Báo cáo khoa học: Natural-abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose–citrate cofermentation by Lactococcus lactis potx

Natural-abundance isotope ratio mass spectrometry as a means
of evaluating carbon redistribution during glucose–citrate
cofermentation by
Lactococcus lactis
Mohamed Mahmoud, Emmanuel Gentil and Richard J. Robins
Groupe de Fractionnement Isotopique de Me
´
tabolismes, Laboratoire d’Analyse Isotopique et Electrochimique de Me
´
tabolismes,
Universite
´
de Nantes, France
The cometabolism of citrate and glucose by growing
Lactococcus lactis ssp. lactis bv. diacetylactis was studied
using a natural-abundance stable isotope technique. By a
judicious choice of substrates differing slightly in their
13
C/
12
C ratios, the simultaneous metabolism of c itrate and
glucose to a range of compounds was analysed. These end-
products include lactate, acetate, formate, diacetyl and
acetoin. All these products have pyruvate as a co mmon
intermediate. With the objective of estimating the degree to
which glucose and citrate metabolism through pyruvate may
be differentially regulated, the d
13
C v alues of the products
accumulated over a wide range of concentrations of citrate
and glucose were compared. It was found that, whereas the

relative accumulation of different products responds to both
the substrate concentration and the r atio between the sub-
strates, the d
13
C values of the products primarily refl ect
the availability o f t he two s ubstrates over the entire r ange
examined. It can be concluded that in actively growing
L. lactis the maintenance of pyruvate homeostasis takes
precedence over the redox status of the cells as a re gulatory
factor.
Keywords: carbon balance; isotope ratio mass spectrometry;
lactic acid bacteria; m etabolic regulation; pyruvate.
A r ange of simple sug ars can be c atabolized anaerobically
by Lactoc occus lactis and other lactic acid bacteria (LAB) in
order to obtain energy for growth. Central to this metabo-
lism is the C3 compound, pyruvate [1]. This metabolite
forms the link between the essentially oxidative reactions of
energy p roduction and those required f or the regeneration
of reducing equivalents NAD
+
or NADP
+
(Fig. 1 ).
However, pyruvate is relatively toxic [1,2], necessitating
strict control over the level to which it accumulates. In
L. lactis it is primarily reduced to lactic ac id by
L
-lactate
dehydrogenase (LDH, EC 1.1.1.27), thus maintaining both
pyruvate homeostasis and redox equilibrium. Under appro-

priate conditions, however, fermentation leads to C1 and C2
compounds, providing alternative routes for pyruvate
catabolism. Thus, varying amounts of acetate, formate
and ethanol can b e produced by the actions of p yruvate
formate-lyase (PFL, EC 2.3.1.54) or pyruvate dehydro-
genase (PDH, EC 1.2.4.1, EC 2.3.1.12, E C 1 .8.1.4). In the
case of ethanol production, this provides an alternative
means for NAD
+
regeneration.
In addition, some strains of L. lactis,suchasL. lac tis ssp.
lactis bv. d iacetylactis, c an metabo lize citrate [ 3,4], w hich
leads to enhanced or prolonged growth [5,6]. Citrate
metabolism impinges on the pyruvate pool without con-
comitant participation in the redox status of the cells
(Fig. 1 ). These strains are unusual in their capacity to
accumulate the C4 products, d iacetyl, acetoin and butan-
2,3-diol. T his correlation led to a number o f reports that
these compounds were products of citrate catabolism [7–9]
but recent work has disproved this assumption [10,11].
Although the metabolism of citrate to pyruvate does not
consume NAD
+
, acetoin and butan-2,3-diol production
can contribute to NAD
+
regeneration. The formation of
acetoin via the unstable intermediate, a-acetolactate [12,1 3],
however, requires 2 mol o f pyruvate, thus providing a less
efficient route to NAD

+
regeneration than lactate, or
indeed ethanol, formation (Fig. 1 ).
Although a cetoin is the most favoured C4 product, it is
diacetyl that is of greater commercial interest as this
compound is responsible for the ÔbutteryÕ flavour notes in
fermented d airy products. Thus, metabolic regu lation that
Correspondence to R. J. Robins, Isotopic Fr actionation in Metabolism
Group, Laboratory for the Isotopic and Electrochemical Analysis of
Metabolism, CNRS UMR6006, University of Nantes, BP 99208,
F-44322 Nantes, France. Fax: +332 51 12 57 12,
Tel.: +332 51 12 57 01,
E-mail:
Abbreviations: IRMS, isotope ratio mass spectrometry; LAB, lactic
acid bacteria; LDH,
L
-lactate dehydrogenase; PDH, pyruvate
dehydrogenase (acetyl-transferring) complex; PFL, pyruvate formate-
lyase; SPME-GC-C-IMRS, solid-phase micro-extraction-GC-
combustion-IRMS.
Enzymes: acetaldehyde dehydrogenase (EC 1.2.1.10); a-acetolactate
decarboxylase (EC 4.1.1.5); a-acetolactate s ynthase (EC 2.2.1.6); ace-
tyl kinase ( EC 2.7.2.1); alcohol de hydrogenase (EC 1.1.1.1); citrate
lyase (EC 4.1.3.6); diacetyl reductase (EC 1.1.1.5);
L
-lactate dehydro-
genase (EC 1.1.1.27); phosphate acetyl transferase (EC 2.3.1.8);
pyruvate dehydrogenase (acetyl-transferring) complex
(EC 1.2.4.1 + EC 2.3.1.12 + EC 1.8.1.4); pyruvate formate-lyase
(EC 2.3.1.54).

(Received 3 0 June 2004, revised 13 A ugust 2004,
accepted 23 September 2004)
Eur. J. Biochem. 271, 4392–4400 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04376.x
results i n diacetyl accumulation has received considerable
interest [14,15] and, as has been previously argued [11], is
complex. What is apparent is that the cometabolism of
citrate and glucose leads to the enhanced production of
these compounds by re-routeing of the metabolic through-
put [3,7–9,16,17]. Because t his is not specifically due to the
metabolism of citrate to the C4 compounds [10,11], it must
reflect an overall shift in the balance between different
routes for pyruvate catabolism. This shift could be a
response to a n altered redo x status or simply an up-shift in
the size o f the pyruvate supply. Currently, it is not clear
which is the more important of these factors.
The regulation of glycolysis in LAB has been studied
extensively and a number of potential regulatory points
proposed ([1,18] and r eferences therein). However, t here is
compelling evidence that neither the [pyruvate] [1,18] nor the
NAD
+
/NADH balance [19], nor the level of ldh expression
[20] regulates glycolysis. Notably, genetic manipulation of
key glycolytic enzymes has failed to identify one specific
control point in the pathway for pyruvate p roduction from
glucose [18]. Furthermore, genetic manipulation of pyruvate
catabolism, such as varied expression of ldh [20–22] or
enhanced a-acetolactate synthase (EC 2 .2.1.6) production
[23], c an substantially alter total metabolic throughput in
the alternative pathways of pyruvate catabolism. Hence,

it may b e suggested that in L. lactis pyruvate throughput
plays a more important regulatory role than does pyruvate
input.
This hypothesis h as been tested by examining the total
carbon redistribution from glucose and citrate during
cofermentation under a range of concentrations of both
cosubstrates. A difficulty in unravelling LAB metabolic
throughput is the continuous change in environment that
takes p lace as cells grow and substrate is consumed. The
study of the redistribution of
13
C-label in nongrowing cells
helps indicate m etabolite t urnover and concentrations [24]
but does not show the throughput during growth conditions
[16]. Similarly, modelling of flux has been restricted to
situations with only a single fermentable substrate p resent
and requires a ssumptions about the steady-state levels of
metabolites [25]. As two pathways are active simultaneously
and both l ead to the key intermediate, pyruvate, it is crucial
to understand the extent to which their i ndividual t hrough-
puts are interdependent.
To overcome these difficulties and to measure directly the
total carbon redistribution in actively growing LAB during
glucose–citrate cofermentation, we have developed an
approach that exploits the small variation in n atural
13
C
content between substrates de rived f rom different biological
sources [10]. These small differences can b e d etermined by
isotope ratio mass spectrometry (IRMS) on the relative

d
13
C scale with an accuracy of at least ± 0.2& [26]. The
relative d
13
C scale is used routinely to compare different
13
C/
12
C ratios. The scale is standardized against a calibrated
reference (R) of known
13
C/
12
C r atio and the value of the
unknown (S) is expressed in & according to the formula:
d
13
C ¼
13
C
12
C
hi
S
13
C
12
C
ÂÃ

R
À 1
0
@
1
A
 100
By fermenting glucose and citrate that differ by % 15&,
intermediate values of d
13
C determined for the various
fermentation products can be used to calculate the
proportion originating from each of these two possible
substrates. B y t his means, w e h ave previously shown t hat,
under one defined set of conditions, glucose an d citrate
contributed to the C4 compound s and lactic acid in
proportions closely represe nting the availability of the two
carbon sources [10]. Thus, their metabolic equivalence at
the level of pyruvate was implied. Further investigation,
however, indicated that the proportional utilization varied
depending on the environment; notably that the relative
availability o f t he substrates and t he le vel of advancement
of the f ermentation could influence the d
13
C determined f or
the p roducts [11].
esoculg 5.0
etartic
etavuryp
etatcal

α etatcaloteca-
etamrof
A
oC-lyteca
HDAN
D
AN
+
HDAN
DAN
+
nioteca
l
y
tecaid
P-lyteca
eta
teca
HDAN
DAN
+
PDA
PTA
HDAN
DAN
+
PTA
PDA
1
2

4
a6,5
7
b6
3
8
9
31
21
01
ed
y
hedlatec
a
D
A
N
+
HD
A
N
HDAN
DAN
+
l
o
n
a
hte
11

HDAN
D
AN
+
l
o
id
-
3
,
2
-
n
a
tu
b
7
Fig. 1. Schematic p athway showing the key metabolic relationships between the substrates and p roducts in glucose–citrate cofermentation. 1, glycolysis;
2, citrate lyase (EC 4.1.3.6); 3,
L
-lactate dehydrogenase (EC 1.1.1.27); 4, a-acetolactate synthase (EC 2.2.1.6); 5, a-acetolactate decarboxylase
(EC 4.1.1.5); 6a, nonenzymatic decarboxylation; 6b, nonenzymatic oxidative decarboxylation; 7, diacetyl dehydrogenase (EC 1.1.1.5); 8, pyruvate
dehydrogenase (acetyl-transferring) c omplex (EC 1.2.4.1, EC 2 .3.1.12, EC 1.8.1.4); 9, pyruvate formate-lyase (EC 2.3.1.54); 10, acetaldehyde
dehydrogenase (acetylating) (EC 1.2.1.10); 11, alcohol dehydrogenase (EC 1.1.1.1); 12, phosphate acetyl transferase (EC 2.3.1.8); 13, acetyl kinase
(EC 2.7.2.1).
Ó FEBS 2004 Carbon redistribution in L. lactis cofermentation (Eur. J. Biochem. 271) 4393
In order to examine the relationship between the
consumption of glucose and of citrate and the accumulation
of the products of pyruvate catabolism, the effect of varying
the glucose and citrate availability has been examined. Both

substrates have been varied over a four-to-fivefold range of
concentration and the relationships between the concentra-
tions and d
13
C values of s ubstrates and products u sed to
construct a balance sheet for carbon redistribution. It is
found that the d
13
C values o f the products primarily reflect
the relative input to the pyruvate pool of the two substrates,
thus co nfirming experimentally the key role proposed for
pyruvate in metabolic regulation in L. lactis [1,25].
Materials and methods
Bacterial strains and culture conditions
Lactococcus l actis ssp. lactis bv. diacetylactis strain B7/2147
was obtained from the Collection of L actic Acid Bacteria
(Institute of Food Research, Norwich, UK: collection no.
B7/2147). This strain has a high capacity to produce
diacetyl. The culture was stored at )80 °C i n M17 medium
[27] with 15% (v/v) glycerol.
Routine culture was in sterile (20 min; 121 °C;
10
5
NÆm
)2
) M17 broth, appropriately supplemented with
glucose or citrate–glucose, in 200 mL Duran bottles, as
described previously [10]. The general fermentation c ondi-
tions were: anaerobic (static, closed), 30 °C, pH initially
6.3 ± 0.1 ( HCl) and left to evolve freely. Cultures were

initiated with an 8-h preculture from t he same medium and
harvested after complete consumption of substrates
(16–22 h), the supernatant being recovered by centrifuga-
tion (4500 g,10min,4°C) and kept at )20 °C.
The d
13
C
initial
values for the citrate and glucose fermented
were
13
C
glucose
¼ )10.7& and d
13
C
citrate
¼ )24.7&.The
concentration of these substrates was varied from 13.9 to
55.5 m
M
for glucose and 0 to 34.8 m
M
for citrate. Reference
conditions used 27.8 m
M
for glucose and 13.9 m
M
for
citrate.

Metabolite analysis and isotopic determinations
Metabolite concentrations in the culture medium were
determined d irectly on the culture filtrate by
1
HNMR
using an external e lectronic reference, as described previ-
ously [28].
The d
13
C
acetoin
and d
13
C
diacetyl
values were determined
by solid-phase micro-extraction-GC-combustion-IRMS
(SPME-GC-C-IRMS) as described previously [29]. The
d
13
C
acetate
value was d etermined under the sam e conditions.
Essentially, these products were recovered from the head-
space above a sample of the f ermentation broth using
polydimethylsiloxane-divinylbenzene-coated fibres (Supe-
lco) and introduced directly into the injector of an
HP6890 gas chromatograph (Agilent T echnologies) linked
on-line to a combustion interface and an IRMS (Finnegan
Mat Delta S, Finnegan). Separation was accomplished

using a Stabilwax column (length, 60 m; i.d., 0.32 mm; film
thickness, 0.5 lm; Restek). Samples w ere i ntroduced via a
split/splitless injector (splitle ss mode, 250 °C) and chroma-
tographed under the following conditions: vector gas, He;
flow rate 2.2 mLÆmin
)1
(constant pressure); temperature
gradient, 50 °C for 0.1 min, followed by an increase of
10 °CÆmin
)1
to 200 °C, then 200 °C for 2 min. Each sample
was analysed at least three times and compounds were
identified by reference to authentic standards. Measured
d
13
C values were corrected for slight shifts to ward the
negative associated with t he use of the SPME-GC-C-IRMS
protocol. Corrections were based on standard solutions
containing acetic acid, acetoin and diacetyl for which d
13
C
values were determined by elemental analyser-IRMS.
Correction factors applied were acetic acid + 0.4&,acetoin
+0.2& and diacetyl +0.6&.
Lactic acid was purified from culture filtrate and the
d
13
C
lactate
values were determined by elemental analyser-

IRMS (Finnegan Mat Delta E, Finnegan) on encapsulated
samples as described previously [10].
Results
In order for th e analysis of
13
C redistribution to b e valid,
three criteria should be fulfilled. First, i t is essential that all
the available s ubstrates ar e consumed. Second, it is neces-
sary to account for all the available carbon among the
products of ferm entation. Third, i t is preferable t hat no or
little catabolism of the initial fermentation products has
taken place. Thus, a d etailed quantitative analysis of the
different metabolites is a prerequisite for interpreting the
d
13
C values in terms of
13
C redistribution and this
information is summarized below.
Influence of the [glucose]/[citrate] ratio on product
accumulation
Fermentation was always conducted under static, closed,
but not strictly anaerobic growth conditions with glucose
and citrate as the only substrates and an initial pH of
6.3 ± 0.1. A range of [glucose]/[citrate] from 0.8 to 4.0 was
used, with concentrations varying from 13.9 to 55.6 m
M
for
glucose and 6.9 to 34.8 m
M

for citrate. With the exception of
the highest [glucose], fermentation of both substrates
present was complete by 16 h. For [glucose] at 41.7 and
51.6 m
M
, fermentation was complete by 18 h. These end
points were used for all further analyses.
Growth and final pH both varied considerably depending
on the quantities of substrates available (Table 1). As
anticipated, increasing glucose availability gave hig her cell
density and high [glucose] led to elevated [lactate] (Table 1)
and a concomitant low pH. Growth was slightly diminished
by high [citrate] (Table 1) and the fall in pH was less evi-
dent, presumably because of the n egative acidity b alance
associated with citrate catabolism (3 · COO
–
giving
2 · COO
–
+CO
2
) [5]. This growth inhibition did not
prevent fermentation as all available substrate was con-
sumed over the whole range of concentrations used
(Table 1). H owever, it was associated with a shift in the
balance of products accumulated.
The fermentation o f glucose alone led entirely t o lactate
and a low level of acetate, no other product being detected.
In cofermentation, lactate was always the principal product
and showed a strong correlation with the available glucose

(Table 1). In all cases, the lactate p roduced was between 80
and 95% of the theoretical yield from glucose consumed but
never exceeded 100% even at the higher [citrate]. Indeed, an
4394 M. Mahmoud et al. (Eur. J. Biochem. 271) Ó FEBS 2004
increased availability of citrate had no influence on the total
lactate produced. This i s compatible w ith the tightly linked
redox balance of g lucose metabolism, glucose to p yruvate
producing 2 mol of NADH per mol of glucose that are then
consumed by pyruvate to initiate metabolism. As citrate
metabolism to pyruvate does not generate NADH, this
cannot be linked to lactate production because the total
lactate produced cannot exceed the maximum theoretical
yield from glucose due to the redox constraints.
The picture for acetate accumulation is the reverse
(Table 1). As expected from the known metabolism o f
citrate (Fig. 1), [acetate] showed a strong correlation with
available citrate. However, in all cases, the acetate accumu-
lated was significantly higher than 100% of t he theoretical
yield f rom t he activity of citrate lyase (EC 4.1.3.6), indica-
ting that a proportion was derived from pyruvate. No
significant influence of the [glucose] was seen, the [acetate]
remained constant (19.1 ± 0.4 m
M
) over a fourfold change
in [glucose], indicating that % 30% of the acetate was
derived from pyruvate w hen [citrate] ¼ 13.9 m
M
.Increas-
ing [citrate] led to a slight overall increase in net acetate
derived from pyruvate, from 3.5 m

M
at 0 m
M
citrate to
7.5 m
M
at 34.8 m
M
citrate. Hence, it is evident that neither
lactate nor acetate production from pyruvate is strongly
affected by citrate catabolism.
Acetate production from pyruvate can have two main
consequences. It can act to diminish the pool of pyruvate
under conditions in which the major mechanism –
reduction to lactate – is inadequate. It can also act to
provide ATP via the acti on of acetyl kinase (EC 2.7.2.1).
Anaerobic conditions favour pyruvate catabolism to
acetate via PFL (Fig. 1), which will result in a 1 : 1
ratio for formate/acetate. At low or zero [citrate], n o
formate was detected (Table 1), even though pyruvate-
derived acetate was present, whereas at 13.9 m
M
citrate,
the [formate] was consistently 65–70% of the pyruvate-
derived acetate. This indicates t hat, although PFL activity
was the princ iple source of acetate, either acetate from an
alternative catabolism was also being produced or
formate was being degraded. As L. lactis lacks formate
dehydrogenase, the latter option is ruled out. The most
likely source of this additional acetate is PDH, which,

although typically associated with aerobic conditions,
does show some activity in anaerobic fermentation [30].
Although PDH-mediated acetyl-CoA production is unfa-
vourable as it produces NADH, it fulfils both objectives
of decreasing the pyruvate pool and providing acetyl-P
for ATP generation.
Because ethanol was not detected by
1
HNMR inthe
medium from any o f these experiments ( data not shown),
acetyl-CoA formation was not linked to N AD
+
regener-
ation. This strongly suggests that both PFL and PDH are
primarily involved in regulating the size of the pyruvate
pool, rather than in maintaining the redox status of the
cells. In effect, the production of pyruvate from citrate
does not generate NADH. If this enhanced pyruvate
production led to enhanced lactate production, the
NADH/NAD
+
balance w ould be disequilibrated. Acetate
production by PFL effectively utilizes pyruvate without
interfering with the NADH/NAD
+
balance. By contrast,
excess acetate production can also be detrimental to cell
growth.
What, then, is the role of the alternative pathways of
pyruvate catabolism that lead t o the formation of t he C4

compounds, acetoin and diacetyl? The availability of citrate
rather than of glucose was, as found previously, the
determining factor for the accumulation of these com-
pounds (Table 1). Under all conditions, about twice the
amount of acetoin was found than of diacetyl but no butan-
2,3-diol was d etected, either by
1
H N MR or by GC (data
not shown). At constant 13.9 m
M
citrate, a fourfold increase
in [glucose] (13.9–55.6 m
M
) caused both [diacetyl] a nd
[acetoin] to increase % twofold. Increasing the [citrate] 2.5-
fold, however, led to an % 3.5-fold increase in both acetoin
and diacetyl. This pathway has the potential to contribute to
both the pyruvate homeostasis and t he redox status of the
cells, as 2 mol pyruvate can be used for the regeneration of
1mol NAD
+
via diacetyl reductase (EC 1 .1.1.5) [31,32].
However, this route to NAD
+
regeneration appears to have
been negligible in this study, as strains containing diacetyl
reductase generally produce butan-2,3-diol, because of
the activity of the same enzyme on acetoin [32]. Under
anaerobic culture conditions, oxidation of acetoin to
diacetyl is extremely unlikely [3]. Consequently, ac etoin

Table 1. Product accumulation profiles for fermentation of Lactococcus l actis with diffe ring initial amounts of glucose and citrate. N, number of repeat
fermentations in these conditions. A is the o ptical dispersion at 550 n m.
Duration
(h) N
a
Initial
concentration
Ratio
G/C
Final
pH
Final
A
Lactate
(m
M
)
Acetate
(m
M
)
Formate
(m
M
)
Diacetyl
(m
M
)
Acetoin

(m
M
)
Balance
(%)
Glucose
(m
M
)
Citrate
(m
M
)
16 4 13.9 13.9 1.0 5.9 1.02 31.33 ± 2.26
b
18.97 ± 0.60 3.62 ± 0.25 1.15 ± 0.18 2.48 ± 0.57 112.9 ± 7.9
16 5 27.8 13.9 2.0 5.1 1.53 53.26 ± 2.47 19.10 ± 0.90 3.67 ± 0.18 1.25 ± 0.13 2.92 ± 0.38 101.5 ± 8.2
18
a
1 41.7 13.9 3.0 4.6 1.64 83.96 ± 7.47 19.31 ± 0.40 4.00 ± 0.07 2.22 ± 0.19 3.56 ± 0.40 107.8 ± 3.3
18
a
1 56.6 13.9 4.0 4.6 1.66 90.63 ± 4.75 20.38 ± 0.80 4.20 ± 0.45 2.85 ± 0.19 4.16 ± 0.92 90.6 ± 4.1
16 2 27.8 0.0 – 4.4 1.32 54.90 ± 0.85 3.45± 0.78 0.00 0.00 0.00 107.6 ± 3.8
16 5 27.8 6.99 4.0 4.8 1.52 55.16 ± 1.19 11.46 ± 0.44 0.00 0.00 0.00 95.6 ± 2.0
16 4 27.8 20.9 1.3 5.4 1.38 58.60 ± 3.06 27.10 ± 0.73 1.92 ± 1.14 2.50 ± 0.58 4.70 ± 0.93 106.0 ± 7.0
16 4 27.8 34.8 0.8 6.0 1.35 56.15 ± 1.63 42.11 ± 1.88 5.96 ± 2.23 4.62 ± 0.20 9.45 ± 0.21 107.9 ± 2.8
a
At high [glucose], fermentation was not complete at 16 h but no substrates remained at 18 h.
b

Combined SD is given for the number of
fermentations and for the replicate measurements in each fermentation.
Ó FEBS 2004 Carbon redistribution in L. lactis cofermentation (Eur. J. Biochem. 271) 4395
must have been produced by the d irect decarboxylation o f
a-acetolactate, a conclusion confirmed by the absence of
any accumulation of butan-2,3-diol. Both products accu-
mulate in strains lacking a-acetolactate decarboxylase
[22,33,34] and the high diacetyl/acetoin ratio observed i n
our experiments s uggests that L. lactis B7/2147 has dimin-
ished a-acetolactate decarboxylase activity (C. Monnet,
INRA, Paris-Grignon, France, personal communication).
The extent to which nonenzymatic decarboxylation of
a-acetolactate lead s to acetoin or to diacetyl is strongly
dependent on the p revailing c onditions of culture, notably
pH [35], O
2
[14,36] and the presence of metal ions [37] or
other oxidizing agents [38] in the medium.
Nevertheless, irrespective of the route by which acetoin
and diacetyl a re formed, their biosynthes is appears to p lay
no role in the control of the redox status of the cells.
Rather, it appears that, once again, the major role of this
alternative pathway is to regulate the size of the pyruvate
pool. If this is the case, pyruvate catabolism should be
independent of the substrate supplying the p yruvate: if it is
not, then a link should b e seen between the amount of each
substrate being metabolized and the redistribution of
carbon into th e different products of pyruvate metabolism.
These alternatives can be tested by relating the d
13

Cvalues
in the products accumulated to those of t he substrates
supplied.
Influence of the [glucose]/[citrate] ratio on d
13
C values
and substrate redistribution between products
Although t he final concentrations of products indicate the
total throughput for different catabolic routes, these cannot
discriminate between th e utilization of alternative substrates
for the different products. H owever, this can be deduced
from the relationship between the initial d
13
C
glucose
and
d
13
C
citrate
values of the substrates and the d
13
C
lactate
,
d
13
C
acetate
, d

13
C
diacetyl
and d
13
C
acetoin
values a t term. (Dat a
for d
13
C
formate
could not be obtained, as this product was
not sufficiently w ell r esolved i n t he GC-C-IRMS.) Prelim-
inary data for a limited range of substrate conditions
showed that some of these f actors are r elated [11] althou gh,
notably, no data for acetate were presented. In Table 2 are
presented values for d
13
C for cultures using various
concentrations of citrate (initial d
13
C
citrate
¼ )24.7&)and
of glucose (initial d
13
C ¼ )10.7&).
The d
13

C
lactate
produced in the absence of citrate had a
value of )12.5&, showing a Dd
13
C
lactate
¼ )2&, as f ound
previously [10,11]. In the r eference conditions (27.8 m
M
glucose, 13.9 m
M
citrate), the d
13
C
lactate
¼ )14.8& is also in
good agreement with previous values. In contrast to the
effect on [lactate], both [glucose] and [citrate] influenced the
value of d
13
C
lactate
. Thus, as [citrate] increased, the d
13
C
lactate
steadily became more negative, reaching )16.2& at
34.8 m
M

(Fig. 2 A). In contrast, the influence of citrate
was diminished as [ glucose] increased, a value of
d
13
C
lactate
¼ )13.6& being f ound at 56.6 m
M
glucose. Thus
it is clear that both substrates were being used in all
conditions to give rise to lactate, even though [ citrate] had
no influence on [lactate].
The d
13
C
acetate
produced in the absence of citrate had a
value of )10.9&,evenclosertothatofglucosethanthe
d
13
C
lactate
.However,asexpected,thed
13
C
acetate
was strongly
influenced by [c itrate] (Fig. 2B). Thus, at the lowest [citrate]
of 6.9 m
M

,thed
13
C
acetate
()19.7&) had shifted significantly
closer to d
13
C
citrate
, reflecting the fact that even in these
conditions at least 60% of the a cetate comes from citrate
(Table 1 ). As [citrate] further increased, the d
13
C
acetate
became more negative and a t 34.8 m
M
citrate the d
13
C
acetate
was not significantly d ifferent from the initial d
13
C
citrate
.In
contrast, even the highest [glucose] (55.6 m
M
)hadno
significant influence o n the d

13
C
acetate
value, in agreement
with at least 68% of the a cetate being derived from citrate
(Table 1).
In comparing the data in Table 1 and Fig. 2, it appears
that the availability of each substrate, t he concentrations of
products and the d
13
C values o f t he products do not bear
direct relationships to each other. For example, although
[lactate] shows a strict correlation with [glucose], w ithout
any influence of [citrate], the d
13
C
lactate
values evolve in
relation to the r elative a vailability o f citrate. At first sight,
this might indicate metabolic control interaction between
the two pathways supplying pyruvate. However, a different
interpretation emerge s when these parameters are related to
the throughput of the pyruvate pool. For cultures grown on
glucose alone, d
13
C
lactate
()12.5&)andd
13
C

acetate
()10.9&)
can b e determined in the absence of c itrate. The d
13
C
lactate
is close t o the d
13
C
glucose
, the difference probably being due
to pyruvate conversion to lactate, while the d
13
C
acetate
is
insignificantly different. That these values only differ slightly
from the d
13
C
glucose
indicates that fractionation between
glucose and pyruvate is small or negligible. Thus, it is
possible to model the d
13
C
pyruvate
values using the molar
production ratios for p yruvate f rom g lucose (2 : 1) or from
citrate (1 : 1), the concentration ratios, and the known

d
13
C
glucose
and d
13
C
citrate
:
d
13
C
calc
pyr ¼
2:mol
glc
2:mol
glc
þ mol
cit

Á d
13
Cglc þ
mol
cit
2:mol
glc
þ mol
cit


Á d
13
Ccit
where pyr ¼ pyr uvate, glc ¼ glucose, cit ¼ citrate and
calc ¼ calculated. The resulting predicted d
13
C
pyruvate
val-
ues are plotted w ith the measured values for the products
in Fig. 3.
It can be seen from Fig. 3A that, even though the
evolution of the d
13
C
lactate
follows th e same tendency as the
d
13
C
pyruvate
, there is a divergence for high [glucose] at
constant [citrate]. This indicates that t he relative input from
glucose at high [glucose] is lower than theoretically expected.
This could indicate that glycolytic pyruvate production is
saturated or is b eing downregulated at the higher [glucose]
but that simultaneous pyruvate production from citrate
shows n o such constraint. This explanation is supported by
therelativeevolutionofthed

13
C values i n conditions of
constant [glucose] and increasing [c itrate] (Fig. 3B). Here,
the tendency is for the d
13
C
lactate
to approach the theoretical
d
13
C
pyruvate
as [citrate] i ncreases. Thus, e ven though [citrate]
has no influence on [lactate] it influences the carbon
redistribution from the common pool of pyruvate. This
supports a model in which [pyruvate] does regulate g lyco-
lytic input to the pyruvate pool [1].
In contrast, t he d
13
C
acetate
shows a lack of correlation
with the d
13
C
pyruvate
(Fig. 3 A,B). Only w hen no citrate is
4396 M. Mahmoud et al. (Eur. J. Biochem. 271) Ó FEBS 2004
present is d
13

C
acetate
close to d
13
C
pyruvate
.Withconstant
[citrate], [acetate] is unchanging irrespective of [glucose]
(Table 1), t he proportion derived from pyruvate is invari-
able, and the d
13
C
acetate
does not significantly vary (Table 2).
Obviously, in t he absence of citrate the d
13
C
acetate
is close to
the calculated d
13
C
pyruvate
but as [citrate] increases, there is a
rapid trend towards the d
13
C
citrate
. In fact, however, the
measured values in these two series of conditions are exactly

as predicted by a proportionation model in which the
d
13
C
acetate
is broken down into the part derived from
pyruvate and that derived directly from citrate:
d
13
C
calc
acetate
¼
mol
ac
À mol
cit
mol
ac

Á d
13
C
calc
pyruvate
þ
mol
cit
mol
ac


Á d
13
C
citrate
where calc ¼ calculated, a c ¼ acetate and c it ¼ citrate.
The measured d
13
C
acetate
values are seen to follow closely the
values obtained b y calculation (Fig. 3C). Hence, it c an be
concluded that acetate production, even more so than
lactate production, simply follows the input to th e pyruvate
pool.
As acetoin a nd diacetyl are not present in the absence of
citrate, no values for d
13
C
acetoin
and d
13
C
diacetyl
produced
exclusively from glucose could be obtained. At 6.9 m
M
citrate, when no acetoin or d iacetyl could be detected by
1
H N MR, the concentrating e ffect of the S PME fibre did

allow d
13
C
acetoin
and d
13
C
diacetyl
values to be d etermined,
although the low concentrations mean that the values
should be treated with caution. However, the fact that they
are both close to the initial d
13
C
glucose
indicates that the
a-acetolactate pathway is being supplied with pyruvate
from a common pool, which, as already shown for these
conditions, is strongly dominated by glucose. As the
[citrate] increases, so the d
13
C
acetoin
and d
13
C
diacetyl
values
are displaced towards the initial d
13

C
citrate
(Fig. 3 B).
Similarly, augmenting [glucose] leads to values that tend
towards
13
C
glucose
(Fig. 3 A). Both values retain approxi-
mately the same r elationship t o t he calculated d
13
C
pyruvate
,
indicating that the source of carbon used for their synthesis
is directly related to the metabolism of both available
6.9
13.9
20.9
34.8
13.9
41.7
–25
–20
–15
–10

δδ
δδ

δδ
δδ
13
C
Acetoin
Citrate (mM)
Glucose
(mM)
6.9
13.9
20.9
34.8
13.9
41.7
–25
–20
–15
–10
13
C
Diacetyl
Citrate (mM)
Glucose
(mM)
0
6.9
13.9
20.9
34.8
13.9

41.7
–25
–20
–15
–10
δδ
δδ
13
C
Lactate
Citrate (mM)
Glucose
(mM)
0
6.9
13.9
20.9
34.8
13.9
41.7
–25
–20
–15
–10
δδ
δδ


13
C

Acetate
Citrate (mM)
Glucose
(mM)
AB
DC
(‰)
(‰)
(‰)
(‰)
Fig. 2. The e ffect of varying the citrate and
glucose c oncentrations. (A) Final d
13
C
lactate
,
(B) final d
13
C
acetate
,(C)finald
13
C
acetoin
,(D)
final d
13
C
diacetyl
.Eachd

13
C value ( &)repre-
sents the mean of one to five fermentations
each analysed in triplicate, for which the
appropriate standard error is given in Table 2.
Table 2. Values o f d
13
C(&) d etermined for products of fermentation of Lact ococcu s l ac tis with differing initia l a moun ts of glucose and citrate. ND, not
determined.
Glucose
initial
a
(m
M
)
Citrate
initial
a
(m
M
) Ratio
d
13
C lactate
(&)
d
13
C acetate
(&)
b

d
13
C diacetyl
(&)
b
d
13
C acetoin
(&)
b
13.9 13.9 1.0 )15.82 ± 0.24
c
)23.20 ± 0.14 )17.3 ± 0.09 )21.3 ± 0.08
27.8 13.9 2.0 )14.45 ± 0.40 )23.53 ± 0.04 )15.57 ± 0.14 )17.91 ± 0.05
41.7 13.9 3.0 )13.87 ± 0.13 )22.10 ± 0.12 )12.19 ± 0.12 )17.52 ± 0.08
56.6 13.9 4.0 )13.63 ± 0.18 )23.56 ± 0.18 )12.65 ± 0.29 )15.24 ± 0.18
27.8 0 – )12.51 ± 0.61 )10.9 ± 0.19 ND ND
27.8 6.9 4.0 )13.67 ± 0.22 )19.17 ± 0.04 )8.90 ± 0.41 )13.30 ± 0.11
27.8 20.9 1.3 )15.09 ± 0.19 )23.11 ± 0.17 )13.60 ± 0.13 )19.60 ± 0.35
27.8 34.8 0.8 )16.23 ± 0.35 )23.70 ± 0.04 )14.70 ± 0.17 )19.38 ± 0.11
a
Initial d
13
C, glucose ¼ )10.7&, citrate ¼ )24.7&.
b
d
13
C Corrections were acetate +0.4&, diacetyl +0.6&, acetoin +0.2& (see
Materials and methods).
c

Combined SD is given for the number of fermentations and for the replicate measurements in each fermentation;
for N, see Table 1.
Ó FEBS 2004 Carbon redistribution in L. lactis cofermentation (Eur. J. Biochem. 271) 4397
substrates. I n t his, these c ompounds mimic the pyruvate-
derived acetate.
However, as noted previously [10,11], the d
13
Cvalues
are influenced not only by the [citrate]/[glucose] r atio
(Table 2), but also by the availability of citrate. Thus,
at 13.9 : 56.6 m
M
theyaremorenegativethanat
6.9 : 27.8 m
M
, despite the r atio of 4 : 1 being maintained.
The d
13
C
lactate
, in contrast, shows the same value for both
sets of concentrations. This indicates that, although there is
a strong influence of the inputs to the pyruvate pool, there is
a secondary influence of the [citrate]. This could result from
the production rate of diacetyl and acetoin varying
throughout the fermentation, reflecting variations in the
rate of citrate metabolism relative to t hat of g lucose. Such
variation could be induced, for example by c hanges in pH,
as citrate transport (but not glucose transport) is sensitive to
this factor [39]. F urther analysis of a range of ratios and of

the kinetics of the evolution of the d
13
C
acetoin
and d
13
C
diacetyl
values is required to define this effect.
The d
13
C
diacetyl
value is consistently 3–4& more positive
than the d
13
C
acetoin
(Table 2), a difference varying only
slightly with changes in the availability of g lucose and
citrate. This difference has a lso b een found to be retained
throughout the time-course of the fermentation for L. lactis
B7/2147 [11]. F urthermore, d
13
C
diacetyl
is consisten tly close
to the theoretical d
13
C

pyruvate
value, whereas d
13
C
acetoin
is
always 3– 6& more negative. Under anaerobic c onditions,
diacetyl is produced only through the nonenzymatic
decarboxylation of a-acetolactate, whereas acetoin may be
derived by either the nonenzymatic or the enzymatic
decarboxylation of a-acetolactate (Fig. 1). The high accu-
mulation of diacetyl and the lack of butan-2,3-diol indicates
that diacetyl dehydrogenase activity is negligible. It is
proposed that the strain B7/2147 accumulates unusually
high levels of diacetyl because of a deficiency in a-aceto-
lactate d ecarboxylase ( C. Monnet, INRA, Paris-Grignon,
France, personal communication). The discrepancy in the
d
13
C values may indicate, however, t hat L. lactis strain B7/
2147 has diminished, rather than deleted, a-acetolactate
decarboxylase activity because strains characterized as
lacking a-acetolactate decarboxylase [22] do not show a
similar large D(d
13
C
diacetyl
–d
13
C

acetoin
) [40]. Nonenzymatic
decarboxylation shows a range of isotope effects [ 41],
whereas enzymatic decarboxylation g enerally selects against
13
C [42,43]. Furthermore, previous evidence indicates t hat
biologically produced acetoin, as opposed to chemically
synthesized acetoin, is impoverished in
13
C in the hydroxy-
methylene g roup relat ive to the k eto g roup [4 4]. H ence, the
data (Table 2) support t he hypothesis that the acetoin is
derived by both enzymatic and n onenzymatic decarboxyla-
tion of a-acetolactate, whereas the diacetyl is produced only
nonenzymatically. Further work is required to clarify this
aspect of pyruvate metabolism.
Discussion
The role of pyruvate and the regulation of pyruvate
metabolism have b een much discussed in terms of the
overall regulation of LAB metabolism [1,3]. By following
the simultaneous cometabolism of glucose and citrate in
actively growing c ells of L. lactis, our data show that the
accumulation of pyruvate-derived metabolites depends
principally on the t hroughput of the pyruvate pool. With
glucose as sole substrate, throughput is apparently regu-
lated with reference to the maximal glycolytic capacity.
Thus, the pool of pyruvate is limited by glycolysis and
only small amounts of products other than lactate are
observed. This is in agreement with the known relative
affinities of LDH, PDH, PFL and a-acetolactate synthase

([45] and refs therein). As recently suggested, the role of
glycolysis is almost exclusively t o supply ATP and
throughput is probably maximal in rapidly growing
anaerobic c ultures [18], the utilization of pyruvate by
LDH being balanced by its supply. Thus, pyruvate does
not accumulate and problems with its toxicity are avoided.
A
–25.0
–20.0
–15.0
–10.0
–5.0
Glucose (mM)
δδ
δδ
13
C (‰)
δδ
δδ
13
C (‰)
B
–25.0
–20.0
–15.0
–10.0
–5.0
Citrate (mM)
C
–25.0

–20.0
–15.0
–10.0
–5.0
Concentration (mM)
δδ
δδ
13
C acetate (‰)
Fig. 3. The relationship between calculated d
13
C
pyruvate
values and
reaction products. Th e relationship b etween calculated d
13
C
pyruvate
and
measured d
13
C
lactate
, d
13
C
acetate
, d
13
C

acetoin
,andd
13
C
diacetyl
(A) at
constant [citrate] and variable [glucose], (B) at constant [glucose] and
variable [citrate]. The c alculated d
13
C
pyruvate
values are obtained from
the data in Tables 1 and 2 and the known m olar participation of each
substrate to pyruvate formation. Legend: c alculated pyruvate (r),
lactate (j), d iacetyl (m), acetoin (n), acetate (d). (C) The relationship
between the c alculated d
13
C
acetate
(line) and the measured d
13
C
acetate
(symbol). The calculated d
13
C
acetate
values are obtained from the data
in Table 1 and the calculated d
13

C
pyruvate
values. Legend: at constant
[glucose] and variable [citrate] (broken lin e, d); constant [citrate] a nd
variable [glucose] ( solid line, j).
4398 M. Mahmoud et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Recent studies have indicated that none of a number of
proposed control factors – the NAD
+
/NADH ratio [19],
the glyceraldehyde-phosphase dehydrogenase activity [46],
the LDH activity [20] or the phosphofructokinase activity
[18] – actually controls glycolytic flux. That glycolysis is
essentially unregulated under low to moderate [glucose] is
shown by our data, wh ich demonstrate that the availab-
ility of citrate leads to a net increase in the pyruvate
productive capacity without a ny concomitant inhibition of
glycolytic input. When the d
13
C values of the products of
these pathways – lactate, acetate, diacetyl and acetoin –
are examined, it is clear that they primarily reflect the
relative input to the pyruvate p ool, in this case governed
by the relative availability of glucose and citrate. While
there is some i ndication of limited feedback regulation on
glycolysis at high [glucose], no Ôcross-talkÕ between citrate
and glucose metabolism was detected. Rather, pyruvate
production is essentially unchecked and alternative path-
ways of pyruvate catabolism are required to maintain
pyruvate homeostasis and p revent pyruvate toxicity. T his

directly supports the p ropositions of Koebmann et al.[18]
and Neves et al. [19] that input to and ou tput from the
pyruvate pool are regulated by factors external to the
primary metabolic pathways.
Increasing throughput into pyruvate from citrate leads
to a progressive increase in the activity of alternative
pathways. Even so, it is found that the d
13
C v alues f or all
the products reflect the i nput into the pyruvate pool. That
augmenting [citrate ] le ads to an increase in pyruvate-
derived products i n the alter native pathways i ndicates that
the LDH capacity becomes limiting. This is confirmed b y
flux control analysis, which suggests that the LDH
capacity in wild-type L. lactis cells is only % 70% in
excess of the glycolytic rate [20], and by strains with
diminished LDH activity, which a ccumulate higher levels
of other products, even at low su bstrate supply or in the
absence of citrate [21,22,40]. There appears to be no
correlation between the activity of the given alternative
pathways and a need to regenerate NAD
+
, as indicated
by the l ack of ethanol accumu lation in the current system.
Rather, it appears that lactate prod uction is sufficient to
satisfy this need and the metabolism of acetate to
regenerate NAD
+
is not required. Instead, acetate
production from pyruvate can be seen as an ATP-

generating process. As high [acetate] occurs, ethanol
production could be inhibited [47] but, because L. lactis
ldh
–
can accumulate ethanol even in th e presence of citrate
(C. Monnet, INRA, Paris-Grignon, France, personal
communication) [40], this appears improbable. Therefore,
it can be argued that the most important role of the
alternative c atabolic uses of pyruvate is to m aintain a low
[pyruvate]. Hence, pathways in which no N ADH con-
sumption occurs but in which ATP generation is possible
(acetate via PDH and PFL) are favoured over those
which consume NADH (ethanol and butan-2,3-diol)
because t o consume NADH would t end to disequilibrate
the glucose-to-lactate redox balance (Fig. 1). In these
studies, neither product was found, suggesting that both
acetoin a nd acetyl-CoA reduction were absent.
In conclusion, this study shows t hat carbon redistribu-
tion from multiple substrates can effectively be followed
by IRMS by measuring
13
C at natural abundance. This
approach allows insights into metabolism that are
difficult to obtain b y other techniques. It enables the
study of the concurrent consumption of substrates and
the quantification of the orientation of their carbon
towards a range of products that can arise from more
than one route. Its application to following how the
utilization of glucose and citrate for products other th an
lactate is affected at the genetic level is the subject of

current studies.
Acknowledgements
WearegratefultoHughGriffinandHaroldUnderwood(IFR,
Norwich, UK) for supplying the Lactococcus lactis B7/2147 culture
and advice on growth conditions, to Christophe Monnet (INRA,
Paris-Grignon, France), Helena Santos (ITQB, Oeiras, Portugal) and
a number of o ur colleagues in Nantes for advice a nd discussion, and
to the Human Nutri tion Research C entre (Nantes) f or the use of the
GC-C-IRMS apparatus. Mohamed Mahmoud acknowledges the
financial support of the Arab Republic of Egypt for a doctoral
bursary.
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