Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

Authors


  • Editor: Isak Pretorius

Correspondence: Jean-Michel Salmon, UMR INRA, ‘Sciences Pour l'Oenologie’, bât 28, 2 Place Viala, 34060 Montpellier Cedex 1, France. Tel.: +33 499 61 25 05; fax: +33 499 61 28 57; e-mail: jmsalmon@supagro.inra.fr

Abstract

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Δ mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.

Introduction

Sauvignon Blanc wine is widespread all over the world. It has a characteristic aromatic profile: grapefruit and passion fruit are the most appreciated descriptors for this type of wine. The compounds responsible for this typical aroma of Sauvignon Blanc wines possess a free thiol group (Darriet et al., 1995; Tominaga et al., 1998a). Other compounds such as methoxypyrazines also play a major role in the aroma profile of this particular grape variety (Allen et al., 1991).

Sauvignon Blanc aromatic thiols are released during vinification. The yeast releases them from S-cysteine conjugate precursors initially present in the must (Tominaga et al., 1998b; Tominaga & Dubourdieu, 2000). 3-Mercapto-hexanol (3MH) and 3-mercaptohexylacetate (3MHA) are two thiols that strongly contribute to the flavor of Sauvignon white wines (Tominaga et al., 1998a). They are characterized by low perception thresholds. The R and S forms of 3MH, when in a hydroalcoholic model solution, display a slightly different flavor, reminiscent of grapefruit and passion fruit, respectively, but with similar perception thresholds (50 and 60 ng L−1, respectively, Dubourdieu et al., 2000). The two enantiomers of 3MHA, produced from acetylation of 3MH (Swiegers et al., 2005; Swiegers & Pretorius, 2007), exhibit different aromas and perception thresholds: the R form is less odoriferous (perception threshold of 9 ng L−1) and is reminiscent of passion fruit, while the S form has a more herbaceous odour of boxwood with a perception threshold of 2.5 ng L−1 (Tominaga et al., 2006). 4-Mercapto-4-methyl-pentan-2-one (4MMP), reminiscent of boxtree, has also been shown to be an important compound for Sauvignon Blanc wine aroma. Of all thiols, 4MMP has the lowest perception threshold of 0.8 ng L−1 (Darriet et al., 1995).

An understanding of the mechanisms of transformation of nonvolatile precursors into aromatic thiols may allow winemakers to improve the aromatic potential present in the grape musts. One approach is based on genetic engineering and tends to generate 4MMP and 3MH from their respective precursors S-4-(4-methylpentan-2-onel)-l-cysteine (Cys-4MMP) and S-3-(hexan-1-ol)-l-cysteine (Cys-3MH), by identifying the genes encoding enzymes with carbon–sulfur lyase activity (Darriet et al., 1995; Tominaga et al., 1995, 2006). The mechanism of 4MMP release from Cys-4MMP has been studied by deleting genes encoding putative yeast carbon–sulfur lyases (Howell et al., 2005). The results indicate the involvement of multiple genes. Overexpression of the genes thus identified does not result in an increased 4MMP release (Swiegers & Pretorius, 2007). This suggests that the main control point of thiol release in wine yeast strains might not be at the level of Cys-4MMP cleavage. More recently, the overexpression of a heterologous carbon–sulfur lyase gene in a commercial wine yeast strain allowed a significant increase of the 3MH and 4MMP concentration in model medium, resulting in an overpowering passion fruit aroma in Sauvignon Blanc wine (Swiegers et al., 2007). Other approaches focus on fermentation parameters with the aim to assist the winemakers. It has been demonstrated that the production of volatile thiols from their corresponding precursors in must depends on the wine yeast strain used to conduct the fermentation (Murat et al., 2001; Howell et al., 2004; Swiegers et al., 2006). Nevertheless, even the better thiol producers rarely transform more than 5% of the cysteinylated precursor potential initially contained in the must into thiols (Swiegers & Pretorius, 2007). Also, the impact of fermentation temperature on the concentration of volatile thiols was investigated. Even though this parameter was shown to have a significant impact, the results are quite variable depending on the fermentation scale (Masneuf-Pomarede et al., 2006; Swiegers et al., 2006).

The approach here has been to study Cys-3MH transformation during fermentation in a defined medium rather than in Sauvignon Blanc must, because nutritional parameters can be varied individually. In addition to being the precursor of 3MHA, our interest in 3MH production is due to the fact that, among the thiols present in Sauvignon Blanc wine, its perception threshold is higher. The results demonstrate the influence of the nitrogen status of the medium, identifies at least one transporter of the cysteinylated precursor and describes the properties of mutant strains insensitive to the nitrogen catabolite repression (NCR).

Materials and methods

Chemicals

Analytical reagents have been purchased from Sigma-Aldrich.

3MH, 3MH-d2, 3MHA and 3MHA-d5

3MH and 3MHA were purchased from Interchim, Montlucon (France). 3MH-d2 and 3MHA-d5 were synthesized as reported in a previous paper (Kotseridis et al., 2000).

Synthesis of Cys-3MH and Cys-3MH-d8

Cys-3MH is synthesized according to the method reported previously (Dagan, 2006) by addition of N-Boc-cysteine to (E)-hex-2-enal. Cys-3MH-d8 is also synthesized according to this method, by addition of N-Boc-cysteine to (E)-hex-2-enal-d8. (E)-hex-2-enal-d8 is synthesized from butanol-d10 following the protocol of Schneider et al. (2006).

Yeast strains

Two commercial wine yeast strains of Saccharomyces cerevisiae-IS1 and -IS2 were used in this study. IS1 mutant strains that exhibit a relief of NCR were isolated on selective media as described below.

All the S. cerevisiae laboratory strains used in this study (Iraqui et al., 1999b) are isogenic to the wild-type Σ1278b strain (Bechet et al., 1970). Their genotypes are detailed in Table 1.

Table 1.   Laboratory strains used in this study
StrainGenotypeReferences
Σ1278b ura3MATα ura3Iraqui et al. (1999b)
Σ1278b ura3 gap1ΔMATα gap1Δ∷kanMX2 ura3Iraqui et al. (1999b)

Culture media and fermentation conditions

Synthetic media and grape musts were used in this study.

The basis of the synthetic media consisted in 0.17% yeast extract without amino acid and ammonium (Difco), 10–20%d-glucose and variable nitrogen source as described below. The synthetic fermentation media used in this study are symbolized as SMi in the text, where i represents the nature of the assimilable nitrogen. Yeast nitrogen base (YNB) media and sugar were heat-sterilized (110 °C, 20 min). Stock solutions of the nitrogen source were sterilized by filtration (Ø 0.45 μm).

For experiments conducted to investigate the impact of the nitrogen source, strain IS1 was allowed to ferment on 15%d-glucose, with urea or diammonium phosphate (DAP) (5 or 10 mM) as the sole source of nitrogen (SMUrea and SMDAP , respectively). For experiments conducted to investigate the impact of GAP1 deletion, strain Σ1278b ura3 and its deletion mutant gap1Δ were allowed to ferment on 10%d-glucose, with urea or DAP (10 mM) as the sole source of nitrogen (SMUrea and SMDAP, respectively). In all cases, strains were precultured in the same medium as the one used for fermentation.

For strain IS1 and its mutants that exhibit a relief of NCR, experiments were conducted on 20%d-glucose and an available nitrogen concentration of 300 mg N/L (SMDAP+AA: 60% DAP and 40% complete amino acid mix (Luparia et al., 2004). Strains were precultured in standard nutrient medium YPD [1% yeast extract (Difco), 2% Peptone (Difco) and 2% glucose].

All precultures were conducted anaerobically for 24 h in Erlenmeyer flasks (50 mL working volume) at 28 °C with continuous shaking. Cys-3MH is added at 293 μg L−1 (which represents about 10-fold the highest concentration found in Sauvignon Blanc musts) or at a concentration specified in the Figure legend. All fermentations were conducted in handmade glass fermentors (250 mL working volume) fitted with fermentation capillary locks. The culture medium was inoculated with 2 × 106 cells mL−1 after centrifugation (1000 g for 5 min). Incubation was performed under isothermal conditions (25 °C) with shaking at 180 r.p.m. The progress of fermentation was regularly checked by weighing the fermentors.

Two Sauvignon Blanc grape musts were used: one from Languedoc of vintage 2004 [169 g L−1 of sugars, 6.15 g L−1 of total acidity (H2SO4 equivalent), 158 mg L−1 of nitrogen corresponding to c. 102 mg L−1 of assimilabe nitrogen, 6 NTU] containing 11.5 μg L−1 of Cys-3MH, and one from Gers of vintage 2006 [188 g L−1 of sugars, 4.7 g L−1 of total acidity (H2SO4 equivalent), 224 mg L−1 of nitrogen corresponding to c. 145 mg L−1 of assimilabe nitrogen, 15 NTU (2% of mires were added for fermentation)] containing 35 μg L−1 of Cys-3MH.

Experiments on grape must from Languedoc were conducted at laboratory scale in handmade glass fermentors with a working volume of 1.1 L. Fermentors were fitted with fermentation locks (CO2 bubbling outlets filled with water), and fermentations were conducted under anaerobic conditions with permanent stirring under isothermal conditions (22 °C). For inoculation, IS1 active dry yeasts (ADY) of were simply rehydrated as recommended by the manufacturer. Briefly, 2 g of dry yeast was suspended in 20 mL of warm water (37 °C) containing glucose (50 g L−1). This suspension was incubated for 30 min at 37 °C with strong agitation every 15 min. Two milliliters of this suspension were used to inoculate 1 L of the fermentation medium, giving a cell concentration of about 2 × 106 cells mL−1. The progress of fermentation progress was followed by fermenter weighing: the amount of CO2 released was determined by automatic measurement of fermenter weight loss every 20 min (Sablayrolles et al., 1987).

Fermentations of the grape must from Gers were carried out at a pilot scale, in stainless-steel tanks (100 L working volume) with fermentation locks. Fermentations were conducted under anaerobic and isothermal conditions (22 °C). The must was de-aerated at 22 °C by bubbling pure nitrogen before inoculation (initial oxygen concentration lower than 0.1 mg L−1). IS2 ADY were simply rehydrated as recommended by the manufacturer: 20 g of dry yeast was suspended in 200 mL of warm water containing 10 g of glucose. This suspension was incubated for 20 min with strong stirring every 10 min. The entire suspension was used to inoculate the 100-L fermenters, giving a cell concentration of about 4 × 106 cells mL−1. The progress of fermentation progress was followed by measurement of the released CO2 with a gas massic flowmeter, which allowed the calculation of the CO2 production rate.

Completed fermentations were clarified by centrifugation (1800 g for 10 min). Supernatants (200 mL for fermented synthetic media and 500 mL for wines) were treated for thiol extraction, immediately after fermentation for experiments at the laboratory scale, and 1 year after bottling for experiments at the pilot scale. Twenty-five milliliters of the supernatant was stored at −20 °C for further analysis of Cys-3MH. All experiments were conducted in duplicate.

Genetic methods: mutagenesis and mutant selection

In order to isolate IS1 mutant strains with a relief of NCR, the selection protocol used methylamine, d-histidine and l-citrulline. Methylamine, a susbstitute for ammonia, enters the cell through ammonium transporters and therefore induces NCR, but its accumulation is lethal for yeasts. Transport of d-histidine into yeast occurs through the only GAP1p (general amino acid permease). d-Histidine is lethal for the cell. Citrulline has only one transporter, GAP1p, and can substitute for ammonium as a nitrogen source, when NCR on GAP1 is relieved (Stanbrough et al., 1995).

Ethyl methane sulfonate (EMS) mutagenesis was performed on growing cells of IS1. After EMS mutagenesis, cells were spread at various dilutions on SMCit+Met plates (0.17% YNB without amino acids and ammonium sulfate, 2% glucose, 2% agar, 0.38% citrulline) containing twice the minimal inhibitory concentration of methylamine [2% (v/v)] to estimate mortality. About 90% of the cells were killed. To obtain the desired phenotype, the 300 survivors were screened on plates containing SMDAP+D−Hist (0.17% YNB without amino acids and ammonium sulfate, 2% glucose, 2% agar 0.2% DAP and 0.5%d-histidine). Fourteen clones exhibited no growth on this medium; they had the correct lethal phenotype. These selected clones were spread again on plates of SMDAP+D−Hist, and on plates of SMCit+Met at three different dilutions (1, 10−1, 10−2). Clones that were the most rapidly inhibited on SMDAP+D−Hist and that showed growth on SMCit+Met were then tested on liquid SMDAP+Cit (0.17% YNB without amino acids and ammonium sulfate, 2% glucose, 0.2% DAP and 0.38% citrulline) for citrulline consumption. Two clones (IS1 c1 and IS1 c2) showing a higher citrulline consumption rate than the parental strain were finally chosen.

Cell counting

Yeast culture samples are diluted with Isoton II® (Beckman-Coulter, Margency, France) for cell enumeration (1000–2500 times). After sonication (35 s, 10 W), cells were counted with an electronic Coulter counter (model Z2, Beckman-Coulter, Margency, France) fitted with a 100-μm-aperture probe.

Citrulline uptake measurements

Yeast cells were harvested during early growth on 0.17% YNB without amino acids and ammonium sulfate, 2% glucose, 0.2% DAP and 0.38% citrulline. Consumption of citrulline was determined by following citrulline concentration in the supernatant every 2 h for 8 h (Biochrom 30 amino acid analyser, Cambridge, UK).

Ammonia quantification

Quantitative analysis of ammonium ions in grape must was performed using the UV method at 340 nm with the R-Biopharm commercial test: in the presence of glutamate dehydrogenase and reduced nicotinamide-adenine dinucleotide (NADH), ammonium ions react with 2-oxoglutarate to generate l-glutamate, whereby NADH is oxidized in NAD+. The amount of NAD+ in this reaction is stoichiometric to the amount of ammonium ions. The NADH left is determined by means of its light A340 nm.

Analysis of 3MH and 3MHA

Extraction of the volatile thiols and purification of the extracts were performed according to the method reported previously (Schneider, 2001; Schneider et al., 2003) and slightly modified as follows: volatile thiols were recovered from 200 mL of fermented media by liquid–liquid extraction [with 2 × 50 mL of pentane/dichloromethane (2/1 (v/v)) azeotrope], and then purified by covalent chromatography on Affi-Gel 501 [synthesized from Affi-Gel 10 (Biorad)] as described previously (Schneider, 2001). 3MH-d2 and 3MHA-d5 were used as internal standards for stable isotope dilution assay. Typically, 500 ng L−1 of 3MH-d2 and 50 ng L−1 of 3MHA-d5 were added to 200 mL of fermented synthetic media. The final purified extracts were analyzed by GC coupled with ion trap tandem MS (GC-ITMS-MS) (Dagan, 2006). New calibration solutions were prepared for each series of experiments. The peak area ratios for the selected quantifiers were plotted against the concentration ratios. Calibration concentration ratio and absolute area value were adapted to the results obtained on the extract analysis. The SEs for quantification of 3MH and 3MHA under these conditions (extraction on 200 mL of synthetic medium, range of concentration of 0.2–10 nM for 3MH and of 0.05–0.5 nM for 3MHA) are 10% and 5%, respectively.

Analysis of Cys-3MH

Cys-3MH-d8 was added to the defrosted supernatant as internal standards (typically, 200 μg L−1 of Cys-3MH-d8 was added to 25 mL of the synthetic medium). Initial or fermented media were extracted on cation exchange Dowex resin (50wX4-100; Sigma Aldrich); derivatization of the extract with ethyl chloroformate and analysis of the final derivatized extract by GC–Electronic Impact MS (GC–ECMS) was performed as described (Dagan, 2006). Calibration curves were plotted for the target compound, i.e. natural Cys-3MH. After concentration to dryness under a nitrogen flow at 45 °C, calibration solutions containing the target compounds at serial dilutions and the fixed amount of Cys-3MH-d8 used in the samples were derivatized with ethyl chloroformate (Dagan, 2006). The peak area ratios for the selected quantifiers were plotted against the concentration ratios. Calibration concentration ratio and absolute area value were adapted to results obtained on the extract analysis.

Statistical analysis

The kyplot (version 2.15) free software (http://www.qualest.co.jp/Download/KyPlot) was used to perform anova and Tukey's tests (pairwise comparisons for one-way layout design) were used to classify the data into homogeneous groups.

Results

An experimental design strategy was used initially to assay various fermentation parameters in their effect on thiol production. Nitrogen, oxygen, sugar levels, vitamins and sterols were compared (data shown in supplementary material). Nitrogen was found to have a significant influence. This parameter was thus further investigated in more detail.

Effect of nitrogen source on cysteinylated precursors' consumption

The influence of DAP and urea, as sole nitrogen sources, on the release of 3MH and 3MHA, was compared. When Strain IS1 was grown on SMUrea and SMDAP complemented with 295 μg L−1 of Cys-3MH, its consumption was significantly different on SMUrea and SMDAP at high nitrogen concentrations (Fig. 1, P value=6.7 × 10−6). On SMUrea, there was no precursor left in the medium at the end of the fermentation contrary to SMDAP. These results suggest that, in the presence of an excess of the preferred nitrogen source such as DAP, Cys-3MH assimilatory pathways were not completely activated. It is known that a preferred nitrogen source exerts a negative effect on the expression of many genes involved in the use of nonpreferred nitrogen sources. This phenomenon is known as NCR (Dubois et al., 1973, 1974, 1977; Ter Schule et al., 1995a, b, 1998). Thus, in the presence of a preferred nitrogen source, less cysteinylated precursors are metabolized whereas in the presence of urea, as the sole source of nitrogen, NCR is not active (Dubois & Wiame, 1976; Dubois et al., 1977; Stanbrough & Magasanik, 1995; Roberg et al., 1997; Magasanik & Kaiser, 2002) and consequently more precursors were consumed.

Figure 1.

 Effect of nitrogen source on the consumption of Cys-3MH by industrial IS1 strain. IS1 strain was grown at 25°C on MSUrea (urea 5 and 10 mM) and MSDAP (DAP 5 and 10 mM) complemented with Cys-3MH. T0 represents the initial culture medium at the time of inoculation. Means and SEs of duplicate experiments are given. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

The thiol quantifications are depicted in Fig. 2 and show a significant difference between both nitrogen sources: IS1 strain produces significantly more 3MH on SMUrea than on SMDAP, especially when the nitrogen supply was not growth-limiting (at 10 mM DAP, P value=1.6 × 10−4). Qualitatively similar results were obtained with various industrial strains (data not shown). The nitrogen source also seems to have a significant impact on the release of the thiols. The effect was lower at a low nitrogen (5 mM) concentration as expected because NCR is released when the nitrogen source becomes depleted. Although increased nitrogen availability is known to increase the formation of acetate esters by enhancing the expression of the ATF1 gene (Yoshimoto et al., 2002) encoding the alcohol acetyltransferase activity responsible for the major part of volatile acetate ester production during fermentation (Mason & Dufour, 2000; Vilanova et al., 2007), no significant conversion of 3MH into its corresponding acetate ester 3MHA could explain the differences observed in 3MH releases (Fig. 2).

Figure 2.

 Effect of nitrogen source on the production of 3MH (white boxes), 3MHA (black boxes) and molar conversion yield of 3MH+3MHA (black stars) by industrial IS1 strain. IS1 strain was grown at 25°C on MSUrea (urea 5 or 10 mM) or MSDAP (DAP 5 or 10 mM) complemented with Cys-3MH. Molar conversion yields were calculated on the basis of the initial cysteinylated precursor content of the fermentation medium. Means and SEs of duplicate experiments are given. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

The molar conversion yields of Cys-3MH into 3MH, on SMUrea and SMDAP, calculated on the basis of the Cys-3MH actually consumed by the yeast, gave identical values close to 0.7% (data not shown). Thus, the actual 3MH conversion yield was constant and the absolute amount of 3MH produced was directly dependent on the amount of Cys-3MH consumed. In the absence of NCR (growth on urea), when Cys-3MH was entirely consumed, the molar conversion yields remained quite low. Nevertheless, limitation of the uptake of the cysteinylated precursor induced by the presence of a large quantity of a preferred nitrogen source also resulted in a further decrease in thiol production.

Because the GAP1 gene is known to be particularly responsive to NCR (Jauniaux & Grenson, 1990; Coffman et al., 1995, 1996), its role in volatile thiol production was further investigated.

Impact of gap1 deletion on volatile thiol production

Strain Σ1278b ura3 was chosen as the reference strain, because it has been widely used in studies on nitrogen regulation (Wiame et al., 1985), with ammonium being a preferred nitrogen source for strain Σ1278b (Dubois et al., 1973, 1974; Iraqui et al., 1999a). To investigate the role of GAP1 (general amino acid permease) in the release of volatile thiols, strains Σ1278b ura3 and its deletion mutant gap1Δ were grown on SMUrea. For comparison, strain Σ1278b ura3 was also grown on SMDAP. All fermentation media were complemented with 250 μg L−1 of Cys-3MH. The general amino acid permease (GAP1p) transports all amino acids. GAP1 expression is affected by both the availability of the nitrogen source and its quality: its transcription is repressed in glutamine or ammonia medium (preferred nitrogen sources), but also by elevated intracellular levels of glutamate or any other amino acid (Stanbrough et al., 1995; Magasanik & Kaiser, 2002; Boer et al., 2007; Godard et al., 2007). GAP1 is repressed by preferred nitrogen sources, through a sorting process in the late secretory pathway (Chen & Kaiser, 2002). Finally, GAP1p activity is high on a nonpreferred nitrogen source like urea or when the total intracellular amino acid levels are low.

Thiol production is depicted in Fig. 3. As observed with strain IS1, the 3MH and 3MHA production by strain Σ1278b ura3 was significantly lower on DAP than on urea (molar yield × 0.3, P values=0.015 and 0.009 for 3MH and 3MHA, respectively). The gap1Δ mutant produced significantly less 3MH and 3MHA than reference strain Σ1278b ura3 (molar yield × 0.6, P values=0.010 and 0.004 for 3MH and 3MHA, respectively) on urea. Still, the production of 3MH and 3MHA by strain Σ1278b ura3 on DAP was lower than the production of the mutant gap1Δ on urea (molar yield × 0.5). The absence of GAP1p limited the uptake of Cys-3MH into the cell and consequently the corresponding thiol production. This showed that the GAP1p transporter – when present – is responsible for the uptake of the major part of the precursor. GAP1p is not the only transporter, because the gap1Δ mutant still produced 3MH. This indicated that other transporters are involved in the uptake of the precursor.

Figure 3.

 Effect of GAP1 deletion on 3MH (white boxes) and 3MHA (black boxes) releases and on the molar conversion yield of 3MH+3MHA (black stars) at 25°C. Reference strain Σ1278b ura3 was grown on MSUrea and MSDAP, and the Σ1278b ura3 gap1Δ mutant strain was grown on MSUrea (all nitrogen sources at 10 mM). All media were complemented with Cys-3MH. Molar conversion yields were calculated on the basis of the initial cysteinylated precursor content of the fermentation medium. Means and SEs of duplicate experiments are given. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

If the uptake of Cys-3MH through GAP1p is limiting, relief of NCR on GAP1 may increase thiol production. This was investigated next.

Impact of the relief of NCR

IS1 mutant strains insensitive to NCR were isolated and confirmed on different media. The mutants are able to grow on citrulline (SMCit+Met), in the presence of the gratuitous NCR inducer, methylamine. In these mutants, the use of citrulline as the sole nitrogen source is completely dependent on GAP1p activity, when GAP1 is no longer subject to NCR. Candidates were double-checked for their ability to grow on SMDAP+D−Hist. The presence of a ‘constitutive’ GAP1p allows the transport of the toxic d-histidine into the cell in the presence of ammonium ions. Mutants exhibiting a lethal phenotype on SMDAP+D−Hist confirmed the relief of NCR on GAP1. Finally, 14 mutant clones of strain IS1 were isolated. Consumption of citrulline by these mutants as measured on liquid SMDAP+Cit designated two mutants of strain IS1 (IS1c1 and IS1c2) with the higher consumption rates of citrulline as compared with the parental strain (Table 2). Thus, in the presence of ammonium, the transport of amino acids through GAP1p is increased in these mutants. Consequently, the production of thiol from Cys-3MH was analyzed on SMDAP+AA complemented with 100 μg L−1 of Cys-3MH. A mixture of DAP in excess and amino acids (SMDAP+AA) was used in this case to assure strong NCR conditions. The addition of amino acids to the mixture also mimics the nitrogen content of a grape must.

Table 2.   Measurement of citrulline assimilation by IS1 wild type and IS1c1 and IS1c2 mutant strains in the presence of ammonium
 Maximum citrulline
assimilation rate
(μmol 10−9cells h−1)
Ammonium
concentration
at time of
harvest (μM)
Cell
population
at time of
harvest
(109 cells L−1)
  1. Data are expressed as the means ± SDs of three different experiments.

  2. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

IS1 wild-type industrial parental strain0.17 ± 0.02 (a)141730.1 ± 0.1
IS1c1 mutant strain1.00 ± 0.04 (b)151110.8 ± 0.2
IS1c2 mutant strain0.52 ± 0.02 (c)15786.1 ± 0.1

Mutants IS1c1 and IS1c2 produced double the amount of 3MH than the IS1 wild-type strain (Fig. 4). Interestingly, IS1c1, the strain that metabolized citrulline faster, was also the better 3MH producer. These results showed that when nitrogen-induced repression of GAP1 was abolished, the yeast could produce more 3MH and 3MHA. It is noteworthy that the molar conversion yields were lower than those obtained during the previous experiments. It is suggested that Cys-3MH uptake is reduced due to competition of the amino acids for uptake by GAP1p. Indeed, the molar ratio of cys-3MH/amino acids present is largely in disfavor for uptake of the thiol precursor.

Figure 4.

 3MH (white boxes) and 3MHA (black boxes) production and molar conversion yield of 3MH+3MHA (black stars) by wild-type strains IS1 and its corresponding mutant strains with a constitutive relief of NCR IS1c1, IS1c2. Fermentations were performed at 25°C on MSDAP+AA complemented with 100 μg L−1 Cys-3MH. Molar conversion yields were calculated on the basis of the initial cysteinylated precursor content of the fermentation medium. Means and SEs of triplicate experiments are given. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

However, the gap1Δ mutant still produced 3MH. This indicated that other transporters are involved in the uptake of the precursor.

Results obtained in grape musts

On the Sauvignon Blanc must from Gers, neither the mutant strain insensitive to NCR IS1 nor the deletion mutant gap1Δ gave significant differences in their thiol production, compared with their respective parental strains (data not shown). It seemed therefore that on actual grape must the role of GAP1 is not as important as it was observed on synthetic media.

Nevertheless, the complementation of the grape musts (from Languedoc and Gers) with 2.5 mM of DAP led to a significant decrease in thiol production [Fig. 5, P values (3MH)=0.008 and 0.006 for Languedoc and Gers musts, respectively]. In grape must, it is thus possible that NCR indeed exerts a negative effect on thiol production, but not directly related to the repression of GAP1. Further experiments on different grape musts (which contain different initial levels of nitrogen) should be conducted to investigate the possible consequences of addition of DAP in musts on thiol production.

Figure 5.

 Effect of DAP addition (2.5 mM) on the 3MH (white boxes) and 3MHA (black boxes) production and molar conversion yield of 3MH+3MHA (black stars) on actual grape musts. Two Sauvignon Blanc musts originating from Languedoc and Gers were used. Wild-type strains IS1 and IS2 were used for fermentation of grape musts from Languedoc and Gers, respectively. Fermentations were performed at 22°C as described in Materials and methods. Molar conversion yields were calculated on the basis of the initial cysteinylated precursors present in the grape musts. Means and SEs of duplicate experiments are given. The same letters in parenthesis indicate homogeneous groups at the 95% confidence level, as tested by Tukey's statistical test.

Discussion

In grape juice, the two main sources of yeast-assimilable nitrogen compounds are amino acids and ammonium ions, ammonium ions representing up to 30% of the total assimilable nitrogen fraction (Henschke & Jiranek, 1993). Because S. cerevisiae yeasts require a relatively high level of nutrients to complete a grape juice fermentation, assimilable nitrogen is a key nutrient that can become growth-limiting in some grape musts (Salmon & Barre, 1998). Insufficient nitrogen leads to lower biomass yield, which, in turn, slows the fermentation rate with an increased risk of sluggish or stuck fermentation. Therefore, in order to supplement for the nitrogen requirements, DAP is often added to the grape juice. However, only recently has the relationship between nitrogen availability and supplementation been analyzed from the perspective of the formation of volatile and nonvolatile compounds that are important for the organoleptic qualities of wine (Vilanova et al., 2007).

In wine fermentations, the cells evolve from a nitrogen-repressed situation at the beginning of the process to a nitrogen-derepressed situation as the nitrogen is consumed (Salmon & Barre, 1998). These nitrogen-repressed/derepressed conditions determine the different patterns of ammonium and amino acid consumption (Beltran et al., 2004). At the beginning of wine fermentation, GAP1 expression for example is repressed by the presence of ammonium ions in the medium. This repression can be extended throughout the whole fermentation under nitrogen-rich conditions (Beltran et al., 2004, 2005). The same research group has shown that GAP1 expression is derepressed only when the ammonium concentration is below 50 mg N L−1 (Beltran et al., 2004). Consequently, we hypothesized that prolongation of NCR by DAP addition could delay cysteinylated precursor uptake through GAP1p and that this limitation may impact the final organoleptic quality of the wine. On synthetic media and actual grape musts, we showed that addition of DAP could indeed lead to a decrease in thiol production. Nevertheless, the direct effect of GAP1 expression or derepression on thiol production from a cysteinylated precursor was only demonstrated on synthetic media. The relation between ammonium increase and thiol decrease in grape must should be further studied to identify the key step(s) on which NCR eventually acts.

However, on synthetic media and grape musts, the cysteinylated precursor potential is far from being totally used. On the synthetic media used in the present study, most molar conversion yields were below 1% at full precursor consumption. Ninety-nine percent of the consumed cysteinylated precursors were thus not transformed into the corresponding volatile thiols. On the Sauvignon Blanc musts, the molar conversion yields calculated from the Cys-3MH quantified in the musts were 7% and 25% (Languedoc and Gers grape musts, respectively). Thiol conversion yields, when mentioned in the literature, tend to be considerably lower, typically around 3% on actual grape musts (Swiegers & Pretorius, 2007). Clearly, exploitation of the aromatic potential by yeast of Sauvignon Blanc must, requires further studies.

Acknowledgements

This work was funded by Pernod-Ricard, France (CIFRE fellowship). We thank Stephan Vissers, from the Laboratoire de Physiologie Moléculaire de la Cellule (Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, Belgium), for the gift of strain Σ1278b ura3 and its mutant gap1Δ, and Stéphane Guézenec (INRA) for the isolation of mutants with a constitutive relief of NRC.

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