RESEARCH ARTICLE
Nitrogen catabolite repression modulates the production of
aromatic thiols characteristic of Sauvignon Blanc at the level of
precursor transport
Maeva Subileau1, Rémy Schneider2, Jean-Michel Salmon2 & Eric Degryse1
1
Centre de Recherches de Pernod Ricard, Créteil Cedex, France; and 2UMR INRA, ‘Sciences Pour l’Oenologie’, Montpellier Cedex 1, France
Received 18 January 2008; revised 22 April
2008; accepted 30 April 2008.
First published online 11 June 2008.
DOI:10.1111/j.1567-1364.2008.00400.x
Editor: Isak Pretorius
Keywords
Saccharomyces cerevisiae ; volatile thiols;
fermentation; S -3-(hexan-1-ol)-L-cysteine
(Cys-3MH); GAP1 .
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 gap1D 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, Cys3MH 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-Mercaptohexanol (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,
FEMS Yeast Res 8 (2008) 771–780
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-4methyl-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
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
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Correspondence: Jean-Michel Salmon, UMR
INRA, ‘Sciences Pour l’Oenologie’, bât 28, 2
Place Viala, 34060 Montpellier Cedex 1,
France. Tel.: 133 499 61 25 05; fax: 133 499
61 28 57; e-mail: jmsalmon@supagro.inra.fr
772
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).
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
Table 1. Laboratory strains used in this study
Strain
Genotype
References
S1278b ura3
S1278b ura3
gap1D
MATa ura3
MATa gap1D<kanMX2 ura3
Iraqui et al. (1999b)
Iraqui et al. (1999b)
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-2enal-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 S1278b
strain (Bechet et al., 1970). Their genotypes are detailed in
Table 1.
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 1C,
20 min). Stock solutions of the nitrogen source were sterilized by filtration (Ø 0.45 mm).
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 S1278b ura3
and its deletion mutant gap1D 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 (SMDAP1AA:
60% DAP and 40% complete amino acid mix (Luparia et al.,
FEMS Yeast Res 8 (2008) 771–780
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precursors S-4-(4-methylpentan-2-onel)-L-cysteine (Cys4MMP) 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).
M. Subileau et al.
773
Precursor transport and aromatic thiols production
FEMS Yeast Res 8 (2008) 771–780
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 1C 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 Lcitrulline. 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 SMCit1Met 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 SMDAP1D 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 SMDAP1D Hist, and on plates of
SMCit1Met at three different dilutions (1, 10 1, 10 2). Clones
that were the most rapidly inhibited on SMDAP1D Hist and
that showed growth on SMCit1Met were then tested on liquid
SMDAP1Cit (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 IIs (BeckmanCoulter, Margency, France) for cell enumeration (1000–2500
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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 1C with
continuous shaking. Cys-3MH is added at 293 mg 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 1C) 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 mg 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 mg 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 1C). 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 1C) containing glucose (50 g L 1). This suspension
was incubated for 30 min at 37 1C 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 1C).
The must was de-aerated at 22 1C 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
774
times). After sonication (35 s, 10 W), cells were counted
with an electronic Coulter counter (model Z2, BeckmanCoulter, Margency, France) fitted with a 100-mm-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
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.
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
Analysis of Cys-3MH
Cys-3MH-d8 was added to the defrosted supernatant as
internal standards (typically, 200 mg 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 1C, calibration solutions containing the target compounds at serial dilutions and the fixed amount of Cys3MH-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 mg 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,
FEMS Yeast Res 8 (2008) 771–780
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Quantitative analysis of ammonium ions in grape must was
performed using the UV method at 340 nm with the RBiopharm 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
NAD1. The amount of NAD1 in this reaction is stoichiometric to the amount of ammonium ions. The NADH left is
determined by means of its light A340 nm.
M. Subileau et al.
Precursor transport and aromatic thiols production
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.
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 growthlimiting (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)
FEMS Yeast Res 8 (2008) 771–780
Fig. 2. Effect of nitrogen source on the production of 3MH (white
boxes), 3MHA (black boxes) and molar conversion yield of 3MH13MHA
(black stars) by industrial IS1 strain. IS1 strain was grown at 25 1C 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.
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).
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 Cys3MH 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.
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Fig. 1. Effect of nitrogen source on the consumption of Cys-3MH by
industrial IS1 strain. IS1 strain was grown at 25 1C 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.
775
776
M. Subileau et al.
Impact of gap1 deletion on volatile thiol
production
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 (SMCit1Met), 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
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
Fig. 3. Effect of GAP1 deletion on 3MH (white boxes) and 3MHA (black
boxes) releases and on the molar conversion yield of 3MH13MHA (black
stars) at 25 1C. Reference strain S1278b ura3 was grown on MSUrea and
MSDAP, and the S1278b ura3 gap1D 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.
grow on SMDAP1D 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 SMDAP1D 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 SMDAP1Cit 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 Cys3MH was analyzed on SMDAP1AA complemented with
100 mg L 1 of Cys-3MH. A mixture of DAP in excess and
amino acids (SMDAP1AA) 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.
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.
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Strain S1278b 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 S1278b (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
S1278b ura3 and its deletion mutant gap1D were grown on
SMUrea. For comparison, strain S1278b ura3 was also grown
on SMDAP. All fermentation media were complemented with
250 mg 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 S1278b
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 gap1D mutant produced significantly less
3MH and 3MHA than reference strain S1278b 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 S1278b ura3 on DAP was lower than the
production of the mutant gap1D 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
gap1D mutant still produced 3MH. This indicated that other
transporters are involved in the uptake of the precursor.
If the uptake of Cys-3MH through GAP1p is limiting,
relief of NCR on GAP1 may increase thiol production. This
was investigated next.
777
Precursor transport and aromatic thiols production
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
(mmol 10 9cells h 1)
IS1 wild-type 0.17 0.02 (a)
industrial
parental strain
IS1c1 mutant 1.00 0.04 (b)
strain
IS1c2 mutant 0.52 0.02 (c)
strain
Ammonium
concentration
at time of
harvest (mM)
Cell
population
at time of
harvest
(109 cells L 1)
1417
30.1 0.1
1511
10.8 0.2
1578
6.1 0.1
Data are expressed as the means SDs of three different experiments.
The same letters in parenthesis indicate homogeneous groups at the
95% confidence level, as tested by Tukey’s statistical test.
However, the gap1D 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 gap1D gave significant differences in their thiol
production, compared with their respective parental strains
(data not shown). It seemed therefore that on actual grape
FEMS Yeast Res 8 (2008) 771–780
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.
Discussion
In grape juice, the two main sources of yeast-assimilable
nitrogen compounds are amino acids and ammonium ions,
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
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Fig. 4. 3MH (white boxes) and 3MHA (black boxes) production and
molar conversion yield of 3MH13MHA (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 1C on MSDAP1AA
complemented with 100 mg 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.
Fig. 5. Effect of DAP addition (2.5 mM) on the 3MH (white boxes) and
3MHA (black boxes) production and molar conversion yield of
3MH13MHA (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 1C 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.
778
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd. No claim to original French government works
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 S1278b ura3 and
its mutant gap1D, and Stéphane Guézenec (INRA) for the
isolation of mutants with a constitutive relief of NRC.
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Supplementary material
The following supplementary material for this article is
available online:
Appendix S1. Experimental design strategy to determine the
effect of various fermentation parameters on thiol production.
Table S1. Description of the high (1) and low ( ) levels of
the fermentation parameters and their respective contents in
the eight fermentation media, based on SMust, used for the
experimentation.
Table S2. Amino acids stock solution.
Table S3. Vitamins stock solution.
Fig. S1. 3MH (white boxes) and 3MHA (black boxes)
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different synthetic media.
Fig. S2. Quantification of the effect of five controllable
fermentation parameters (oxygen (white circles), sugar
(black stars), ammonium (black squares), vitamins (black
diamonds) and sterols (black triangles) on 3MH production
by strain ES7 at 22 1C, following the fractional factorial
design.
This material is available as part of the online article
from: http://www.blackwell-synergy.com/doi/abs/10.1111/
j.1567-1364.2008.00400.x (this link will take you to the
article abstract).
Please note: Blackwell Publishing is not responsible for
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material) should be directed to the corresponding author for
the article.
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