePEP branching point in an attempt to define the
glutamicum during l-glutamic acid fermentation, was evaluated by relative contribution of each anaplerotic enzyme for TCA
quantitative analysis of the evolution of intracellular metabolites and cycle during growth and amino acid synthesis (Park et al.,
key enzyme concentrations. Glutamate production was induced 1997b; Peters-Wendisch et al., 1996). Much of this work has
by an increase of the temperature and a final concentration of involved either growth of the organism on various sub-
80 g l was attained. During the production phase, various other strates or l-lysine production. The metabolic constraints
compounds, notably lactate, trehalose, and DHA were secreted to
associated with l-glutamic acid production have not been
the medium. Intracellular metabolites analysis showed important
variations of glycolytic intermediates and NADH, NAD coenzymes
as thoroughly characterized. However, NMR experiments
levels throughout the production phase. Two phenomena occur (Ishino et al., 1991; Sonntag et al., 1995; Marx et al., 1997),
during the production phase which potentially provoke a decrease in have reported significant modifications of metabolic flux
the glutamate yield: Both the intracellular concentrations of between exponential growth and l-glutamate production.
glycolytic intermediates and the NADH NAD ratio increase signi- Most important are a diminished flux through the pentose
ficantly during the period in which the overall metabolic rates pathway and an increased flux through isocitrate dehydro-
decline. This correlates with the decrease in glutamate yield due genase during the phase of l-glutamate production. The
in part to the production of lactate and also to the period of the smaller contribution of the pentose pathway was assumed
fermentation in which growth no longer occurred.  1999 Academic Press to occur because of the diminished requirement of NADPH
in glutamic acid formation, postulating a correlation
between the carbon flux distribution and the energetic
INTRODUCTION demand though this has proved difficult to exploit (Marx et
al., 1999). However, these experiments though elegant in
Corynebacterium glutamicum is widely used for the produc- their laboratory conception often used either poor produc-
tion of amino acids, such as l-glutamic acid and l-lysine. tion strains or fermentation strategies somewhat different
Because of the economic importance of these fermentations, from those suitable for industrial use. Little attempt was
a great deal of effort has been made, in the last decade, to made to see how flux distribution varied throughout the
improve our working knowledge of the physiology of this production phase, notably at glutamate concentrations of
bacterium. Investigations have focused on the regulation of industrial interest. In this study, a temperature-triggered
amino acid biosynthetic pathways and the carbon flux process of glutamate overproduction has been used and
distribution within the central metabolism. Various dynamic variations in intracellular metabolite pools have
groups have examined the fate of glucose in the central been correlated to whole cell kinetic behavior. Important
pathways, using NMR (Rollin et al., 1995; Marx et al., 1996; variations of metabolites levels were measured during the
Dominguez et al., 1998), enzymatic (Cocaign-Bousquet fermentation indicating that the production phase cannot
et al., 1996) and mathematical modelling (Vallino and be assumed to be a period of quasi-steady-state behavior.
Stephanopoulos, 1993; Pons et al., 1996; Park et al., 1997a) This dynamic analysis suggests that strain improvement
1
strategies based upon data obtained during the early phase
To whom correspondence should be addressed at centre de
Bioingenierie Gilbert Durand, INSA, Complexe Scientifique de Rangueil,
of production may fail to perform up to expectations due to
31077 Toulouse cedex 4, France. Fax: (33) 5 61 55 94 00. E-mail: the modified metabolic constraints intervening later in the
lindleyinsa-tlse.fr. fermentation.

1096-717699 30.00 224


Copyright  1999 by Academic Press
All rights of reproduction in any form reserved.
Metabolic Constraints during Glutamate Fermentation Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122

MATERIALS AND METHODS extracts were prepared as follows: cell were harvested,
washed twice with 0.20 KCl, and resuspended in Tris-
Organism and Cultivation tricarballylate buffer (250 mM, pH 7.8) containing glycerol
(300), and MgCl 2 (5 mM). Cells were disrupted by sonica-
The strain used in this work was Corynebacterium tion and cell debris was removed by centrifugation at
glutamicum 2262. Cultivation was done in a 3.5-liter reactor 10,000g, for 10 min at 4%C. The supernatant was used for
(Chemap). The pH was maintained at 7.6 by automatic enzyme assay and the protein concentration of the extract
addition of NH 4 (12 N). During the production phase, the was determined by the Lowry method with bovine serum
culture was pulsed with a concentrated glucose solution albumin as standard.
(500 gl) to avoid periods of glucose limitation. The Glucose 6-phosphate dehydrogenase was assayed by a
medium was based on MCGC medium (Von der Osten et method based on that of Sugimoto and Shiio (1987a) in
al., 1989) although citrate was replaced by deferoxamine. a reaction mixture containing Tris-HCl buffer (100 mM,
The medium contained glucose (60 gl), Na 2HPO 4 (3 gl), pH 7.8), MgCl 2 (10 mM), NADP + (0.5 mM), and glucose
KH 2PO 4 (6 gl), NaCl (2 gl), (NH 4 ) 2SO 4 (8 gl), MgSO 4, 6-phosphate (2 mM) as the substrate. 6-phosphogluconate
7H 2O (0.4 gl), FeSO 4, 7H 2O (40 mgl), FeCl 3 (4 mgl), dehydrogenase was assayed by a method based on that of
ZnSO4, 7H 2O (1 mgl), CuCl 2, 2H 2O (0.4 mgl), MnSO 4, Sugimoto and Shiio (1987b) using the same reaction
H 2O (2 mgl), (NH 4 ) 6Mo 7O 24, 4 H 2O (0.2 mgl), Na 2B 4O 7 , mixture as above, except that 6-Phosphogluconate (1 mM)
10 H 2O (0.4 mgl), CaCl 2 (84 mgl), biotin (2 mgl), was added as the substrate instead of glucose 6-phosphate.
thiamine (20 mgl), desferoxamine (3 mgl), and betaine Phosphoenolpyruvate carboxylase was assayed by a method
(2 gl). The onset of glutamate production was induced by based on that of Mori and Shiio (1984) in a reaction
increasing the growth temperature from 33 to 39.5%C over a mixture containing Tris-HCl buffer (100 mM, pH 7.8),
5 min period when the biomass concentration reached MnSO 4 (5 mM), KHCO 3 (10 mM), NADH 2 (0.15 mM),
5.6 gl. acetyl coenzyme-A (0.1 mM), 10 +g } ml &1 malate dehy-
drogenase, and PEP (2 mM). The reaction was started
Analytical Methods by the addition of PEP. Glyceraldehyde-3-phosphate
Biomass was estimated by absorbance at 650 nm and by dehydrogenase was assayed according to the method of
a direct gravimetric method following drying of washed cells Crow and Wittenberger (1979) with the following reaction
to constant weight under partial vacuum at 60%C for at least mixture: NAD (1 mM), sodium arsenate (5 mM), cysteine
24 h. In this manner, potential errors in biomass estimation HCl (5 mM), triethanolamine buffer (125 mM, pH 7.8),
linked to morphological changes were avoided. Sugars and dl-glyceraldehyde-3-P (4 mM), and diluted enzyme extract.
organic acids were determined on the culture supernatant Malic enzyme was assayed by the method described by
by HPLC using a HPX87H column (Biorad) maintained at Mori and Shiio (1987) with an optimized reaction mixture
48%C with H 2SO 4 (5 mM) as eluant. Detection was per- consisting of phosphate buffer (100 mM, pH 7.8),
formed by UV and refractometer detectors. Gas phase MgCl 2(5 mM), NADP + (0.6 mM), and malate (40 mM) as
composition was determined by gas chromatography with a the substrate. Assays were initiated by the addition of
Porapak Q column maintained at 40%C with helium as the malate. Isocitrate dehydrogenase was assayed with the
carrier gas and catharometer detection. Glutamate and following reaction mixture : Tris-HCl buffer (100 mM,
other amino acid concentrations was determined with an pH 7.8), MnSO 4 (5 mM), NADP + (0,6 mM), and
AminoQuant 1090 high-pressure liquid chromatography isocitrate (10 mM). Assays were initiated by the addition of
(HewlettPackard) after derivatization by orthophthalade- isocitrate. Glutamate dehydrogenase was assayed using a
hyde in the presence of 3-mercaptopropionic acid, separation reaction mixture containing Tris-HCl buffer (100 mM,
with a C 18 column, and spectrophotometric detection at pH 7.8), NH 4Cl (40 mM), NADPH 2 (0.6 mM), and
338 nm. :-ketoglutarate (10 mM). Nonspecific NADH oxidation
was assayed using a reaction mixture containing Tris-HCl
Enzymatic Assays buffer (100 mM, pH 7.8), MnSO 4 (5 mM), and NADH 2
(0.3 mM).
Most enzyme activities were assayed by spectrophoto-
metric measurement of variation in NAD(P)H concentration Extraction and Estimation of Intracellular Metabolite
at 340 nm (==6.223 M &1 cm &1 ). One unit of activity was Concentrations
the amount of enzyme required to produce 1 nmol of
product per min. All enzyme activity were measured in The extraction procedure optimized by Dominguez et al.
crude cell-free extracts obtained by sonication. Crude (1998) was used in which cell samples (10 ml) of known cell

225
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

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Metabolic Constraints during Glutamate Fermentation Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122

FIG. 3. Instantaneous yields relative to glucose consumption


FIG. 2. Evolution of specific rates throughout the fermentation. (A) throughout the fermentation. The vertical division lines indicate distinct
Glucose consumption ( - - ), glutamate production (   ), and growth phases during the glutamate production phase as mentioned in the text.
(www). (B) trehalose production ( - ), lactate production (  ), and (A) Glutamate (   ), biomass (www), and CO 2 ( - - ). (B) trehalose
DHA production (-------). ( -), lactate (  ), and DHA (-------).

was in part associated with a number of minor products carbon conversion efficiency is seen for glutamate with con-
present at trace amounts (succinate, :-ketoglutarate, comitant increases in the molar yields for lactate, trehalose,
acetate, alanine, glutamine, and N-acetyl-glutamine). The and CO 2 .
post-temperature shift part of the fermentation can be
divided into three distinct phases. The first phase which Enzyme Activity Profiles
lasted approximately 2 h is characterized by a rapid shift
from growth to glutamate production. A second phase is The specific activities of various enzymes, representative
then initiated until growth stops after 15 h of fermentation. of different pathways of central metabolism, were assayed
This phase is marked by a reduction of the biomass yield throughout the culture (Fig. 4). A transient decrease of all
associated with co-metabolite production. It can be specu- activities was observed immediately after the temperature
lated that the decrease of the anabolic carbon flux leaving shift, corresponding, to an increased cellular protein content,
glycolysis for biomass synthesis provokes an increase of the probably due to the induced synthesis of stress proteins.
glycolytic flux and probably leads to overflow metabolism. Glutamate dehydrogenase activity remained constant
The glutamate yield remains stable during this phase. throughout the fermentation. The enzyme concentration
A final phase begins after the growth arrest with an increase seems not to be affected by the shift to glutamate pro-
in the yields of lactate and trehalose at the expense of duction. A similar profile was obtained when glutamate
glutamate production. This tendency accelerates in the final production was induced by biotin limitation, addition of
3 h of the fermentation in which a marked decrease in penicillin G or surfactant (Kawahara et al., 1997). The GAP

227
Metabolic Engineering 1, 224231 (1999) Gourdon and Lindley
Article ID mben.1999.0122

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
FIG. 4. Evolution of specific enzyme activities of C. glutamicum
throughout the fermentation. (A) Glyceraldehyde 3-phosphate dehydro- The intracellular concentrations of key phosphorylated
genase (s), isocitrate dehydrogenase (S), and glutamate dehydrogenase glycolytic intermediates, pyruvate and NAD(H) were
(Q). (B) 6-phosphoguconate dehydrogenase (m), glucose 6-phosphate followed during the fermentation. The concentration of all
dehydrogenase (M), phosphoenolpyruvate carboxylase (G), malic enzyme
(g), and NADH oxidizing activity (q). Each value represents the average
glycolytic metabolites increased during the second half of
of three independent determinations with error bars. the production phase indicating a progressive limitation
of the flux through glycolysis (Fig. 5). Both glucose
6-phosphate and fructose 6-phosphate concentrations
dehydrogenase activity increased rapidly during the first remained constant for about 6 h after the temperature
half of the production phase prior to stabilising at a value shift at levels comparable with these measured during
approximately twofold higher than during exponential exponential growth phase, but increased sharply thereafter
growth when growth no longer occurred. There is an throughout the following 12 h until the end of the fermenta-
apparent contradiction between the increase of GAP tion to concentrations more than fourfold higher than seen
dehydrogenase specific activity and DHA production in exponential growth. Fructose 1,6-diphosphate concentra-
indicative of a possible flux limitation through this enzyme tion increases sharply during the initial phase of glutamate
though clearly this will depend upon the in vivo regulation production and then more slowly throughout the remainder
of this enzyme activity. The specific activity of isocitrate of the production phase. Triose-Ps (GAP and DHAP) levels
dehydrogenase increased progressively throughout the increase linearly from the onset of the production phase to
production phase. stabilize transiently at a value of 20 +molg coinciding with
The two dehydrogenation reactions (G6P dehydrogenase the peak of DHA production. Thereafter, the pool concen-
and 6PG dehydrogenase) constituting the initial entry into tration increased sharply to reach a maximal value of
the pentose pathway showed different variations in specific 80 +molg when growth ceased: value maintained through-
activity profiles: 6PG dehydrogenase behaved similarly to out the remainder of the fermentation. Phosphorylated
GAP dehydrogenase while G6P dehydrogenase activity intermediates situated downstream of GAP but upstream of
decreased markedly once growth ceased. PEP were at all times lower than the analytical precision

228
Metabolic Constraints during Glutamate Fermentation Metabolic Engineering 1, 224231 (1999)
Article ID mben.1999.0122

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 a progressive but small increase in concentration
PEP evolved throughout the production phase was similar throughout the production phase until the final hours of the
to that observed for sugar phosphates (glucose 6-phosphate fermentation during which the pyruvate concentration
and fructose 6-phosphate) possibly involving the type of increases rapidly, concomitant with the increased rate of
coordinated regulation of phosphofructokinase and lactate production.
pyruvate kinase activities described for Lactobacillus Although the intracellular concentration of NAD remained
bulgericus (Le Bras et al., 1998). The pyruvate pool shows approximately constant throughout the fermentation

229
Metabolic Engineering 1, 224231 (1999) Gourdon and Lindley
Article ID mben.1999.0122

the NADH concentration increased in a pattern similar to of the specific metabolic rates and even more clearly in the
that observed for many of the glycolytic intermediates, i.e., shift from glutamate production to alternative products in
a slight increase throughout the initial period of the pro- the final phase of the fermentation. This is particularly
duction phase with a more marked increase in the later noticeable for lactate production which accompanies the
period of glutamate production. This leads to a significant increased NADHNAD ratio. Whole cell kinetics and
modification of the NADHNAD ratio, known to play a intracellular analysis of metabolites and enzyme activities
major role in the in vivo regulation of glycolysis and notably are similar to what would be expected under oxygen limit-
as an inhibitor of pyruvate and GAP dehydrogenases ing conditions though only trace amounts of succinate,
(Snoep et al., 1992; Garrigues et al., 1997; Dominguez et al., indicative of anoxic conditions (Dominguez et al., 1993),
1998) while such an increase in intracellular NADH concen- were measured during the production phase. Indeed the
tration is also known to activate lactate dehydrogenase situation appears to be more closely akin to the behavior of
activity (Garrigues et al., 1997) which is constitutively C. glutamicum during growth on fructose in which both
expressed in C. glutamicum but increases progressively DHA and lactate accumulate due to the modified
throughout the production phase to reach activities intracellular flux partition and the resulting increase in
threefold higher than those measured in exponentially glycolysis (Dominguez et al., 1998). This was attributed to
growing cells (results not shown). It would appear likely an increased NADHNAD ratio and the control exerted
that this increase in NADHNAD ratio, probably due to a over dehydrogenation reactions such that glycolysis
decreased respiratory capacity since NADH oxidation becomes saturated. In this case, the overflow of lactate and
decreases rapidly after the temperature shift, provokes DHA is again correlated to an increased NADHNAD ratio
many of the intracellular modifications of metabolite pools though in this case this would appear to be associated to the
and the consequences this has on whole cell behavior. diminished capacity to oxidize NADH. Future work needs
to establish to what extent respiratory activity is affected
DISCUSSION under such conditions.
Clearly increasing product yields in such a fermentation
Metabolic engineering strategies attempt to obtain a will probably depend upon our capacity to apply correctly
realistic view of the metabolic constraints associated with the metabolic knowledge that can be gained in physiological
the specific operating conditions used in industrial fermen- studies. Analysis of the initial period of glutamate produc-
tations to pragmatically identify targets for rational tion would no doubt lead to credible targets for improving
improvement of either the process or the biological compo- carbon conversion efficiency, but the loss of metabolic
nent of the process. However, this approach frequently activity observed throughout the second half of the fermen-
assumes that metabolite overproduction obeys a rigid tation would most probably attenuate the anticipated
steady-state type behavior and that rate-controlling reac- gains. An alternative approach, derived directly from the
tions remain so over the entire production phase despite the metabolic analysis used in this study would be to modify
frequently observed changes in kinetic behavior. Fermenta- the process constraints so as to maintain a higher level of
tion processes are intrinsically changing systems, or at least metabolic activity and thereby prolong the period of high
in the more commonly used batch systems, in which the rate and high yield of glutamate production, retarding the
physicochemical environment seen by the cells is being con- increased NADHNAD ratio and associated consequences
stantly modified, not least by the accumulation of desired for product formation and sugar consumption rate.
metabolites. During the fermentation, the temperature-
induced overflow of glutamate leads to an important ACKNOWLEDGEMENTS
accumulation of products (notably glutamic acid) with
associated osmotic stress. This progressive increase in The authors thank Mrs. Monique Suderie for valuable technical
osmolarity is associated with trehalose production whose assistance. This work was made possible by financial assistance from
intracellular accumulation is known to contribute to ORSAN-Amylum France, the CNRS, and as part of the work package
supported by the European Union Grant BIO4-CT96-01145.
osmoprotective mechanisms (Guillouet and Engasser,
1995) but is frequently reported to be excreted into the
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