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J. Biol. Chem., Vol. 277, Issue 31, 28088-28098, August 2, 2002
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From the
Received for publication, March 16, 2002, and in revised form, April 29, 2002
The involvement of nicotinamide
adenine nucleotides (NAD+, NADH) in the regulation of
glycolysis in Lactococcus lactis was investigated by using
13C and 31P NMR to monitor in vivo
the kinetics of the pools of NAD+, NADH, ATP, inorganic
phosphate (Pi), glycolytic intermediates, and end products
derived from a pulse of glucose. Nicotinic acid specifically labeled on
carbon 5 was synthesized and used in the growth medium as a precursor
of pyridine nucleotides to allow for in vivo detection of
13C-labeled NAD+ and NADH. The capacity of
L. lactis MG1363 to regenerate NAD+ was
manipulated either by turning on NADH oxidase activity or by knocking
out the gene encoding lactate dehydrogenase (LDH). An LDH Lactococcus lactis plays an essential role
in the manufacture of a wide range of dairy products. The relative
simplicity of L. lactis metabolism that converts sugars via
the glycolytic pathway to pyruvate, generating energy mainly through
substrate level phosphorylation, makes it an attractive model organism
to test metabolic engineering strategies. Moreover, the large number of genetic tools available for L. lactis (1) and the recent
release of the complete genome sequence are additional incentives to
study the physiology of this organism in great depth (2).
Despite numerous studies, a satisfactory answer to the question, What
controls the glycolytic flux in L. lactis? has not been put
forward. During homolactic fermentation, regulation of the carbon flux
has been associated with high levels of fructose 1,6-bisphosphate (FBP),1 which activates
lactate dehydrogenase (LDH; EC 1.1.1.27) and pyruvate kinase (PK; EC
2.7.1.40), directing the flux toward the production of lactate (3). A
metabolic shift from homolactic (lactate production) to mixed acid
fermentation (ethanol, acetate, and formate production) was observed in
glucose-limited chemostat cultures (4). A deviation from homolactic
fermentation was also reported under aerobic conditions (5) or during
the metabolism of galactose (6). The formation of end products other
than lactate in glucose-limited cultures was rationalized as being due
to the reduction of LDH activity caused by lower levels of the
effector, FBP, and to the relief of pyruvate formate lyase inhibition
by the concomitant decrease of dihydroxyacetone phosphate and
glyceraldehyde 3-phosphate; on the other hand, the oxygen-induced metabolic shift would be explained by activation of the pyruvate dehydrogenase complex under aerobic conditions (3). Recently, Garrigues
et al. (7) downplayed the role of FBP, proposing that
control of glycolysis was dependent on modulation of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) by
NADH/NAD+ (7), and earlier work based on inhibitor
titrations also suggests GAPDH as a major site for glycolytic control
(8).
L. lactis shows a remarkable versatility in regard to
metabolic routes used to regenerate NAD+, a crucial process
in this typical fermentative organism. The presence of NADH oxidase
activity enables this organism to utilize oxygen, when available in the
environment, as an acceptor for the reducing power in NADH, inducing a
shift of the carbon flux to the production of acetate and acetoin (9).
On the other hand, mutants with disruption of LDH, the main catalytic
site for NAD+ regeneration, produced mannitol or ethanol as
alternatives to lactate (10, 11).
The ubiquitous role of pyridine nucleotides in the energy-transforming
and redox reactions and their potential involvement in regulation of
carbon fluxes has demanded the determination of the intracellular
levels of NAD+ and NADH. However, reliable data on
intracellular pools of NAD+ and NADH are hard to obtain.
Most studies rely on analysis of cell extracts, which may not represent
the composition of living cells in a specific metabolic state due to
compound instability and/or unsatisfactory quenching of metabolism.
NADH and NAD+ are acid and alkali labile, respectively, and
we found that NADH was not fully stable under ethanol extraction
conditions. Therefore, measurement of both oxidized and reduced forms
requires that extraction be carried out in separate samples,
with consequent impairment of reproducibility. These are probably the
most important factors responsible for the discrepancies found in the
literature for NAD+ and NADH concentrations in L. lactis (12-15) and other organisms (16-21).
Therefore, the development of techniques for monitoring these pools in
a non-invasive way is mandatory. The fluorescence of NAD(P)H has been
used for the characterization of intracellular pyridine nucleotide
metabolism (22), but this methodology has the disadvantage of detecting
the reduced forms only, and data obtained with this technique in whole
cells should be regarded as qualitative rather than quantitative (Ref.
23 and references therein).
In vivo 13C NMR was used by Unkefer et
al. to follow pyridine nucleotide metabolism in Escherichia
coli and yeast cells (24). The ability of this method to monitor
both the reduced and oxidized pools non-invasively allied to the
concomitant observation of intracellular metabolites derived from
13C substrates is expected to provide a more comprehensive
and reliable insight into cell metabolism. In this study, we used this
methodology to monitor the kinetics of the intracellular pools of
NAD+, NADH, and several metabolic intermediates in the
living cells of L. lactis without interfering with the
cellular processes. Manipulation of the cell capacity to regenerate
NAD+ was achieved either by turning on NADH oxidase
activity (switching from anaerobic to aerobic conditions) or by
knocking out the gene encoding lactate dehydrogenase, the main site for
NADH oxidation in this organism. Regulation of glycolysis in L. lactis is discussed in the light of these in vivo measurements.
Microbial Strains and Growth Conditions--
L.
lactis MG1363 (parent strain) and FI9630, harboring an
ldh gene deletion, hereafter denoted as LDH Construction of an LDH Synthesis of [5-13C]Nicotinic
Acid--
[5-13C]Nicotinic acid was synthesized starting
from [1,3-13C]acetone (Cambridge Isotope
Laboratories, MA) and o-aminobenzaldehyde. A modification of
the method previously used to prepare [6-13C]nicotinamide
was followed (29). Base-catalyzed condensation of
o-aminobenzaldehyde and
[1,3-13C2]acetone afforded labeled quinaldine
in 90% yield. Bromination was carried out as described in Vogel (30),
also in high yield (96%), to produce tribromoquinaldine. The
alternative method for this bromination (29) was not satisfactory in
our hands. Hydrolysis of the tribromomethyl group was performed by
heating in sulfuric acid until no more bromine vapors were observed.
Oxidative degradation of the resulting acid with nitric acid had to be
carried out under very careful control in a temperature-controlled oil
bath. Heating below 260 °C resulted in the formation of several
products, and heating even marginally above this temperature resulted
in the formation of several products or the complete carbonization of the product. The acidic residue from this reaction was heated in the
presence of methanol and trimethylorthoformate for 18 h to give
the required diester in 75% yield for the two steps. Selective hydrolysis of the 2-carboxylate was carried out by refluxing the diester in a solution of potassium hydroxide (1.01 eq.) in methanol. The monoacid (80% yield) formed on acidifying was then decarboxylated by heating to reflux in anisole for 6 h. Without purification, the
methyl ester was hydrolyzed by potassium hydroxide in water to yield
nicotinic acid, which was extracted from the acidified aqueous
phase continuously with dichloromethane. At least two extractions of up
to a week were found necessary for optimum yields. Pure nicotinic acid
was obtained in 90% overall yield from the monoacid. The overall yield
for the entire process was 47%.
NMR Experiments--
Cells were harvested in mid-logarithmic
growth phase (A600 = 2.2), centrifuged, washed
twice, and suspended to a protein concentration of 16.5 mg/ml in 50 mM KPi or Mes/KOH buffer, pH 6.5, for
13C or 31P NMR experiments, respectively. To
determine whether the metabolism was affected by the presence or
absence of phosphate in the buffer, experiments using PIPES for
13C NMR were also run. No changes in the dynamics and
levels of intracellular metabolites, namely FBP, phosphoenolpyruvate
(PEP), and 3-phosphoglycerate (3-PGA) were observed when PIPES instead of KPi was used (data not shown). Anaerobic experiments
were performed as described before (31). For experiments under
O2 atmosphere, a micro pO2 probe (Lazar
Research Laboratories, Inc.) was included in the experimental set-up
previously described (31). To ensure an adequate level of oxygenation,
an air-lift system (32) with pure oxygen was used inside the NMR tube,
and in addition, pure oxygen was continuously bubbled through the cell
suspension in the bioreactor. Spectra were acquired sequentially before
and after addition of [1-13C]glucose (40 or 80 mM). After glucose exhaustion and when no changes in the
resonances due to end products and intracellular metabolites were
observed, an aliquot of the cell suspension was passed through a French
press; the resulting cell extract was incubated at 80 °C (10 min) in
a stoppered tube, cooled down on ice, and cell debris and denatured
macromolecules were removed by centrifugation. The supernatant,
designated herein as NMR sample extract, was used for quantification of
end products and minor metabolites as previously described (10).
Although individual experiments are illustrated in each figure, each
type of in vivo NMR experiment was repeated at least twice,
and the results were highly reproducible. The values reported are
averages of 2-4 experiments, and the accuracy varied from 2% (end
products) to 15% in the case of intracellular metabolites with
concentrations below 5 mM.
Preparation of Ethanol Extracts for Purification, Identification,
and Quantification of Intracellular
[5-12C]NAD+--
A cell suspension of
L. lactis MG1363 grown under anaerobiosis was prepared as
described for in vivo NMR, except that ~1 g of cells was
suspended in a volume of 60 ml in ultrapure water. The suspension was
transferred to 1.2 liters of cold ethanol 70% (v/v) and extracted as
described by Ramos et al. (33). During the extraction
procedure, the pH was kept below 7.5 to avoid NAD+
degradation. To substantiate the assignment to
[5-13C]NAD+ of the resonance at 129.1 ppm
observed in the 13C NMR spectrum of L. lactis
before substrate addition, cell components present in the extract were
separated by anion-exchange chromatography, the extract was loaded onto
a QAE-Sephadex column equilibrated with 5 mM acetate
buffer, pH 6.0, and the elution was performed with a linear gradient of
5 mM to 1 M acetate buffer, pH 6.0. Aliquots of
each fraction were analyzed by 13C NMR, and fractions
originating at the resonance at 129.1 ppm eluting between 0.5 and 0.6 M acetate were pooled, lyophilized, and dissolved in
2H2O. Assignment of
[5-13C]NAD+ was performed by
1H NMR and 13C NMR spectroscopy.
The concentration of [5-13C]NAD+ was also
measured in cell extracts by 1H NMR to provide a basis for
comparison with in vivo determinations. Cell suspensions
(anaerobic cultures) were prepared as described for NMR experiments,
incubated at 30 °C for 15 min under an argon atmosphere, and
extracted with ethanol as above. The lyophilized extracts were
dissolved in 2H2O containing 5 mM
EDTA; the concentration of [5-13C]NAD+ was
determined by comparing the areas of its resonance in 1H-
or 13C-relaxed spectra with the area of the resonance due
to formate or [3-13C]lactate, added as internal standards
for analysis by 1H NMR or 13C NMR, respectively.
Quantification of Products--
Lactate, acetoin, acetate,
2,3-butanediol, ethanol, and formate were quantified in NMR sample
extracts by 1H NMR (10). The concentration of minor
products (e.g. pyruvate, ethanol, diacetyl) and metabolic
intermediates that remained inside the cells (PEP, 3-PGA) was
determined from the analysis of 13C spectra of NMR sample
extracts as previously described (31). The concentration of labeled
lactate determined by 1H NMR was used as a standard to
calculate the concentration of the other metabolites in the sample, and
the total amount of lactate was confirmed by enzymatic methods.
Quantification of Intracellular Metabolites in Living Cells by
13C NMR--
Because of the fast pulsing conditions used
for acquiring in vivo 13C spectra, correction
factors have to be determined to convert peak intensities into
concentrations. The correction factor for the C5 of
NAD+ (0.64 ± 0.01) was determined as follows. A cell
suspension was prepared as described above for 13C NMR
experiments, 2.5 mM [3-13C]lactate was added,
and circulation was started. In the absence of glucose, the intensity
of the resonance due to C5 of NAD+ remained
constant for a long period of time, enabling the acquisition of fully
and partially relaxed spectra and the determination of correction
factors. For the resonances due to C1 and C6 of
FBP (0.48 ± 0.02), mannitol, and mannitol 1-phosphate (Mtl1P)
(0.48 ± 0.01), the factors were determined from spectra of a
perchloric acid extract, which was circulated through the NMR tube at a
similar rate to that used for cell suspensions. Extracts were derived from suspensions of LDH NMR Spectroscopy--
All NMR spectra of living cells were run
at 30 °C with a quadruple-nucleus probe head on a Bruker DRX500
spectrometer. Acquisition of 31P NMR and 13C
NMR spectra was performed as described by Neves et al. (31); however, acquisition parameters for 13C NMR were modified
as follows: data size, 16,000; recycle delay, 0.3 s; number of
transients, 480. Carbon and phosphorus chemical shifts are referenced
to the resonances of external methanol or H3PO4
(85%) designated at 49.3 ppm and 0.0 ppm, respectively.
Enzyme Activity Measurements--
All enzymes were assayed at
30 °C immediately after mechanical disruption of a cell suspension
by passage through a French press (twice at 120 MPa) and centrifugation
for 15 min at 30,000 × g to remove cell debris. The
protein concentration was determined by the method of Bradford (35).
L-LDH was assayed as described by Garrigues et
al. (7). GAPDH was assayed by the method of Even et al.
(36). NADH oxidase activity was determined as described by Lopez de
Felipe et al. (9). Mtl1P dehydrogenase (EC 1.1.1.17), leading to the formation of Mtl1P, was measured using 0.3 mM NADH, and the reaction was initiated by the addition of
3 mM fructose 6-phosphate (10).
Chemicals--
[1-13C]Glucose (99% enrichment)
and [1,3-13C]acetone (99% enrichment) were obtained from
Campro Scientific and Cambridge Isotope Laboratories,
respectively. [3-13C]Lactate (99%) was supplied by
A.R.C. Formic acid (sodium salt) was purchased from Merck. QAE-Sephadex
A25 was obtained from Amersham Biosciences. All other chemicals were
reagent grade.
13C Labeling of Intracellular Pools of Pyridine
Nucleotides--
In most microbial systems NAD+ can be
synthesized from aspartate (de novo pathway) or nicotinic
acid (salvage pathway) (37). Because of the lack of information about
the route of NAD+ synthesis in L. lactis, it was
necessary to ascertain which precursor would achieve maximal labeling
of the intracellular pool. Therefore, L. lactis was grown in
chemical-defined medium without nicotinic acid or without aspartate and
asparagine. In the absence of these amino acids, a slight decrease in
the growth rate (µ) was observed: µ = 0.71 h
A 13C spectrum of an anaerobic cell suspension of MG1363
cells grown on [5-13C]nicotinic acid is shown in Fig.
1A. The spectrum showed a
single 13C resonance at 129.1 ppm, tentatively assigned to
C5 of NAD+ by comparison with 13C
NMR spectra of standards of NAD+, NADH, NADP+,
and nicotinic acid (all at 20 mM in 50 mM
KPi buffer, pH 6.5). Chemical shift values of
C5 for the latter compounds were 129.1, 105.8, 129.1, and
124.5 ppm, respectively. To confirm the assignment, an ethanol extract
of cells grown on [5-13C]nicotinic acid was prepared.
After passage through an anion-exchange column, the fractions
containing a 13C resonance at 129.1 ppm were pooled and
lyophilized. The resulting sample was analyzed by 13C NMR
(Fig. 1B), and the resonance at 129.1 ppm was firmly
assigned to carbon 5 (C5) of NAD+ by spiking
the sample with the pure compound. A 1H NMR spectrum of the
same sample confirmed the 13C NMR data (Fig.
1C). In addition, analysis of the proton spectrum showed
that the NAD+ pool was totally labeled at position
C5. No signal due to the 12CH was observed, and
the two multiplet resonances (highlighted with an
asterisk in Fig. 1C), with half the intensity of
the other single proton signals, separated by ~0.60 ppm (coupling
constant, J13C-1H = 179 Hz) are due to the
methine proton bound to carbon 5 (13C5).
No resonance attributable to NADH was detected in the spectrum of
starved cells. Resonances due to NADP+ or NADPH were not
detected in vivo or in ethanol extracts of starved cells,
albeit a resonance due to carbon 5 of NAD+ was readily
observed. NADP+ is stable in ethanol and at the pH used
during the extraction procedure, and therefore, lack of detection due
to degradation can be excluded. Despite the existence of a typical
NADP+-dependent 6-phosphogluconate
dehydrogenase (38), the flux through the pentose phosphate pathway when
compared with the glycolytic flux is very low in L. lactis
(39), and no transhydrogenase activities have ever been reported;
hence, NADP(H) concentrations in this organism are expected to be very
low and, therefore, not amenable to detection by in vivo
NMR.
Characterization of Pyridine Nucleotides Kinetics during Anaerobic
Metabolism of Glucose by MG1363--
A selection of 13C
NMR spectra showing the evolution of NAD+ and NADH pools
before and after the addition of 80 mM
[1-13C]glucose to an anaerobic cell suspension of MG1363
is presented in Fig. 2. Before the
addition of glucose, a single resonance at 129.1 ppm was detected and
assigned to [5-13C]NAD+. When glucose became
limiting (~18 min after addition), a resonance appeared at 105.8 ppm
that was assigned to [5-13C]NADH. The kinetics of
consumption of glucose, the build-up and consumption of FBP, and
evolution of NAD+ and NADH were also monitored (Fig.
3A). After glucose addition, the FBP pool increased rapidly, reaching a steady level of 51.1 ± 0.3 mM; before and during this steady state period, no
changes were observed in the NAD+ level, which was
maintained at ~4.7 ± 0.3 mM. At the onset of glucose exhaustion, the NAD+ dropped to 1.4 ± 0.1 mM, whereas the FBP pool declined sharply, and the NADH
pool increased from undetectable levels to a maximum of 2.7 ± 0.3 mM. Also, the 3-PGA and PEP pools started to increase, leveling off at 9.2 ± 0.7 and 5.6 ± 0.4 mM,
respectively (Fig. 3B). Subsequently, the FBP decline slowed
down, the NAD+ pool recovered very slowly to its initial
concentration, and the NADH pool became undetectable.
It is well known that NMR is able to detect freely mobile metabolites
and that cofactors tightly bound to macromolecules originate very broad
lines that are not detected. To assess the extent of detection achieved
by in vivo NMR, the pool of NAD+ was also
quantified in ethanol extracts of cell suspensions grown under
anaerobic conditions (see "Materials and Methods"). We found an
NAD+ concentration of 4.2 ± 0.7 mM
(average of three independent determinations), which compares well with
the value measured by in vivo NMR. Therefore, the total cell
pool of coenzyme is visible by NMR. Also, no evidence for NADH
invisibility was found at the different metabolic stages examined since
the sum of NAD+ and NADH agreed well with the initial pool
of NAD+ in starved cells. The half-height line-width for
NAD+ and NADH in the spectra of living cells was 25 and 35 Hz, respectively.
The major product from glucose metabolism under anaerobic conditions
was lactate (145.6 ± 0.7 mM), although minor amounts of acetate (1.3 ± 0.1 mM) and 2,3-butanediol (0.3 mM) were detected (not shown). The carbon balance (from
glucose) was 95% taking into consideration both extracellular and
intracellular metabolites.
The NTP and intracellular Pi levels during the metabolism
of glucose (80 mM) as well as the evolution of
intracellular pH were determined by 31P NMR (Fig.
3C). The concentration of NTP increased from undetectable levels to a maximum of 8.6 ± 0.3 mM, remaining high
for a few minutes beyond glucose depletion. After glucose exhaustion
(at t Pyridine Nucleotide Levels during Glucose Metabolism Under Aerobic
Conditions--
A selection of 13C NMR spectra showing the
evolution of pyridine nucleotide pools before and after the addition of
40 mM [1-13C]glucose to an aerobic cell
suspension of MG1363 (fully saturated in oxygen) is presented in Fig.
4A. The intensity of the
resonance due to [5-13C]NAD+ remained
constant during the time of observation, and NADH was never
detected.
The time courses for glucose consumption, glycolytic intermediates, and
NAD+ evolution are shown in Fig. 4B.
Intracellular FBP increased to a maximal concentration of 48.2 ± 0.1 mM, and the subsequent decay was considerably steeper
than under anaerobic conditions. NAD+ concentration was
constant around 4.8 ± 0.3 mM regardless the presence
or absence of glucose, and NADH was always below the detection limit
(0.5 mM). The build-up of the 3-PGA and PEP pools, which
reached intracellular concentrations of 36.6 ± 1.5 and 15.9 ± 0.4 mM, respectively, was concomitant with the FBP
decline upon glucose depletion (Fig. 4C).
Under these conditions, a considerable shift from lactate to acetate
production was observed (Table I). LDH
activity of cells grown under aerobiosis was about half that measured
in extracts of cells grown under anaerobiosis, whereas NADH oxidase
activity was 3.4-fold higher (Table
II).
Biochemical parameters derived from 31P NMR spectra during
the metabolism of glucose (40 mM) under aerobic conditions
are shown in Fig. 4D. The time courses for the variation of
intracellular pH, Pi, and NTP were generally similar,
except for the decline in NTP after substrate exhaustion, which was
clearly faster under aerobic conditions.
Pyridine Nucleotides and Other Intracellular Metabolite Pools in
Strain FI9630 (LDH
In contrast to MG1363, an oxygen atmosphere was not sufficient to
maintain a constant NAD+ pool in the LDH Pattern of End Products Derived from the Metabolism of Glucose in
Strain FI9630 (LDH
Under anaerobic conditions the glucose consumption rate of the
LDH Regulation of LDH and GAPDH by NAD+ and NADH--
The
effect of the NADH/NAD+ ratio on GAPDH and LDH activities
(forward reactions) was investigated using crude cell extracts prepared
from cell suspensions of MG1363 grown under anaerobiosis. Maximal
activities of 30.6 ± 0.6 and 29.7 ± 0.8 µmol
min
Equivalent data for GAPDH were obtained; setting NADH at a constant
level resulted in a specific degree of inhibition, which was
independent of the NADH/NAD+ ratio. We concluded that under
the conditions examined inhibitory effects on LDH and GAPDH were due to
NAD+ or NADH concentrations, respectively, rather than the
NADH/NAD+ ratio. At the maximal physiological
NAD+ concentration (~5 mM) and disregarding
other possible effectors, about 50% of the maximal LDH activity was
retained. GAPDH activity was 50% inhibited by 0.4 mM NADH,
meaning that the activity of this enzyme in vitro would be
severely inhibited when the NADH concentration rises to values such as
those detected in vivo in the present study.
It is generally accepted that pyridine nucleotides play a key role
in metabolic control and that reliable data on their pool sizes are
essential for an accurate description of cell metabolism. In this work
we used in vivo 13C NMR to determine NADH and
NAD+ levels in L. lactis. Despite the
disadvantage of a low sensitivity that hampers determinations in
growing cultures, this technique has invaluable advantages: in a single
sample, reduced and oxidized forms of pyridine nucleotides can be
quantified on line along with glycolytic intermediates and end
products. Furthermore, information on levels of NTP, intracellular pH,
and inorganic phosphate is accessible from experiments monitored by
31P NMR; in this way, it was possible to obtain a global
picture of glucose metabolism in L. lactis in real time
through the measurements of several intracellular parameters in
vivo.
NAD+ was constant and maximal during glucose metabolism in
the parent strain, but at the onset of glucose depletion and under anaerobic conditions, it decreased sharply with a concomitant increase
of NADH. Under aerobic conditions, however, the NAD+ level
remained maximal throughout all the stages. Therefore, we conclude that
when glucose was not limiting and metabolism proceeded at a full rate,
LDH activity alone (under anaerobic conditions) was sufficient to
promptly oxidize NADH formed by GAPDH, leading to a quasi-steady state
condition where the level of NAD+ remained maximal.
However, when glucose became limiting, there was a sharp decline in
NAD+ and a consequent rise of NADH, attributed to the
decrease in the flux through LDH, the main site for NADH oxidation
under anaerobic conditions. This decrease in flux through LDH resulted
directly from the reduced availability of the substrate, pyruvate,
caused by (i) cessation of glucose transport via the
PEP-phosphotransferase system, which releases pyruvate efficiently, and
(ii) inhibition of PK by the combined action of high levels of
Pi, which accumulates at this stage, and decreased levels
of FBP. The antagonistic effects of FBP and Pi in the
modulation of PK activity have been put forward by other authors (40,
41).
NADH oxidase activity present in L. lactis provided an extra
route for the oxidation of NADH, when oxygen was available. Therefore, under aerobic conditions, the NAD+ level was maximal,
regardless of glucose being available or exhausted or, in other words,
regardless of PK inhibition; the activity of NADH oxidase allowed for
an efficient regeneration of NAD+, counteracting the
constriction of flux through PK (and consequently through LDH).
In the construct with a disruption in the ldh gene, the
NAD+ pool dropped sharply immediately after the supply of
glucose regardless of anaerobic or aerobic conditions (Fig. 5), showing
that NADH oxidase activity was insufficient to compensate for the
severe LDH deficiency. A proportional increase in NADH concentration was observed, which reached 2.5 mM under anaerobic
conditions and up to 1.5 mM in the presence of oxygen.
Under anaerobic conditions, the glycolytic flux was strongly depressed
(7-fold lower than that of the parent strain, MG1363), but
interestingly, in the presence of oxygen the glucose consumption rate
increased significantly, amounting to 60% that of MG1363. This result
clearly shows that a high glycolytic flux is compatible with a high
concentration of NADH in the cell (higher than 1 mM and
higher than the concentration of NAD+, Fig. 5); this
concentration would inhibit GAPDH to a considerable extent on the basis
of in vitro data for this enzyme (Supplemental Fig. 1).
Therefore, it appears that GAPDH activity in vivo is not as
sensitive to inhibition by NADH as in vitro (Ref. 36 and
this study) or that the excess activity of the enzyme in the cell
allows for a residual flux comparable with that of the parental strain
even when the extent of inhibition is extremely high. In fact, we have
found about 100-fold excess GAPDH activity in extracts of MG1363.
The strong accumulation of FBP during the initial stage of glucose
metabolism (activation stage) is a feature common to all the strains
and conditions examined. Immediately before glucose addition the level
of Pi is invariably high; therefore, PK is expected to be
virtually inoperative. As a consequence of this initial bottleneck, FBP
builds up at the expense of the Pi pool, resulting in the
relief of PK inhibition and the establishment of a quasi-steady state
characterized by a constant level of FBP (in the 50 mM
range) and maximal rate of glucose consumption. This high FBP level was
important to counteract the negative effect of Pi on PK,
allowing for reasonably high flux even at high intracellular Pi concentration.
At the onset of glucose depletion, a reversion of the events occurring
during the activation stage was observed. FBP was depleted at a rate
dependent on the cell capacity to regenerate NAD+ (higher
rate under aerobic conditions), and the pool of Pi
increased sharply, resulting in the inhibition of PK. This constriction was responsible for the strong accumulation under aerobic conditions of
3-PGA and PEP derived from the metabolism of residual FBP; in fact,
when oxygen is available, NADH oxidases provide an alternative pathway for the regeneration of NAD+, which disregards PK,
thus enabling the build up of 3-PGA and PEP pools to higher levels and
the fast depletion of FBP (compare Figs. 3B and
4C). In this respect, the behavior of the LDH In all cases examined the maximum level of NTP was around 6-7
mM, except for the LDH The presence of a bottleneck at the level of LDH in the LDH-deficient
strain was also evidenced by the clear accumulation of pyruvate and
aspartate. The limited capacity to oxidize NADH led to the accumulation
of this coenzyme (up to 2.5 mM), which forced the operation
of the rescue pathway to oxidize NADH through Mtl1P dehydrogenase with
production of mannitol (10). The accumulation of pyruvate, only
observed in this strain, indicates flux constriction at
pyruvate-utilizing enzymes rather than at the level of GAPDH that could
be severely inhibited by the high NADH content.
The comparison of the metabolic profiles of the LDH Recent work using a series of mutant strains of L. lactis
with a wide range of LDH activities has shown convincingly that LDH has
no control on the glycolytic flux, reflecting the excess capacity of
this pivotal enzyme in the cell (42). Manipulation of the PFK activity,
however, had a negative effect on the glycolytic flux, but the
interpretation of results is complicated by co-lateral effects on the
activity of the other enzymes encoded by the las operon
(43). It has been shown that the in vitro activities of the
two main dehydrogenases in L. lactis are highly
sensitive to the levels of NADH or NAD+, and the
NADH/NAD+ ratio has been proposed to play a predominant
role in the control of flux through glycolysis (7). Our in
vivo measurements of intracellular pools of pyridine nucleotides
and several other metabolites in MG1363 show that under steady state
conditions of glucose metabolism, NADH was kept at undetectable levels
by the luxurious LDH activity in the wild type strain. Thus, it is unlikely that the control of the glycolytic flux resides primarily at
the level of GAPDH, since the activity of this enzyme was sufficient to
support a high flux even at concentrations of NADH in the range of 1.5 mM. It is conceivable that, as for the other main
dehydrogenase (LDH), the activity of GAPDH in the cell is in great
excess, because the activities of LDH and GAPDH measured in
vitro in MG1363 and other strains are comparable (this study and
Ref. 13). On the other hand, overproduction of PK in L. lactis did not lead to enhancement of the glycolytic
flux.2 At this point we
speculate that glycolytic flux could be controlled by ADP availability
or ATP surplus, as previously proposed for yeast and other organisms
(31, 44-47). High levels of ATP were determined in L. lactis, but ADP was not detected in 31P NMR spectra of
starved cells, suggesting restricted motion, probably due to binding to
proteins (48). Therefore, it is conceivable that the free ADP pool
could be a limiting factor when glucose is abundant.
Despite the fact that a final answer to the complex question of what
controls glycolytic flux in L. lactis cannot be given at
this stage, it is clear that our data do not support the existence of
major control at the level of GAPDH in the wild type strain. The unique
in vivo measurements accomplished in this work provided an
interpretation of glucose metabolism based on direct observations rather than on unreliable extrapolations from in vitro data
or speculative reasoning derived from the pattern of end products. Although a more substantiated proposal cannot be put forward, we favor
the view that glycolytic control resides mainly outside glycolysis,
probably in the processes that affect the ATP and/or ADP content rather
than the NADH/NAD+ ratio.
We thank A. Mingote for technical assistance
in the purification of NAD+.
*
This work was supported by Commission of the European
Communities Contracts BIO4CT-96-0498 and QLK1-CT-2000-01376 and by
Fundação para a Ciência e Tecnologia, Portugal,
Contract PRAXIS/P/BIA/11072/1998.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by research fellowships of Fundação para a
Ciência e Tecnologia, Portugal.
**
To whom correspondence should be addressed: Instituto de Tecnologia
Química e Biológica, Universidade Nova de Lisboa, Rua da
Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal. Tel.: 351-21-4469828; Fax: 351-21-4428766; E-mail: santos@itqb.unl.pt.
Published, JBC Papers in Press, May 13, 2002, DOI 10.1074/jbc.M202573200
2
A. Ramos, A. R. Neves, P. Lopez, and H. Santos, unpublished observations.
The abbreviations used are:
FBP, fructose
1,6-bisphosphate;
Mtl1P, mannitol 1-phosphate;
PEP, phosphoenolpyruvate;
3-PGA, 3-phosphoglycerate;
Pi, inorganic phosphate;
KPi, potassium phosphate;
LDH, L-lactate dehydrogenase;
LDH
Is the Glycolytic Flux in Lactococcus lactis
Primarily Controlled by the Redox Charge?
KINETICS OF NAD+ AND NADH POOLS DETERMINED
IN VIVO BY 13C NMR*,
§,,
,,§, and**
Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa and Instituto de Biologia
Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal and ¶ Institute of Food Research,
Norwich Laboratory, Norwich Research Park, Colney,
Norwich NR4 7UA, United Kingdom
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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deficient strain was constructed by double crossover. Upon supply of
glucose, NAD+ was constant and maximal (~5
mM) in the parent strain (MG1363) but decreased abruptly in
the LDH strain both under aerobic and anaerobic
conditions. NADH in MG1363 was always below the detection limit as long
as glucose was available. The rate of glucose consumption under
anaerobic conditions was 7-fold lower in the LDH strain
and NADH reached high levels (2.5 mM), reflecting severe limitation in regenerating NAD+. However, under aerobic
conditions the glycolytic flux was nearly as high as in MG1363 despite
the accumulation of NADH up to 1.5 mM.
Glyceraldehyde-3-phosphate dehydrogenase was able to support a high
flux even in the presence of NADH concentrations much higher than those
of the parent strain. We interpret the data as showing that the
glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by
NADH. The ATP/ADP/Pi content could play an important role.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain, were grown at 30 °C in a 2-liter vessel (B. Braun Biostat® MD) in the chemical-defined medium described by Poolman and Konings (25) modified as follows. Aspartate and asparagine were omitted, and
nicotinic acid (1 mg/liter) was replaced by
[5-13C]nicotinic acid (5 mg/liter). Glucose was added to
a final concentration of 1% (w/v), and the pH was kept at 6.5 by
automatic addition of 10 N NaOH. Anaerobiosis was attained
by flushing sterile argon through the medium in the fermenter for
1 h preceding inoculation. Dissolved oxygen was monitored with an
oxygen electrode (Ingold). A specific air tension of 90% was
maintained by automatic control of the airflow during aerobic growth.
Strain by Double Crossover
(FI9630)--
An L. lactis LDH-deficient strain was
constructed by double crossover to ensure full stability of the gene
knock-out and to avoid antibiotic selection. The pOri/pVE6007
two-plasmid system (26) was used to generate a food grade deletion of
the ldh gene in MG1363. The upstream flanking fragment was
cloned from pFI2320 as a 1.24-kb EcoRI-DraI
fragment (equivalent to 1785-3025 bp; accession number L07920 (27)).
This fragment was cloned into pOri280 cut with EcoRI and
SmaI to give pFI2277. The DNA sequence downstream of the
ldh gene was determined from pFI516 (28) and used to design
primers ldh3 (5'-CCGTCAGCCCGCGGCGCAATTGC-3') and ldh4
(5'-GCAATCAAAGCTTCAGAAGCC-3'). These primers amplified the 950-bp
downstream fragment, which was cloned into pCR2.1TOPO
(Invitrogen) to give pFI2323. pFI2277 was cut with BglII, and the 1.98-kb BamHI-BglII fragment from pFI2323
was inserted. The orientation of the upstream and downstream
ldh fragments was confirmed by PCR analysis and sequencing
across the junction. This construct, named pFI2278, was electroporated
into FI8645 (MG1363 pVE6007) to give FI9619. This strain was taken
through the temperature shift protocol for single and double crossovers (26), giving FI9620 and FI9630, respectively. FI9630 was confirmed as a
ldh strain by PCR analysis, Southern blotting, and
sequencing of the PCR product amplified across the deletion junction.
The stability of the LDH deletion was assessed as described previously (10).
(Mtl1P and mannitol) or MG1363
cells (FBP) actively metabolizing [1-13C]glucose. The
correction factors for the resonances due to C3 of 3-PGA
and PEP (0.60 ± 0.02) were determined in a cell suspension of
L. lactis after the metabolism of
[1-13C]glucose, since after glucose depletion the
intracellular pools of these metabolites were stable. The quantitative
kinetic data for intracellular metabolites were calculated from the
areas of the relevant resonances by applying the correction factors and comparing with the intensity of the lactate resonance in the last spectrum of the sequence. For calculation of the correction factors 13C NMR spectra were acquired with a 60° flip angle and a
recycle delay of 0.3 s (saturating conditions) or 60.3 s
(relaxed conditions). Average values derived from at least three
independent experiments were used. The lower limit for in
vivo NMR detection of NAD+ or NADH under these
conditions was 0.5 mM. Intracellular metabolite concentrations were calculated using a value of 2.9 µl/mg protein for
the intracellular volume of L. lactis (34).
RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 as
compared with µ = 1.15 h1 for the complete
chemical-defined medium under anaerobiosis. No growth was detected when
nicotinic acid was omitted. These data and the previous observation of
the production of labeled aspartate from the metabolism of
[1-13C]glucose (10) led to the conclusion that nicotinic
acid, but not aspartate, was essential for growth and, therefore, was
the compound of choice to supply as a biosynthetic precursor of
NAD+. In this work, aspartate and asparagine were omitted
from the medium to ensure maximal labeling of the pyridine nucleotide
pool. [5-13C]Nicotinic acid was synthesized, with an
overall yield of 47% as described under "Materials and Methods"
and used as the only nicotinic acid source for the growth of L. lactis. The purity of the labeled compound was evaluated both by
13C NMR and 1H NMR spectroscopy, and no
contaminants were detected in a 3.5 mM
[5-13C]nicotinic acid solution (data not shown).
View larger version (24K):
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Fig. 1.
13C NMR spectrum of an anaerobic
suspension of L. lactis cells grown in the presence of
[5-13C]nicotinic acid (A); carbon
(B) and proton (C) spectra of the
purified [5-13C]NAD+ at pH 6.5. The
resonance in spectrum A is due to the C5 of
NAD+. Resonances labeled with R and A
are assigned to protons in ribose and adenine moieties,
respectively.
View larger version (22K):
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Fig. 2.
Sequence of 13C spectra showing
the evolution of NAD+ (dark gray) and NADH
(light gray) pools in MG1363 before and after the
addition of glucose under anaerobic conditions. Cells were grown
in chemical-defined medium containing 5 mg/liter
[5-13C]nicotinic acid and suspended in 50 mM
KPi, pH 6.5, at a protein concentration of 16 mg/ml.
Glucose (80 mM) was added at time 0; each spectrum was
acquired within 2.2 min and processed with 25/35 Hz
line-broadening. The two unknown resonances (129.8 and 130.1 ppm)
downfield of [5-13C]NAD+ were assigned to
minor products of glucose metabolism.
View larger version (23K):
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Fig. 3.
Glycolytic dynamics of L. lactis MG1363 under anaerobic conditions assessed
using in vivo 13C and 31P
NMR. Consumption of [1-13C]glucose (80 mM) and evolution of FBP, NAD+, and NADH
(A) and lactate, FBP, 3-PGA, and PEP (B)
monitored by 13C NMR is shown. Biochemical parameters
determined by 31P NMR during the metabolism of glucose (80 mM) is shown. Black diamond, glucose; pink
square, lactate; red triangle, FBP; green
square, 3-PGA; blue circle, PEP; yellow
square, NAD+; aqua circle, NADH;
burgundy square, NTP; blue triangle,
Pi; orange diamond, intracellular pH. The
shaded area indicates glucose availability. Fitted
lines are simple interpolations.
19.5 min) a sudden raise of intracellular
Pi to about 30 mM was followed by a gradual
increase in concentration, reaching values in the range of the initial
values (45 mM). We verified that a lower concentration of
[1-13C]glucose (40 mM) did not affect the
maximum intracellular concentration of FBP nor the general pattern of
other intracellular metabolite pools or cytoplasmic pH (data not
shown); however, the maximal NTP concentration was lower (~6.1
mM).
View larger version (30K):
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Fig. 4.
Metabolism of glucose (40 mMM) in L. lactis
MG1363 under aerobic conditions at 30 °C, pH 6.5. Sequence of 13C spectra showing the evolution of
NAD+ (and NADH) pools in MG1363 (grown in chemical defined
medium containing 5 mg/liter of [5-13C]nicotinic acid)
before and after a glucose pulse under an oxygen atmosphere
(A). Glucose was added at time 0 min; conditions were
similar to those described in Fig. 2. Shown is the time course for the
consumption of 40 mM [1-13C]glucose and
evolution of FBP, NAD+, and NADH pools (B) and
end product formation, FBP, 3-PGA, and PEP pools (C) as
monitored by 13C NMR and biochemical parameters, determined
by 31P NMR during the metabolism of glucose (D).
Black diamond, glucose; pink square, lactate;
gray triangle, acetate; red triangle, FBP;
green square, 3-PGA; blue circle, PEP;
yellow square, NAD+; aqua diamond,
NADH; burgundy square, NTP; blue triangle,
Pi; orange diamond, intracellular pH. Glucose
availability is indicated by the shaded area (D).
Fitted lines are simple interpolations.
End products (mM) from the metabolism of 40 mM
glucose by MG1363 and LDH strains under anaerobic or aerobic (100%
oxygen saturated) conditions as determined by 13C NMR
1 mg of protein1. Mean ± S.D.
(n 
2). ND, not detected. Formate was determined by
1H NMR.
Comparison of enzyme activities determined in crude cell extracts of
the MG1363 and LDH strains grown under anaerobic or
aerobic conditions
1 mg of
protein1. Mean ± S.D. deviation (n 6). ND, not detected; 
, not determined.
)--
Given the central role played
by LDH in the regeneration of NAD+ during glucose
metabolism by L. lactis, it was deemed important to measure
NAD+ and NADH pools in an LDH-deficient strain, which has a
severely reduced capacity to regenerate NAD+. Under
anaerobic conditions, the NAD+ was 4.6 ± 0.6 mM before glucose addition and decreased suddenly to
1.9 ± 0.6 mM upon glucose addition, whereas NADH
raised from undetectable levels to a mean value of 2.5 ± 0.4 mM (Fig. 5A). Meanwhile, the FBP pool increased, reaching a maximal concentration of
36.8 ± 2.3 mM, and the total mannitol pool (Mtl1P and
mannitol) raised gradually (maximal concentration 256 ± 7 mM) as long as glucose was available. A similar pattern of
glycolytic intermediates was previously reported for L. lactis FI7851, a distinct LDH-deficient strain that also uses the
reduction of fructose 6-phosphate to Mtl1P as a rescue pathway to
regenerate NAD+ (10). Likewise, in this LDH
construct, all the mannitol produced was metabolized to the usual end
products after glucose exhaustion. Mannitol depletion (at time 73.7 min) and consumption of Mtl1P caused a slow decrease in the NADH
levels, which became undetectable after Mtl1P exhaustion. The reverse
trend was observed for the NAD+ pool, which slowly
recovered to its initial average value of 4.6 ± 0.6 mM. The buildup of 3-PGA and PEP pools was slow and started
immediately after mannitol exhaustion, reaching maximal concentrations
of 57.0 ± 3.1 and 23.2 ± 0.6 mM, respectively; these concentrations are significantly higher than those observed for
MG1363.
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Fig. 5.
Metabolism of glucose (40 mM) in
the LDH strain. Shown are
[1-13C]glucose consumption, FBP, mannitol/Mtl1P, and
pyridine nucleotide pools evolution, as determined by 13C
NMR under anaerobic (A) or aerobic (B)
conditions, and biochemical parameters, determined by 31P
NMR during the metabolism of glucose under anaerobic (C) or
aerobic (D) conditions. Black diamond, glucose;
red triangle, FBP; green square, 3-PGA;
blue circle, PEP; green circle, mannitol + mannitol 1-phosphate; yellow square, NAD+;
aqua circle, NADH; burgundy square, NTP;
blue triangle; Pi, orange diamond,
intracellular pH. For the calculations, mannitol was assumed
intracellular. The black arrow indicates the depletion of
mannitol (A). Glucose availability is indicated by the
shaded box (C and D).
strain; NAD+ dropped from 3.5 ± 0.6 to 1.2 ± 0.5 mM shortly after glucose addition (Fig. 5B).
NADH raised from undetectable levels to ~1.5 ± 0.6 mM. FBP and mannitol/Mtl1P increased rapidly to maximal concentrations of 50.1 ± 0.9 and 5.7 ± 0.4 mM.
Both NAD+ and NADH pools returned to their initial state at
the time FBP started to decline. Simultaneously, 3-PGA and PEP raised
to maximal values of 45.7 ± 0.6 and 21.2 ± 1.8 mM, respectively. No enhancement of NADH oxidase activity
was detected in extracts of LDH cells grown under
aerobiosis. Additionally, Mtl1P dehydrogenase activity, which acts as
an alternative enzyme in NAD+ regeneration, was strongly
depressed (about 8-fold) in oxygen grown cells (Table II).
Surprisingly, NTP was not detected during glucose consumption by the
LDH strain under anaerobic conditions, and the
intracellular pH did not surpass 6.8 (Fig. 5C). Under
aerobic conditions, NTP reached a maximal concentration of 6.8 ± 0.2 mM, decreasing to undetectable values immediately after
glucose exhaustion, and the profile of intracellular pH was similar to
that of MG1363 (Fig. 5D). Regardless of the gas atmosphere,
the intracellular Pi levels were considerably higher than
those observed during glucose consumption by MG1363, implying a
stronger inhibitory effect on PK.
)--
A comparison of end products
and glucose consumption rates during the metabolism of
[1-13C]glucose (40 mM) by the
LDH and parent strains under anaerobic or aerobic
conditions is shown in Table I. Under anaerobiosis, the
LDH strain produced a mixture of ethanol, formate,
2,3-butanediol, and also lactate; however, no LDH activity could be
detected using the standard spectrophotometric assay coupled to NADH
oxidation (Table II). We propose that an alternative LDH with different kinetic parameters must be responsible for lactate production; in fact,
three other putative ldh genes were found in the genome of
L. lactis IL1403 (2). In the LDH strain,
lactate production was severely inhibited under aerobic conditions, and
glucose metabolism was switched toward the production of acetoin and
acetate. Significant amounts of the flavor compound diacetyl were
observed as well. Pyruvate accumulation was detected in the
LDH, the concentration being 2.5-fold higher under
aerobic conditions (5.8 ± 0.3 mM). Under anaerobic
conditions, aspartate production was observed in MG1363 (5.2 ± 0.4 mM) and in the LDH (10.8 ± 0.4 mM). The presence of oxygen induced a 2-fold increase in
the concentration of aspartate and accumulation of citrate up to
2.6 ± 0.2 and 33.3 ± 0.6 mM in MG1363 and the
LDH strain, respectively.
strain was 7-fold lower than that of the parent
strain, whereas under aerobic conditions the glucose consumption rate
was only 2-fold lower (Table I). The growth rate of this strain was
0.70 h1, whereas strain MG1363 had growth rates of 1.15 and 1.00 h1 under anaerobic or aerobic conditions,
respectively. The lower consumption rate of glucose by
LDH reflects the difficulty to oxidize NADH. Under
aerobic conditions, regeneration of NAD+ by NADH oxidases
relieves this obstruction, allowing for a faster glucose uptake.
1 mg of protein1 were determined for LDH
and GAPDH, respectively. LDH activity was not affected by the
NADH/NAD+ ratio, when a constant NAD+
concentration (5 mM) was used and the concentration of NADH
varied between 0 and 0.5 mM. The NAD+
concentration was set at 5 mM to mimic the maximal pool of
this metabolite determined in vivo in L. lactis.
Conversely, when NADH was set constant (0.3 or 0.5 mM) and
the NAD+ concentration was varied between 0 and 10 mM, the LDH activity decreased as the concentration of
NAD+ increased.
DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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strain deserves further comment, since the levels of 3-PGA and PEP were
high even under anaerobic conditions despite the low intrinsic capacity
of this strain to oxidize NADH upstream the pyruvate node; in this
case, oxidation of NADH after glucose depletion was coupled to
reduction of acetoin, as evidenced by the decline in the concentration
of this end product (Supplemental Fig. 2).
strain under anaerobic
conditions in which NTP was too low to be detected in vivo
(less than 1 mM). It is likely that the NTP formed was
rapidly used to maintain transmembrane gradients in this mutant strain,
which has considerably reduced rates of lactate production and efflux.
As a consequence of this low energizing level, the intracellular pH
during glucose metabolism was also lower (by about 0.4 pH units) than
in the parent strain.
strain under anaerobic and aerobic conditions is particularly
informative. The presence of oxygen induces a remarkable increase in
the glycolytic flux (3-fold) despite the high NADH and Pi
(>10 mM) concentrations observed during the utilization of
glucose. The results suggest that when glucose is not limiting, the
flux through GAPDH is relatively insensitive to NADH levels as high as
1.5 mM, and there is lack of evidence for strong toxicity
associated with NADH concentrations in this range. Moreover, it is
interesting to point out the substantial flux sustained via PK despite
the high level of Pi (Fig. 5), a known inhibitor of the
enzyme. The absence of significant constriction at this enzymatic step
is further supported by the inability to detect 3-PGA and PEP in fully
active cells and by the higher concentrations of pyruvate, aspartate,
and citrate that accumulated under aerobiosis, a direct consequence of
pyruvate accumulation due the higher glycolytic flux. An overview of
the main metabolic features of the MG1363 and the LDH-deficient
construct is shown in Fig. 6.
View larger version (25K):
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Fig. 6.
Overview of main metabolic features observed
for L. lactis MG1363 and the mutant
deficient in LDH, highlighting major regulation sites invoked under
"Discussion." Case sizes are intended to reflect pool sizes.
Stripped arrows indicate steps activated under aerobic
conditions. Plus and minus signs refer to
positive or negative effectors of enzyme activities. 2,3-Bd,
2,3-butanediol; F6P, fructose-6-phosphate.
ACKNOWLEDGEMENT
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 1 and 2.
Supported by a Ph.D. studentship from the Egyptian Government
and National Research Centre. Current address: Food Science and Dairy
Technology, National Research Centre, Tahir St., Cairo, Egypt.
ABBREVIATIONS
, LDH-deficient
strain;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PK, pyruvate
kinase;
Mes, 2-(N-morpholino)ethanesulfonic acid;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
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