1 UMR-Sciences pour l'nologie, Microbiologie et Technologie des Fermentations, Institut National de la Recherche Agronomique, 2 Place Viala, 34060 Montpellier, France
2 Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique et Université d'Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
Correspondence
Carole Camarasa
camarasa@ensam.inra.fr Sylvie Dequin
dequin{at}ensam.inra.fr
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ABSTRACT |
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INTRODUCTION |
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Nevertheless, residual TCA pathway activity is maintained during fermentation. This activity primarily fuels biosynthetic reactions, supplying the cells with C4 and C5 compounds (oxaloacetate and 2-OG), the precursors of aspartate and glutamate, respectively. TCA pathway activity during fermentation also leads to the excretion of organic acids (citrate, malate and succinate). These compounds are of major interest to the food industry and in wine production, in particular, because they affect the organoleptic balance of wines. Succinate, in particular, is one of the most abundant organic acids and plays an important role in wine acidity. Increasing our understanding of the functioning of the TCA pathway during yeast alcoholic fermentation would therefore also provide insight into the pathways involved in the formation of these organic acids.
Several studies have focused on the functioning of the TCA cycle during fermentation. It is widely accepted that, during alcoholic fermentation, the TCA pathway does not operate as a cycle, but as two branches (Fig. 1). One branch is oxidative, leading to 2-OG formation (Nunez de Castro et al., 1970
), whereas the other is reductive, leading to fumarate formation (Atzpodien et al., 1968
; Sols et al., 1971
). This was confirmed by analysing the metabolic network in S. cerevisiae during respiro-fermentative growth on glucose (Gombert et al., 2001
). No net flux from 2-OG to fumarate was observed, but fumarate was formed from oxaloacetate. However, as succinate and succinyl-CoA were not included in the model, it was not possible to identify the point at which the cycle was interrupted (Fig. 1
). The pathways of succinate production were therefore not elucidated. Other studies aiming to identify the point at which the cycle is interrupted have come to different conclusions. Some studies have reported that the OGDH complex is not functional in anaerobic conditions, based on the observation of very little or no activity (Chapman & Bartley, 1968
; Machado et al., 1975
; Wales et al., 1980
). This is consistent with the formation of succinate via a reductive pathway. Two enzymes may be involved in the last step of this pathway: (i) fumarate reductase, which catalyses the irreversible conversion of fumarate into succinate, the activity of which is greater during yeast growth on glucose in anaerobic conditions than during growth on ethanol (Muratsubaki, 1987
), and (ii) the SDH complex, for which basal levels of activity have been detected during yeast fermentative metabolism (Polakis & Bartley, 1965
; Wales et al., 1980
). In contrast, significant levels of OGDH activity have been detected in fermenting S. cerevisiae cells, particularly if glutamate was used as the principal nitrogen source, and it has been suggested that this enzymic complex is involved in the formation of succinate from glutamate by oxidative decarboxylation (Albers et al., 1998
; Heerde & Radler, 1978
).
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METHODS |
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The synthetic medium MS (Bely et al., 1990) mimics the composition of a standard grape must and contains 200 g glucose l-1, 6 g malic acid l-1, 6 g citric acid l-1 and 300 mg nitrogen l-1 in the form of amino acids (180 mg N l-1) and NH4Cl (120 mg N l-1) at pH 3·5. In MG and MA media, the nitrogen source was replaced by glutamate and aspartate, respectively, to give a total of 300 mg N l-1.
Fermentation experiments were performed in fermenters with a working volume of 1·1 l, equipped with fermentation locks and inoculated at a density of 106 cells ml-1. Yeast cells were pre-cultured in MS medium for 24 h. Fermentation was performed at 28 °C, with continuous magnetic stirring (500 r.p.m.). The CO2 released was followed by automatic measurement of the weight lost by the fermenter every 20 min, and the rate of CO2 production (the fermentation rate) was calculated by polynomial smoothing of the CO2 released (Sablayrolles et al., 1987).
For assays with labelled compounds, cells were pre-cultured anaerobically at 28 °C in 50 ml MS medium for 24 h. To remove any trace of the nitrogen source or TCA intermediates before 13C incorporation, cells were harvested by centrifugation (2000 g, 4 °C, 5 min), washed twice and suspended in 15 ml incubation medium (MS medium without nitrogen source, malate or succinate, buffered with 8 g phthalic acid l-1). We incorporated 13C by adding 30 mg of [3-13C]aspartate or [3-13C]glutamate to the concentrated cell suspension (300x106 cells ml-1), and incubating the cells at 28 °C for 24 h. Control experiments were performed by replacing [3-13C]aspartate and [3-13C]glutamate by the unlabelled equivalents of these molecules.
Construction of sdh1 and kgd1 null mutants.
We used the short flanking homology PCR method (Wach et al., 1994) to delete SDH1 or KGD1 in strain V5. A PCR fragment was amplified from pF6A-kanMX4 using the forward and reverse primers 5'-TTCATAGTACGAAGAAGAACGAGAATAAAGATGCTATCGTcgtacgtcgcaggtcgac-3' and 5'-CGTAAAATACAATGAGGTTCAAATTAGTAGGCTCTTACAGgtacgatgaattcgagctcg-3', respectively. The nucleotides in lower case are identical to a region within the pF6A-kanMX4 multiple cloning site and the nucleotides in upper case correspond to the regions -30 to +10 and +1907 to +1947 of SDH1, respectively.
The same strategy was used to delete KGD1, using the forward and reverse primers 5'-TTTTACCGTTATGCTAAGGTTCGTGTCTTCGCAAACCTGCcgtacgtcgcaggtcgac-3' and 5'-CATCTTTAGGATTGTTGGAAAACATCTTTCAAAAAGGCATgatcgatgaattcgagctcg-3', respectively. The nucleotides in lower case are identical to pF6A-kanMX4 sequences, and the 40 nt extensions (upper case) correspond to the regions -10 to +30 and +3011 to +3050 of KGD1.
The PCR fragments were used to transform V5, using the lithium acetate procedure (Schiesti & Gietz, 1989). The correct replacement of the SDH1 and KGD1 ORF by the amplified fragments was verified by PCR analysis of total DNA isolated from kanamycin-resistant transformants, using primers flanking the deleted regions.
NMR spectroscopy.
Samples for NMR spectroscopy were obtained by collecting 1·5 ml of culture at the end of fermentations conducted in the presence of labelled compounds, as described previously. Culture supernatants were passed through cellulose nitrate filters (0·2 µm pores; Whatman) and stored at -20 °C. Before analysis, the pH was adjusted to 7 to 8 with 1 M KOH. Dioxane, diluted in deuterium water and added at a final concentration of 5 mM 13C, was used as a frequency and intensity reference. A solution of Na2/Cr/diethylene/triamine/pentaacetate in D2O was used as a relaxation reagent (final concentration 4 mM).
Spectra were recorded on a Bruker AM-300 NMR instrument at 75·4 MHz in the gated decoupling mode, with a sufficiently long relaxation delay for the obtention of quantitative results. About 1000 transients were accumulated for each sample, using 16 K data points. Signals were zero-filled to 32 K and then processed with a Lorentz Gauss filter before Fourier transformation.
Control experiments were performed, using unlabelled amino acids in the same conditions, and the NMR spectra obtained were compared with those obtained in the presence of a 13C-labelled nitrogen source.
Measurement of intracellular 2-OG levels and OGDH assays.
Intracellular 2-OG concentrations were determined using a previously described quantitative metabolite extraction method (Gonzalez et al., 1997). We sprayed 5 ml of culture into 26 ml of cold quenching solution [60 % (v/v) methanol buffered with 70 mM (final concentration) HEPES pH 7·5, kept at -40 °C]. Cells were rapidly collected by centrifugation at 5000 g (-10 °C, 2 min) and intracellular metabolites were extracted by suspending the pellet in 5 ml of 75 % (v/v) ethanol (diluted in 280 mM HEPES pH 7·5) for 5 min at 80 °C. The ethanol was evaporated off, cell debris was removed by centrifugation (10 000 g, 4 °C, 3 min) and the supernatant was stored at -20 °C for further metabolite determination.
For enzyme assays, cells were collected by centrifugation at 2000 g, washed twice in 10 mM potassium phosphate buffer (pH 7·5) and resuspended in 2 mM MgCl2/1 mM DTT in 100 mM potassium phosphate buffer pH 7·5. Cells were disrupted using 0·5 mm glass beads. The cell homogenate was centrifuged at 15 000 g (4 °C, 5 min) to remove debris and the supernatant was immediately used as a crude cell extract. We determined OGDH activity spectrometrically, as described previously (Reed & Oliver, 1982).
Analytical methods.
Growth was followed by optical density measurements at 660 nm (OD660). Glucose and fermentation products (acetate, succinate, hydroxyglutarate, 2-OG, glycerol and ethanol) were analysed by HPLC on an HPX-87H Aminex column (Bio-Rad). Dual detection was performed with a refractometer and a UV detector (Hewlett Packard). Malate and succinate concentrations were determined by enzymic assays (Boehringer Detection Kit) in samples collected from 13C experiments.
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RESULTS |
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Role of SDH during fermentation
We investigated the role of SDH by deleting the SDH1 gene encoding the flavoprotein subunit of the complex from strain V5. We compared the behaviour of the sdh1 mutant with that of the wild-type strain during fermentation under enological conditions, in MS medium simulating the composition of a typical grape must. Under these conditions, wine yeasts display a short exponential growth phase due to limiting amounts of nitrogen (300 mg l-1) and most of the glucose is consumed by stationary-phase cells. The fermentation rate, expressed as the rate of CO2 production, increased rapidly as the number of cells increased, then progressively decreased during the stationary phase. The sdh1 mutant displayed growth and fermentation profiles similar to those of the wild-type strain V5 (Fig. 5). As the nitrogen composition of the synthetic must used in this experiment differs from that previously used in this study, the behaviour of the mutant was also studied in the conditions used for NMR experiments (MA or MG medium, containing aspartate and glutamate, respectively, as sole nitrogen source). We found that the SDH1 deletion had no effect on yeast cells grown in either of these media (data not shown). We assessed the potential impact of SDH1 deletion on metabolic profiles by comparing the amounts of the main fermentation by-products (ethanol, succinate, glycerol, acetate and pyruvate) and TCA intermediates (2-OG and hydroxyglutarate) after fermentation in MS, MA and MG media for the wild-type and sdh1 strains. No significant difference in the formation of these metabolites was observed between strains V5 and V5 sdh1 (data not shown). The wild-type behaviour of the sdh1 mutant demonstrates that the SDH complex is not active during fermentation.
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DISCUSSION |
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The SDH complex does not operate during fermentation
The non-detection of labelled malate from [3-13C]glutamate strongly indicated that the SDH complex did not operate during glucose fermentation. Consistent with this hypothesis, the growth and metabolite profile of an sdh1 mutant during fermentation were identical to those of the wild-type. Thus, the TCA pathway is interrupted at the level of the SDH complex. Consequently, succinate may be formed either by the oxidative branch of the TCA pathway or by the TCA reductive pathway, via fumarate reductase. The synthesis of succinate via a reductive pathway is thermodynamically feasible and has been suggested to occur during fermentation (Lupiañez et al., 1974; Machado et al., 1975
; Wakaï et al., 1980
). The transformation of the TCA cycle into two half-cycles running in opposite directions has been described in Escherichia coli as an adaptive response to environmental conditions (Gray et al., 1966
; Sauer et al., 1999
). In E. coli, the SDH complex is not functional under anaerobic conditions and its activity is replaced by fumarate reductase activity, which catalyses the final step of anaerobic respiration, using fumarate as a terminal electron acceptor (Iuchi & Lin, 1993
; Kroger, 1978
; Spiro & Guest, 1991
). An identical branched pathway, characteristic of anaerobic metabolism, has been demonstrated in the microaerophilic bacterium Helicobacter pylori (Pitson et al., 1999
).
Pathways for succinate formation
Several lines of evidence suggest that the reductive branch of the TCA pathway is functional up to succinate, regardless of the nitrogen source (aspartate or glutamate). First, similar 13C labelling yields were obtained for malate and succinate during [3-13C]aspartate assimilation, suggesting that these two molecules are produced by the same metabolic pathway. Second, the carbon atoms located in positions 1 and 4 of succinate derived from [3-13C]aspartate were never labelled, indicating that the formation of succinate from oxaloacetate via the oxidative branch of the pathway either does not occur or occurs at levels too low for detection. Third, inactivation of the oxidative TCA pathway by deletion of KGD1 only slightly reduced succinate yield in MA (aspartate) and MS media, and did not abolish succinate production in MG (glutamate) medium.
The second potential route for succinate production is the oxidative branch of the TCA pathway, which was not detected in labelling experiments. However, a small proportion of the succinate produced in MA and MS media is formed by this pathway, as shown by the decrease in succinate formation of the kgd1 mutant. Finally, an additional pathway from glutamate to succinate was observed in medium containing glutamate as the sole nitrogen source. Under these conditions, significantly more succinate was produced, as reported previously (Albers et al., 1996, 1998
). The detection of [2,3-13C]succinate as the only labelled compound from [3-13C]glutamate indicates that succinate was produced by means of a two-step conversion of [3-13C]glutamate, involving transformation into 2-OG by a NAD-dependent glutamate dehydrogenase, followed by oxidative decarboxylation catalysed by the OGDH complex. This reverse pathway from glutamate has been described elsewhere (Albers et al., 1998
).
Contribution of the various pathways according to nitrogen source
The contributions made by the various pathways (reductive branch, oxidative branch and reverse pathway from glutamate) vary considerably according to the nitrogen source used. However, the exact contribution of each pathway to succinate formation cannot be deduced from the NMR data because only a fraction of the succinate and malate was labelled. The low 13C incorporation yields obtained (0·2 and 0·4 for malate and succinate, respectively) are consistent with the TCA pathway continually receiving unlabelled intermediates via the anaplerotic reaction catalysed by pyruvate carboxylase. This flux was probably favoured in our conditions, due to the high concentration of glucose. Other reactions, such as the transaminations involved in amino acid synthesis or the formation of succinate via the GABA pathway, as described by Coleman et al. (2001), may contribute, to a lesser extent, to the formation of unlabelled malate and succinate. The phenotype of the kgd1 mutant provides further information for quantification of the fluxes through the various pathways. In MS and MA media, the deletion of KGD1 decreased succinate production by 25 and 40 %, respectively, indicating that the oxidative branch of the TCA pathway also produces small amounts of succinate. In contrast, in cells cultured on glutamate, the deletion of KGD1 resulted in a 72 % decrease in succinate formation, indicating that succinate is not only produced by oxidative decarboxylation of glutamate but also, to a lesser extent, by the reductive pathway. The reductive pathway produced similar amounts of succinate in MG and MS media (4 and 3·9 mM, respectively). This suggests that the reductive pathway is unaffected by nitrogen source. It has been shown that succinate is derived exclusively from glutamate (Albers et al., 1998
; Heerde & Radler, 1978
). However, it should be noted that the filiation of 14C from glucose, used by Albers et al. (1998)
, did not result in the detection of any labelled minor by-products. This may be due to the small amount of glucose initially present (2 %) or to a dilution of labelling into the major end products. However, we cannot exclude the possibility that differences in medium composition between this work and previous studies, in particular in terms of the amount of sugar present, affected the pathways of succinate production. Significantly higher levels of OGDH activity were observed in MG medium than in other media, suggesting that the excess succinate produced on glutamate is due to activation of this pathway. Hydroxyglutarate was also excreted in larger amounts into MG medium than into the other media, but to a lesser extent than reported previously (Albers et al., 1996
). Glutamate assimilation results in 2-OG accumulation (Lewis & Rainbow, 1963
). Consistent with this, higher intra- and extracellular 2-OG concentrations were obtained in MG than in MS medium. It remains to be determined whether intracellular 2-OG is the induction signal for enhanced OGDH activity. It has been suggested that intracellular 2-OG concentration may regulate PYC1, encoding one isoform of pyruvate carboxylase (Huet et al., 2000
), and might be a key signal for regulation of the retrograde response (RTG)-dependent pathways (Liu & Butow, 1999
).
Consequences for intracellular redox balance
The aforementioned findings have implications for intracellular redox balance and the formation of by-products. The formation of succinate by the reductive or oxidative branch of the TCA pathway results in the synthesis of oxidized (2 molecules of FAD per molecule of glucose) and reduced (5 molecules of NADH2 per molecule of glucose) cofactors, respectively. In the absence of significant amounts of glutamate, the reductive branch of the TCA pathway is the major route of succinate production. In particular, during wine fermentation (MS medium) 75 % of the succinate produced is generated by this pathway. This finding conflicts with the widely accepted view that succinate is formed by the oxidative branch of the TCA pathway, thereby counterbalancing the formation of reduced metabolites during glucose fermentation (Nordström, 1968; Oura, 1977
). This model should be revised, taking into account also the formation of by-products such as 2-OG, 2,3-butanediol and hydroxyglutarate, which may act as redox sinks (Albers et al., 1998
).
The importance of the reductive pathway raises questions concerning its physiological role. The formation of succinate via the reductive pathway is catalysed by an FAD-dependent fumarate reductase, as the SDH complex is inactive, and therefore results in net FAD production. The observation that the reductive pathway operates equally well on glutamate and on other nitrogen sources suggests that this pathway may play an important physiological role during fermentation. A mutant strain devoid of fumarate reductase activity was reported to be unable to grow under anaerobiosis (Arikawa et al., 1998). We also observed that growth of an osm1 frds mutant was abolished during fermentation, and that this defect was not alleviated on glutamate, which allows succinate production by oxidative decarboxylation of 2-OG (C. Camarasa, unpublished data). We therefore suggest that the reductive branch of the TCA pathway plays an essential role during fermentation, maintaining the intracellular pool of FAD.
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Received 5 February 2003;
accepted 12 May 2003.
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