With the objective of estimating the degree to which glucose and citrate metabolism through pyruvate may be differentially regulated, the d13C values of the products accumulated over a wi
Trang 1Natural-abundance isotope ratio mass spectrometry as a means
of evaluating carbon redistribution during glucose–citrate
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
13C/12C ratios, the simultaneous metabolism of citrate 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 common
intermediate With the objective of estimating the degree to
which glucose and citrate metabolism through pyruvate may
be differentially regulated, the d13C values 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 ratio between the sub-strates, the d13C values of the products primarily reflect the availability of the two substrates over the entire range 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 regulatory factor
Keywords: carbon balance; isotope ratio mass spectrometry; lactic acid bacteria; metabolic regulation; pyruvate
A range of simple sugars can be catabolized anaerobically
by Lactococcus 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 production and those required for 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 lactisit is primarily reduced to lactic acid byL-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 be produced by the actions of pyruvate formate-lyase (PFL, EC 2.3.1.54) or pyruvate dehydro-genase (PDH, EC 1.2.4.1, EC 2.3.1.12, EC 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, such as L lactis ssp lactis bv diacetylactis, can metabolize citrate [3,4], which 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, diacetyl, acetoin and butan-2,3-diol This correlation led to a number of 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 [12,13], however, requires 2 mol of pyruvate, thus providing a less efficient route to NAD+ regeneration than lactate, or indeed ethanol, formation (Fig 1)
Although acetoin is the most favoured C4 product, it is diacetyl that is of greater commercial interest as this compound is responsible for the buttery flavour notes in fermented dairy products Thus, metabolic regulation that
Correspondence to R J Robins, Isotopic Fractionation 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: richard.robins@chimbio.univ-nantes.fr
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 synthase (EC 2.2.1.6);
ace-tyl kinase (EC 2.7.2.1); alcohol dehydrogenase (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 30 June 2004, revised 13 August 2004,
accepted 23 September 2004)
Trang 2results in 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 this 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 an altered redox status or simply an up-shift in
the size of 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 references therein) However, there 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 production 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], can substantially alter total metabolic throughput in
the alternative pathways of pyruvate catabolism Hence,
it may be suggested that in L lactis pyruvate throughput
plays a more important regulatory role than does pyruvate
input
This hypothesis has 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 place as cells grow and substrate is consumed The
study of the redistribution of13C-label in nongrowing cells
helps indicate metabolite turnover 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 present and requires assumptions about the steady-state levels of metabolites [25] As two pathways are active simultaneously and both lead to the key intermediate, pyruvate, it is crucial
to understand the extent to which their individual through-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 natural 13C content between substrates derived from different biological sources [10] These small differences can be determined by isotope ratio mass spectrometry (IRMS) on the relative
d13C scale with an accuracy of at least ± 0.2& [26] The relative d13C scale is used routinely to compare different
13C/12C ratios The scale is standardized against a calibrated reference (R) of known13C/12C ratio and the value of the unknown (S) is expressed in & according to the formula:
d13C¼
13 C
12 C
h i S
13 C
12 C
R 1
0
@
1
A 100
By fermenting glucose and citrate that differ by 15&, intermediate values of d13C determined for the various fermentation products can be used to calculate the proportion originating from each of these two possible substrates By this means, we have previously shown that, under one defined set of conditions, glucose and citrate contributed to the C4 compounds and lactic acid in proportions closely representing 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 of the substrates and the level of advancement
of the fermentation could influence the d13C determined for the products [11]
e s o u l g 5 0 e
t a t i c
e t a u r y p
e t a t c a l
α - a e t o l c t a t e
e t a m r f
A o C -l y t e a
H A D
H A D
n i o t e a
l y t e a i d
P -l y t e
e t a t e
D
P D P T A
H A D
P T A P D
1 2
4
a , 5 7 b
3 8 9
3 2 0
e y e l a t e
+
H A
H A D
l o a t e
1
H A D
l o i d -3 , 2 -n t u
7
Fig 1 Schematic pathway showing the key metabolic relationships between the substrates and products 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) complex (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).
Trang 3In 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 d13C values of substrates and products used to
construct a balance sheet for carbon redistribution It is
found that the d13C values of the products primarily reflect
the relative input to the pyruvate pool of the two substrates,
thus confirming experimentally the key role proposed for
pyruvate in metabolic regulation in L lactis [1,25]
Materials and methods
Bacterial strains and culture conditions
Lactococcus lactisssp lactis bv diacetylactis strain B7/2147
was obtained from the Collection of Lactic 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 in M17 medium
[27] with 15% (v/v) glycerol
Routine culture was in sterile (20 min; 121C;
105NÆm)2) M17 broth, appropriately supplemented with
glucose or citrate–glucose, in 200 mL Duran bottles, as
described previously [10] The general fermentation
condi-tions were: anaerobic (static, closed), 30C, pH initially
6.3 ± 0.1 (HCl) and left to evolve freely Cultures were
initiated with an 8-h preculture from the same medium and
harvested after complete consumption of substrates
(16–22 h), the supernatant being recovered by
centrifuga-tion (4500 g, 10 min, 4C) and kept at)20 C
The d13Cinitialvalues for the citrate and glucose fermented
were 13Cglucose¼)10.7& and d13Ccitrate¼)24.7& The
concentration of these substrates was varied from 13.9 to
55.5 mMfor glucose and 0 to 34.8 mMfor citrate Reference
conditions used 27.8 mM for glucose and 13.9 mM for
citrate
Metabolite analysis and isotopic determinations
Metabolite concentrations in the culture medium were
determined directly on the culture filtrate by 1H NMR
using an external electronic reference, as described
previ-ously [28]
The d13Cacetoin and d13Cdiacetyl values were determined
by solid-phase micro-extraction-GC-combustion-IRMS
(SPME-GC-C-IRMS) as described previously [29] The
d13Cacetatevalue was determined under the same conditions
Essentially, these products were recovered from the
head-space above a sample of the fermentation broth using
polydimethylsiloxane-divinylbenzene-coated fibres
(Supe-lco) and introduced directly into the injector of an
HP6890 gas chromatograph (Agilent Technologies) 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 were introduced via a
split/splitless injector (splitless mode, 250C) and
chroma-tographed under the following conditions: vector gas, He;
flow rate 2.2 mLÆmin)1 (constant pressure); temperature
gradient, 50C for 0.1 min, followed by an increase of
10CÆmin)1to 200C, then 200 C for 2 min Each sample was analysed at least three times and compounds were identified by reference to authentic standards Measured
d13C values were corrected for slight shifts toward the negative associated with the use of the SPME-GC-C-IRMS protocol Corrections were based on standard solutions containing acetic acid, acetoin and diacetyl for which d13C 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
d13Clactate values were determined by elemental analyser-IRMS (Finnegan Mat Delta E, Finnegan) on encapsulated samples as described previously [10]
Results
In order for the analysis of13C redistribution to be valid, three criteria should be fulfilled First, it is essential that all the available substrates are consumed Second, it is neces-sary to account for all the available carbon among the products of fermentation Third, it is preferable that no or little catabolism of the initial fermentation products has taken place Thus, a detailed quantitative analysis of the different metabolites is a prerequisite for interpreting the
d13C values in terms of 13C 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 mMfor glucose and 6.9 to 34.8 mMfor 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 mM, 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 higher 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 negative acidity balance associated with citrate catabolism (3· COO– giving
2· COO–+ CO2) [5] This growth inhibition did not prevent fermentation as all available substrate was con-sumed over the whole range of concentrations used (Table 1) However, it was associated with a shift in the balance of products accumulated
The fermentation of glucose alone led entirely to 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 produced was between 80 and 95% of the theoretical yield from glucose consumed but never exceeded 100% even at the higher [citrate] Indeed, an
Trang 4increased availability of citrate had no influence on the total
lactate produced This is compatible with the tightly linked
redox balance of glucose metabolism, glucose to pyruvate
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 of
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 the theoretical
yield from the 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 mM) over a fourfold change
in [glucose], indicating that 30% of the acetate was
derived from pyruvate when [citrate]¼ 13.9 mM
Increas-ing [citrate] led to a slight overall increase in net acetate
derived from pyruvate, from 3.5 mM at 0 mM citrate to
7.5 mMat 34.8 mMcitrate 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 action 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], no
formate was detected (Table 1), even though
pyruvate-derived acetate was present, whereas at 13.9 mM citrate,
the [formate] was consistently 65–70% of the
pyruvate-derived acetate This indicates that, although PFL activity
was the principle 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 1H NMR in the medium from any of these experiments (data not shown), acetyl-CoA formation was not linked to NAD+ 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 would 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 to the formation of the 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 detected, either by1H NMR or by GC (data not shown) At constant 13.9 mMcitrate, a fourfold increase
in [glucose] (13.9–55.6 mM) caused both [diacetyl] and [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 the redox status of the cells, as 2 mol pyruvate can be used for the regeneration of
1 mol 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, acetoin
Table 1 Product accumulation profiles for fermentation of Lactococcus lactis with differing initial amounts of glucose and citrate N, number of repeat fermentations in these conditions A is the optical dispersion at 550 nm.
Duration
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 ± 2.26b 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 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.
Trang 5must have been produced by the direct decarboxylation of
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 in
our experiments suggests 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 leads to acetoin or to diacetyl is strongly
dependent on the prevailing conditions of culture, notably
pH [35], O2[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 are formed, their biosynthesis appears to play
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 pyruvate: if it is
not, then a link should be seen between the amount of each
substrate being metabolized and the redistribution of
carbon into the different products of pyruvate metabolism
These alternatives can be tested by relating the d13C values
in the products accumulated to those of the substrates
supplied
Influence of the [glucose]/[citrate] ratio on d13C values
and substrate redistribution between products
Although the final concentrations of products indicate the
total throughput for different catabolic routes, these cannot
discriminate between the utilization of alternative substrates
for the different products However, this can be deduced
from the relationship between the initial d13Cglucose and
d13Ccitrate values of the substrates and the d13Clactate,
d13Cacetate, d13Cdiacetyland d13Cacetoinvalues at term (Data
for d13Cformatecould not be obtained, as this product was
not sufficiently well resolved in the GC-C-IRMS.)
Prelim-inary data for a limited range of substrate conditions
showed that some of these factors are related [11] although,
notably, no data for acetate were presented In Table 2 are
presented values for d13C for cultures using various
concentrations of citrate (initial d13Ccitrate¼)24.7&) and
of glucose (initial d13C¼)10.7&)
The d13Clactateproduced in the absence of citrate had a
value of)12.5&, showing a Dd13Clactate¼)2&, as found
previously [10,11] In the reference conditions (27.8 mM
glucose, 13.9 mMcitrate), the d13Clactate¼)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 d13Clactate Thus, as [citrate] increased, the d13Clactate
steadily became more negative, reaching )16.2& at
34.8 mM (Fig 2A) In contrast, the influence of citrate
was diminished as [glucose] increased, a value of
d13Clactate¼)13.6& being found at 56.6 mMglucose 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 d13Cacetateproduced in the absence of citrate had a
value of )10.9&, even closer to that of glucose than the
d13C However, as expected, the d13C was strongly
influenced by [citrate] (Fig 2B) Thus, at the lowest [citrate]
of 6.9 mM, the d13Cacetate()19.7&) had shifted significantly closer to d13Ccitrate, reflecting the fact that even in these conditions at least 60% of the acetate comes from citrate (Table 1) As [citrate] further increased, the d13Cacetate became more negative and at 34.8 mMcitrate the d13Cacetate was not significantly different from the initial d13Ccitrate In contrast, even the highest [glucose] (55.6 mM) had no significant influence on the d13Cacetatevalue, in agreement with at least 68% of the acetate being derived from citrate (Table 1)
In comparing the data in Table 1 and Fig 2, it appears that the availability of each substrate, the concentrations of products and the d13C values of the products do not bear direct relationships to each other For example, although [lactate] shows a strict correlation with [glucose], without any influence of [citrate], the d13Clactate values evolve in relation to the relative availability of citrate At first sight, this might indicate metabolic control interaction between the two pathways supplying pyruvate However, a different interpretation emerges when these parameters are related to the throughput of the pyruvate pool For cultures grown on glucose alone, d13Clactate()12.5&) and d13Cacetate()10.9&) can be determined in the absence of citrate The d13Clactate
is close to the d13Cglucose, the difference probably being due
to pyruvate conversion to lactate, while the d13Cacetateis insignificantly different That these values only differ slightly from the d13Cglucose indicates that fractionation between glucose and pyruvate is small or negligible Thus, it is possible to model the d13Cpyruvatevalues using the molar production ratios for pyruvate from glucose (2 : 1) or from citrate (1 : 1), the concentration ratios, and the known
d13Cglucoseand d13Ccitrate:
d13Ccalcpyr¼ 2:molglc
2:molglcþ molcit
d13Cglcþ molcit
2:molglcþ molcit
d13Ccit
where pyr¼ pyruvate, glc ¼ glucose, cit ¼ citrate and calc¼ calculated The resulting predicted d13Cpyruvate val-ues are plotted with the measured valval-ues for the products
in Fig 3
It can be seen from Fig 3A that, even though the evolution of the d13Clactatefollows the same tendency as the
d13Cpyruvate, there is a divergence for high [glucose] at constant [citrate] This indicates that the relative input from glucose at high [glucose] is lower than theoretically expected This could indicate that glycolytic pyruvate production is saturated or is being downregulated at the higher [glucose] but that simultaneous pyruvate production from citrate shows no such constraint This explanation is supported by the relative evolution of the d13C values in conditions of constant [glucose] and increasing [citrate] (Fig 3B) Here, the tendency is for the d13Clactateto approach the theoretical
d13Cpyruvateas [citrate] increases Thus, even 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 glyco-lytic input to the pyruvate pool [1]
In contrast, the d13Cacetateshows a lack of correlation with the d13C (Fig 3A,B) Only when no citrate is
Trang 6present is d13Cacetate close to d13Cpyruvate With constant
[citrate], [acetate] is unchanging irrespective of [glucose]
(Table 1), the proportion derived from pyruvate is
invari-able, and the d13Cacetatedoes not significantly vary (Table 2)
Obviously, in the absence of citrate the d13Cacetateis close to
the calculated d13Cpyruvatebut as [citrate] increases, there is a
rapid trend towards the d13Ccitrate In fact, however, the
measured values in these two series of conditions are exactly
as predicted by a proportionation model in which the
pyruvate and that derived directly from citrate:
d13Ccalcacetate¼ molac molcit
molac
d13Ccalcpyruvateþ molcit
molac
d13Ccitrate where calc¼ calculated, ac ¼ acetate and cit ¼ citrate
The measured d13Cacetatevalues are seen to follow closely the
values obtained by calculation (Fig 3C) Hence, it can be
concluded that acetate production, even more so than
lactate production, simply follows the input to the pyruvate pool
As acetoin and diacetyl are not present in the absence of citrate, no values for d13Cacetoin and d13Cdiacetylproduced exclusively from glucose could be obtained At 6.9 mM citrate, when no acetoin or diacetyl could be detected by
1H NMR, the concentrating effect of the SPME fibre did allow d13Cacetoin and d13Cdiacetylvalues to be determined, although the low concentrations mean that the values should be treated with caution However, the fact that they are both close to the initial d13Cglucose 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 d13Cacetoinand d13Cdiacetylvalues are displaced towards the initial d13Ccitrate (Fig 3B) Similarly, augmenting [glucose] leads to values that tend towards 13Cglucose (Fig 3A) Both values retain approxi-mately the same relationship to the calculated d13Cpyruvate, 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
Acetoin
Citrate (mM)
Glucose
13.9 41.7
–25 –20 –15 –10
13
C Diacetyl
Citrate (mM)
Glucose (mM)
0 6 9 13.9
2 0 9 34.8
1 3 9
4 1 7
– 2 5 – 2 0 – 1 5 – 1 0
δδδδ13 C Lactate
Citrate (mM)
Glucose (mM)
0 6.9 13.9
20.9 34.8 13.9 41.7
– 2 5
– 2 0
– 1 5
– 1 0
δδδδ 13 C Acetate
Citrate (mM)
Glucose (mM)
D C
(‰)
(‰)
Fig 2 The effect of varying the citrate and
glucose concentrations (A) Final d13C lactate ,
(B) final d 13 C acetate , (C) final d 13 C acetoin , (D)
final d 13 C diacetyl Each d 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 of d13C (&) determined for products of fermentation of Lactococcus lactis with differing initial amounts of glucose and citrate ND, not determined.
Glucose
initiala
(m M )
Citrate
initiala
d13C lactate (&)
d13C acetate (&) b
d13C diacetyl (&) b
d13C acetoin (&) b
)23.20 ± 0.14 )17.3 ± 0.09 )21.3 ± 0.08
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.
Trang 7substrates In this, these compounds mimic the
pyruvate-derived acetate
However, as noted previously [10,11], the d13C values
are influenced not only by the [citrate]/[glucose] ratio
(Table 2), but also by the availability of citrate Thus,
at 13.9 : 56.6 mM they are more negative than at
6.9 : 27.8 mM, despite the ratio of 4 : 1 being maintained
The d13Clactate, 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 that of glucose Such variation could be induced, for example by changes in pH,
as citrate transport (but not glucose transport) is sensitive to this factor [39] Further analysis of a range of ratios and of the kinetics of the evolution of the d13Cacetoinand d13Cdiacetyl values is required to define this effect
The d13Cdiacetylvalue is consistently 3–4& more positive than the d13Cacetoin (Table 2), a difference varying only slightly with changes in the availability of glucose and citrate This difference has also been found to be retained throughout the time-course of the fermentation for L lactis B7/2147 [11] Furthermore, d13Cdiacetylis consistently close
to the theoretical d13Cpyruvate value, whereas d13Cacetoinis always 3–6& more negative Under anaerobic conditions, 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 decarboxylase (C Monnet, INRA, Paris-Grignon, France, personal communication) The discrepancy in the
d13C values may indicate, however, that 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(d13Cdiacetyl–d13Cacetoin) [40] Nonenzymatic decarboxylation shows a range of isotope effects [41], whereas enzymatic decarboxylation generally selects against
13C [42,43] Furthermore, previous evidence indicates that biologically produced acetoin, as opposed to chemically synthesized acetoin, is impoverished in13C in the hydroxy-methylene group relative to the keto group [44] Hence, the data (Table 2) support the hypothesis that the acetoin is derived by both enzymatic and nonenzymatic 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 been 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 cells of L lactis, our data show that the accumulation of pyruvate-derived metabolites depends principally on the throughput 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 to supply ATP and throughput is probably maximal in rapidly growing anaerobic cultures [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)
13 C (
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)
13 C
Fig 3 The relationship between calculated d13C pyruvate values and
reaction products The relationship between calculated d13C pyruvate and
measured d13C lactate , d13C acetate , d13C acetoin , and d13C diacetyl (A) at
constant [citrate] and variable [glucose], (B) at constant [glucose] and
variable [citrate] The calculated d 13 C pyruvate values are obtained from
the data in Tables 1 and 2 and the known molar participation of each
substrate to pyruvate formation Legend: calculated pyruvate (r),
lactate (j), diacetyl (m), acetoin (n), acetate (d) (C) The relationship
between the calculated d13C acetate (line) and the measured d13C acetate
(symbol) The calculated d13C 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 line, d); constant [citrate] and
variable [glucose] (solid line, j).
Trang 8Recent 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, which demonstrate that the
availab-ility of citrate leads to a net increase in the pyruvate
productive capacity without any concomitant inhibition of
glycolytic input When the d13C 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 pool, in this case governed
by the relative availability of glucose and citrate While
there is some indication of limited feedback regulation on
glycolysis at high [glucose], no cross-talk 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 prevent pyruvate toxicity This
directly supports the propositions of Koebmann et al [18]
and Neves et al [19] that input to and output 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 d13C values for all
the products reflect the input into the pyruvate pool That
augmenting [citrate] leads to an increase in
pyruvate-derived products in the alternative pathways indicates that
the LDH capacity becomes limiting This is confirmed by
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 accumulate higher levels
of other products, even at low substrate 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 lack of ethanol accumulation in the current system
Rather, it appears that lactate production 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 the 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 catabolic uses of pyruvate is to maintain a low
[pyruvate] Hence, pathways in which no NADH
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 to consume NADH would tend to disequilibrate
the glucose-to-lactate redox balance (Fig 1) In these
studies, neither product was found, suggesting that both
acetoin and acetyl-CoA reduction were absent
In conclusion, this study shows that carbon
redistribu-tion from multiple substrates can effectively be followed
by IRMS by measuring13C at natural abundance This
approach allows insights into metabolism that are difficult to obtain by 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 than lactate is affected at the genetic level is the subject of current studies
Acknowledgements
We are grateful to Hugh Griffin and Harold Underwood (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 our colleagues in Nantes for advice and discussion, and
to the Human Nutrition Research Centre (Nantes) for 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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