# Quantitative 13 C-isotope labelling-based analysis to elucidate the influence of environmental parameters on the production of fermentative aromas during wine fermentation

Stéphanie Rollero, Jean-Roch Mouret, Audrey Bloem, Isabelle Sanchez, Anne Ortiz-Julien, Jean-Marie Sablayrolles, Sylvie Dequin, Carole Camarasa

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## To cite this version:

Stéphanie Rollero, Jean-Roch Mouret, Audrey Bloem, Isabelle Sanchez, Anne Ortiz-Julien, et al. Quantitative 13 C-isotope labelling-based analysis to elucidate the influence of environmental parameters on the production of fermentative aromas during wine fermentation. Microbial Biotechnology, 2017, 14 p. 10.1111/1751-7915.12749. [hal-01606296](https://hal.science/hal-01606296)

HAL Id: hal-01606296

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Submitted on 26 May 2020

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# Quantitative ¹³C-isotope labelling-based analysis to elucidate the influence of environmental parameters on the production of fermentative aromas during wine fermentation

Stéphanie Rollero,<fnref n="1" />,<fnref n="2" />† Jean-Roch Mouret,<fnref n="1" />

Audrey Bloem,<fnref n="1" /> Isabelle Sanchez,<fnref n="1" />

Anne Ortiz-Julien,<fnref n="2" /> Jean-Marie Sablayrolles,<fnref n="1" />

Sylvie Dequin<fnref n="1" /> and Carole Camarasa<fnref n="1" />,*

## Summary

Nitrogen and lipids are key nutrients of grape must that influence the production of fermentative aromas by wine yeast, and we have previously shown that a strong interaction exists between these two nutrients. However, more than 90% of the acids and higher alcohols (and their acetate ester derivatives) were derived from intermediates produced by the carbon central metabolism (CCM). The objective of this study was to determine how variations in nitrogen and lipid resources can modulate the contribution of nitrogen and carbon metabolisms for the production of fermentative aromas. A quantitative analysis of metabolism using ¹³C-labelled leucine and valine showed that nitrogen availability affected the part of the catabolism of N-containing compounds, the formation of α-ketoacids from CCM and the redistribution of fluxes around these precursors, explaining the optimum production of higher alcohols occurring at an intermediate nitrogen content. Moreover, nitrogen content modulated the total production of acids and higher alcohols differently, through variations in the redox state of cells. We also demonstrated that the phytosterol content, modifying the intracellular availability of acetyl-CoA,

can influence the flux distribution, especially the formation of higher alcohols and the conversion of α-ketoisovalerate to α-ketoisocaproate.

## Introduction

Controlling the quality of wines is a major issue in the winemaking sector: in an increasingly competitive market, a key objective is to best meet consumer expectations in terms of the organoleptic profile of the product (Lee and Noble, 2003; Swiegers et al., 2005). Therefore, one of the current major challenges is to optimize the quality of wine, including its properties on a sensory level. The aroma of a wine is defined by a complex mixture of molecules of different origins: varietal aromas and their precursors coming from grapes, fermentative aromas synthesized by yeast and postfermentative aromas released during the ageing of wines. In this study, we are particularly interested in fermentative aroma compounds, which include molecules belonging to different chemical families (higher alcohols, acetate esters, medium-chain fatty acids and ethyl esters). Their syntheses involve a highly interconnected metabolic network (Swiegers et al., 2005). These volatile compounds can be produced from very different metabolic pathways, e.g. central carbon metabolism (CCM), lipid metabolism or amino acid catabolism (nitrogen metabolism); furthermore, some of these fermentative aromas are derived from common precursors, while others share enzymes involved in their metabolic routes (e.g. higher alcohols). In accordance with this complexity, it was shown that the production of fermentative aromas depends on many environmental factors (assimilable nitrogen, availability of lipids, temperature, etc.; Hernandez-Orte et al., 2006; Varela et al., 2012; Mouret et al., 2014; Rollero et al., 2015).

Several studies have assessed the influence of assimilable nitrogen content on the production of fermentative aromas (reviewed in Bell and Henschke, 2005). Generally, a direct relationship between initial nitrogen content and higher alcohol concentrations was observed when the nitrogen content remained low, whereas at moderate to high nitrogen quantities, there was an inverse relationship (Jiménez-Martí et al., 2007; Vilanova et al., 2007, 2012; Carrau et al., 2008; Mouret et al., 2014; Rollero

Received 13 March, 2017; revised 28 April, 2017; accepted 24 May, 2017.

*For correspondence. E-mail carole.camarasa@inra.fr;
Tel. (33) 4 99 61 23 36; Fax (33) 4 99 61 28 57.

†Present address: Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Private Bag X1, Matrieland 7602, South Africa.

Microbial Biotechnology (2017) 0(0), 000–000
doi:10.1111/1751-7915.12749

## Funding information

No funding information provided.

<footnote n="1">¹UMR SPO: INRA, Université Montpellier, Montpellier
SupAgro, 34060 Montpellier, France.</footnote>

<footnote n="2">²Lallemand SAS, 31700 Blagnac, France.</footnote>
et al., 2015). Acetates and ethyl esters showed a more
simple relationship with the nitrogen concentration: an
increase in initial nitrogen content was associated with an
increase in ester production (Hernandez-Orte et al., 2006;
Garde-Cerdán and Ancín-Azpilicueta, 2008; Ugliano
et al., 2010; Torrea et al., 2011; Mouret et al., 2014; Roll-
ero et al., 2015). Conversely, the influence of lipids and
sterols on the synthesis of these molecules has been little
studied. Sterols and fatty acids not only are essential
components for maintaining the integrity of the yeast
membrane but also are involved as precursors in the syn-
thesis of certain aroma compounds (Swiegers et al.,
2005; Styger et al., 2011; Varela et al., 2012). When
exogenous unsaturated fatty acids are available in abun-
dance, cells incorporate them into their membrane, caus-
ing a significant reduction in the de novo synthesis of fatty
acids. Accordingly, the production of aroma compounds
such as ethyl esters and isoamyl acetate is reduced
(Yunoki et al., 2005). These observations were confirmed
by Saerens et al. (2008), who showed that the addition of
unsaturated fatty acids into fermentation medium causes
a decrease in the production of ethyl esters and acetate
esters. This is consistent with the ability of ergosterol to
inhibit the expression of the ATF1 gene, which is the main
gene coding for the alcohol acetyltransferase responsible
for the production of the majority of acetate esters (Mal-
corps and Dufour, 1992; Fujii et al., 1997; Fujiwara et al.,
1998). Moreover, it has been reported that an increase in
availability of phytosterols, which are unsaturated fatty
acids found in grape must, seemed to favour the forma-
tion of higher alcohols but caused a decrease in the pro-
duction of esters (Rollero et al., 2015).

In this context, to define strategies to modulate the
organoleptic characteristics of wines, it is necessary to
better understand the effect of environmental parameters
on the metabolic origins of fermentative aromas. These
aspects are difficult to capture only by monitoring the
metabolites produced, and isotopic filiation approaches
have been identified as one of the tools of choice to
deepen metabolic knowledge (Blank et al., 2005; Frick
and Wittmann, 2005; Christen and Sauer, 2011). An
overview of the management of nitrogen by yeast during
conventional wine fermentation has recently been pro-
vided from an experimental design based on a combina-
tion of ¹³C and ¹⁵N tracer experiments. This approach
revealed that the incorporation of exogenous amino
acids in biomass was limited in favour of de novo syn-
thesis under the study conditions. In addition, the cata-
bolism of amino acids plays a minor role in the formation
of volatile compounds, with the α-ketoacids precursors
required for these neo-syntheses mainly originating from
the carbon metabolism (Crépin et al., 2017).

The objective of this study was to determine how varia-
tions in nitrogen and lipid resources can modulate the

contributions of both nitrogen and carbon metabolisms to
the production of fermentative aromas using filiation
experiments with ¹³C-labelled nitrogen sources. Under-
standing this relationship provided deeper knowledge on
how environmental factors (initial nitrogen and phytosterol
content) can influence the contribution of nitrogen and
carbon metabolisms to the production of fermentative aro-
mas.

Results

To assess the distribution of nitrogen sources in pro-
teinogenic amino acids and volatile compounds, we used
a quantitative approach based on stable isotope tracer
experiments with amino acids labelled on their carbon
skeleton. This ¹³C labelling provides information on the
amino acid fractions directly used for biomass formation
and those leading to the formation of fermentative aro-
mas, which would not be possible using a ¹⁵N-labelling
strategy. Leucine and valine, which are precursors of the
most abundant higher alcohols in wines – isoamyl alco-
hol and isobutanol, respectively – and which have inter-
connected metabolic pathways, were used in this study.

To study the effect of environmental conditions on the
flux partitioning in the whole metabolic network, fermen-
tations were carried out with a different initial nitrogen
(70, 250 or 425 mg I⁻¹) and phytosterol (2 or 8 mg I⁻¹)
content using a commercial wine yeast strain, Lalvin
EC1118®. The different nitrogen concentrations were
made up by a mixture of ammonium and amino acids,
that kept the same proportion. For each fermentation,
only a single nitrogen compound was supplied as a
labelled molecule, either valine or leucine. These amino
acids, each accounting for 1.3% of total yeast assimil-
able nitrogen, are fully consumed by yeasts during fer-
mentation, regardless of nitrogen availability (between
70 to 425 mg N I⁻¹). Because nitrogen sources are
sequentially assimilated during the growth phase of wine
fermentation (Crepin et al., 2012), we investigated the
flux partitioning at different stages of the culture corre-
sponding to a partial (N₁/₂ and N₃/₄) or complete con-
sumption of the nitrogen content (Nₜ) and at the end of
fermentation (EF). We measured the ¹³C isotopic enrich-
ment of proteinogenic amino acids and of volatile com-
pounds (higher alcohols, their acetate esters and acids)
that may be synthesized from leucine and valine through
the Ehrlich pathway (Hazelwood et al., 2008).

Origin of the carbon backbone of proteinogenic valine
and leucine

In the beginning, we quantified the fraction of valine
and leucine present in the biomass that originates
directly from exogenous amino acids. First, in the case
of both amino acids, we observed a substantial imbal-
ance early in the growth phase between the level of
consumption of the exogenous amino acids and the
content of these compounds in proteins that was unre-
lated to the nitrogen and phytosterol content (Figs 1
and 2). For example, the concentration of leucine in the
medium was between 0.060 and 0.367 mmol l⁻¹, while
the content of this amino acid in the biomass varied
between 0.230 and 0.903 mmol l⁻¹ for SM70 and
SM425 respectively. Overall, the proteinogenic amino
acid content in cells was up to three to four times
higher than the assimilated content (Figs 1 and 2). In
accordance with these data, at the end of the growth
phase (when the nitrogen was completely consumed),
the isotopic enrichment of each amino acid that corre-
sponded to the amount directly originating from the

exogenous compound (Figs 3–6, Tables SD1 and SD2)
was lower than 35% at all nitrogen concentrations. All
these observations indicate that the *de novo* synthesis
of amino acids from the CCM plays an important role in
fulfilling anabolic demand, even in a nitrogen-rich envi-
ronment. These observations generalize the observa-
tions made by Crépin *et al.* (2017) for only one content
of assimilable nitrogen to a wider range of N availabil-
ity. Furthermore, unexpectedly and under all environ-
mental conditions, the direct incorporation of consumed
amino acids in proteins was limited, despite the extent
of anabolic requirements (at least two to three times
higher than the concentration provided in the medium).
This finding suggests that an important fraction of the
consumed amino acids was catabolized by the yeast.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Comparison of the concentration (µM) of consumed valine (green) and proteinogenic valine from *de novo* synthesis (blue) and from direct incorporation of consumed valine (pink) at the four stages of fermentation (N₁/₂, N₃/₄, Nₜ and EF) in SM70 (A), SM250 (B) and SM425 (C) at 2 mg/l phytosterols.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Comparison of the amount (µM) of consumed leucine (green) and proteinogenic leucine from *de novo* synthesis (blue) and from direct incorporation of consumed leucine (pink) at the four stages of fermentation (N₁/₂, N₃/₄, Nₜ and EF) in SM70 (A), SM250 (B) and SM425 (C) at 2 mg l⁻¹ phytosterols.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Flux partitioning around the valine metabolism at 2 mg l⁻¹ phytosterols and three different levels of assimilable nitrogen. The distribution of fluxes around these metabolic routes was investigated using isotopic fission experiments with ¹³C-labelled valine. Portion of consumed valine recovered in proteinogenic leucine and valine: the labelled fraction (light colour) corresponds to the consumed valine directly incorporated into proteins, while the unlabelled fraction (dark colour) represents the proteinogenic amino acids *de novo* synthetized from CCM precursors. Portion of the valine consumed converted into volatile compounds: the labelled fraction (light colour) corresponds to the fraction of volatile compounds synthetized using carbon from consumed valine, while the unlabelled fraction (dark colour) represents the portion of volatile molecules synthesized from α-ketoacids through the CCM. Flux partitioning involved in the use of valine during fermentation: the fraction of consumed valine catabolized through a certain pathway was assessed from the molar ratios between the amount of a proteinogenic amino acid or volatile compound labelled and the total amount of consumed amino acid. Isotopic enrichments (defined as the molar ratio between the quantity of labelled compound and the total quantity) are shown above each bar plot. The raw data and details of the calculations are provided in the Supplemental Material. Blue: SM70; orange: SM250; pink: SM425.</figcaption></figure>

In agreement with this hypothesis, ¹³C labelling was detected in leucine and in isobutanol and isoamyl alcohol in cultures with labelled valine (Figs 3 and 5, Tables SD1 and SD2).

Furthermore, regardless of the environmental conditions, we observed a greater decrease during the growth phase in the isotopic enrichment of leucine (a two- to threefold reduction), which was imported into the cells early on (Jiranek et al., 1995; Crepin et al., 2012), compared with that of valine (from 14% to 11%), which was consumed later (Tables SD1 and SD2). These

observations revealed that the fraction of consumed amino acids directly recovered from the biomass was related to their sequential consumption.

### Impact of environmental parameters on the direct recovery of consumed leucine and valine in proteins

Overall, the partitioning between the fractions of exogenous amino acids directly incorporated in biomass and the part that is further catabolized was not influenced by phytosterol availability (Figs 3–6, Tables SD1 and SD2).
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Flux partitioning around the leucine metabolism at 2 mg l⁻¹ phytosterols and three different levels of assimilable nitrogen. The distribution of fluxes around these metabolic routes was investigated using isotopic fission experiments with ¹³C-labelled leucine. Portion of consumed leucine recovered in proteinogenic leucine: the labelled fraction (light colour) corresponds to the consumed leucine directly incorporated into proteins, while the unlabelled fraction (dark colour) represents the proteinogenic amino acids *de novo* synthesized from CCM precursors. Portion of consumed leucine converted into volatile compounds: the labelled fraction (light colour) corresponds to the fraction of volatile compounds synthesized using carbon from consumed leucine, while the unlabelled fraction (dark colour) represents the portion of volatile molecules synthesized from α-ketoacids through the CCM. Flux partitioning involved in the use of leucine during fermentation: the fraction of consumed leucine catabolized through a certain pathway was assessed from the molar ratios between the amount of a proteinogenic amino acid or volatile compound labelled and the total amount of consumed amino acid. Isotopic enrichments (defined as the molar ratio between the quantity of labelled compound and the total quantity) are shown above each bar plot. The raw data and details of the calculations are provided in the Supplemental Material. Blue: SM70; orange: SM250; pink: SM425.</figcaption></figure>

By contrast, we found that this distribution differed on the basis of the initial nitrogen content. For both amino acids (leucine and valine), we observed that the amount of labelled amino acid directly incorporated in proteins at the end of the growth phase was higher in SM250 and SM425 than in SM70 (Figs 3–6, Tables SD1 and SD2).

Nevertheless, the fraction directly recovered in the biomass also depended on the nature of the amino acid. In SM250 and SM425, the amount of labelled leucine in proteins was quite similar to the amount consumed (70–80%), while only 40% of the valine consumed was found in the biomass. In SM70, the exogenous leucine in the
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Flux partitioning around the valine metabolism at 8 mg l⁻¹ phytosterols and three different levels of assimilable nitrogen. The distribution of fluxes around these metabolic routes was investigated using isotopic fission experiments with ¹³C-labelled valine. Portion of consumed valine recovered in proteinogenic leucine and valine: the labelled fraction (light colour) corresponds to the consumed valine directly incorporated into proteins, while the unlabelled fraction (dark colour) represents the proteinogenic amino acids *de novo* synthesized from CCM precursors. Portion of consumed valine converted into volatile compounds: the labelled fraction (light colour) corresponds to the fraction of volatile compounds synthesized using carbon from consumed valine, while the unlabelled fraction (dark colour) represents the portion of volatile molecules synthesized from α-ketoacids through the CCM. Flux partitioning involved in the use of valine during fermentation: the fraction of consumed valine catabolized through a certain pathway was assessed from the molar ratios between the amount of a proteinogenic amino acid or volatile compound labelled and the total amount of consumed amino acid. Isotopic enrichments (defined as the molar ratio between the quantity of labelled compound and the total quantity) are shown above each bar plot. The raw data and details of the calculations are provided in the Supplemental Material. Blue: SM70; orange: SM250; pink: SM425.</figcaption></figure>

biomass accounted for approximately 40% of the consumed amount and around 20–30% for valine.

The non-negligible and constant (approximately 4–5%) isotopic enrichment of leucine during fermentation in the presence of labelled valine (Figs 3 and 5) – by contrast, no valine enrichment was observed in the presence of labelled leucine (Figs 4 and 6) – is consistent with the existence of a leucine biosynthetic pathway from the carbon backbone of valine. Moreover, the fraction of valine catabolized through this metabolic route varied depending on the initial nitrogen content: in SM70 and SM425, 42–45% of consumed valine was converted into α-

ketoisocaproate, while only 35–36% was metabolized in SM250.

*Impact of environmental parameters on the metabolic origin of higher alcohols*

Low isotopic enrichments of isobutanol (between 5% and 15%) and isoamyl alcohol (between 2% and 8%) were found in the presence of labelled valine and leucine irrespective of the initial nitrogen and phytosterol content (Figs 3–6). These enrichments decreased throughout the fermentation process (Tables SD1 and
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Flux partitioning around the leucine metabolism at 8 mg I⁻¹ phytosterols and three different levels of assimilable nitrogen. The distribution of fluxes around these metabolic routes was investigated using isotopic fission experiments with ¹³C-labelled leucine. Portion of consumed leucine recovered in proteinogenic leucine: the labelled fraction (light colour) corresponds to the consumed leucine directly incorporated into proteins, while the unlabelled fraction (dark colour) represents the proteinogenic amino acids *de novo* synthesized from CCM precursors. Portion of consumed leucine converted into volatile compounds: the labelled fraction (light colour) corresponds to the fraction of volatile compounds synthesized using carbon from consumed leucine, while the unlabelled fraction (dark colour) represents the portion of volatile molecules synthesized from α-ketoacids through the CCM. Flux partitioning involved in the use of leucine during fermentation: the fraction of consumed leucine catabolized through a certain pathway was assessed from the molar ratios between the amount of a proteinogenic amino acid or volatile compound labelled and the total amount of consumed amino acid. Isotopic enrichments (defined as the molar ratio between the quantity of labelled compound and the total quantity) are shown above each bar plot. The raw data and details of the calculations are provided in the Supplemental Material. Blue: SM70; orange: SM250; pink: SM425.</figcaption></figure>

SD2), indicating a strong contribution of the CCM to the
formation of the ketoacid precursors of higher alcohols,
which increased as the exogenous amino acids were
depleted. The relationship between nitrogen content and
higher alcohol concentration was not monotonic: there
was a direct relationship at low nitrogen content

(between 70 and 250 mg I⁻¹), whereas an inverse rela-
tionship was found at moderate to high nitrogen concen-
trations (from 250 to 425 mg I⁻¹). It was also interesting
to note that the isotopic enrichment in isobutanol and
isoamyl alcohol increased with the initial nitrogen con-
tent, even if the fraction of consumed amino acids
recovered in these volatile compounds decreased. All of
these results suggested that the contribution of ketoacids
originating from CCM to the synthesis of higher alcohols
varied according to the initial nitrogen content of the
medium, with a higher contribution under conditions of
nitrogen limitation. We also observed an enrichment of
isoamyl alcohol in fermentations conducted with
¹³C-valine (Figs 3 and 5). This finding confirmed that a
part of the valine consumed was transformed into the
α-ketoisocaproate precursor of isoamyl alcohol through
the CCM.

Overall, the phytosterol availability modulated the for-
mation of higher alcohols (Figs 3–6). For example, in
SM425 at the lower phytosterol content, the concentra-
tion of isobutanol was 0.339 mM, while at 8 mg l⁻¹ phy-
tosterols, it was 0.540 mM. However, the extent of the
response to changes in the phytosterol availability also
depended on other factors, such as the nature of the
higher alcohol and the nitrogen content. Thus, an
increase of approximately 20% in the formation of isoa-
myl alcohol occurred regardless of the initial nitrogen
content, while the increase in the amount of isobutanol
synthesized with respect to the phytosterol availability
varied from approximately 46–47% (SM70-SM250) to
59% (SM425). The comparison between the labelling pat-
terns of volatile compounds produced at low and high
phytosterol content revealed the origin of the carbon
backbone used to increase the formation of higher alco-
hols. We first observed that the amount of isobutanol pro-
duced from labelled valine tended to increase together
with the concentration of phytosterols: in SM425, this
amount was equal to 0.052 and 0.065 mM at 2 and
8 mg l⁻¹ phytosterols respectively (Figs 3 and 5). This
finding indicated that a larger fraction of valine was
catabolized towards higher alcohols in the presence of
high phytosterol content. However, the decrease in the
isotopic enrichment of isobutanol with the increase in ini-
tial phytosterol content reflected a higher increase in the
total formation of this compound than that of its labelled
form. Consequently, the synthesis of α-ketoisovalerate
through the CCM appears to be the major contributor to
the increase in the isobutanol production in response to
changes in phytosterol availability. By contrast, no sub-
stantial modifications were observed in the isotopic
enrichment of isoamyl alcohol from labelled leucine
between fermentations with high or low initial phytosterol
content. For this compound, increased phytosterol
availability resulted in an increase in its flux of produc-
tion without affecting the relative contributions of the
CCM and of the catabolism of leucine into ketoacid pre-
cursors. These observations suggest that different regu-
latory effects triggered by the phytosterol availability
control the biosynthetic pathways of these two higher
alcohols.

Impact of environmental parameters on the metabolic
origin of acetate esters and acids

As observed in the case of the higher alcohols, low iso-
topic enrichments of acetate esters (7% maximum) were
found in the presence of labelled valine and leucine irre-
spective of the initial content of nitrogen and phytos-
terols. Overall, the isotopic enrichment of isoamyl
acetate was largely similar to that measured in its higher
alcohol precursor (Figs 4 and 6). This finding is consis-
tent with the fact that acetate esters can be produced
from only higher alcohols without additional contributions
from other pathways. A similar trend emerged for isobu-
tyl acetate, but it was more difficult to observe because
the concentrations were very low; therefore, the assays
were less accurate. However, the formation of acetate
esters and their corresponding higher alcohols was
affected differently by the fermentative parameters
(Figs 3–6, Tables SD1 and SD2). The concentration of
acetate esters increased together with the nitrogen con-
tent in the medium (e.g. at 2 mg l⁻¹ phytosterols, isoa-
myl acetate was at a concentration of 18 µM in SM70
and 141 µM in SM425), as previously reported in the lit-
erature. Regarding the impact of the phytosterol content,
a low lipid content was found to be more favourable for
the production of acetate esters. This information sug-
gests that the production of acetate esters is more
dependent on the enzymatic activity of alcohol acetyl-
transferases (Atf1 and Atf2) than on the availability of
higher alcohols. Indeed, a high lipid content downregu-
lated the expression of the *ATF1* gene (Saerens *et al.*,
2008).

Comparing the isovaleric and isobutyric acid concen-
trations in relation to the nitrogen and phytosterol
availability clearly highlighted the ability of these envi-
ronmental parameters to control the production of acids
(Figs 3–6). Overall, the production of acids decreased
together with the increase in initial nitrogen content,
except for the increase in isobutyric acid formation
between SM70 and SM250. By contrast, the impact of
increasing the initial phytosterol concentration (2 mg l⁻¹)
was dependent on nitrogen availability, with a decrease
in acid production at low nitrogen content (SM70) and
an increase in their formation in SM250 and SM425.
The production of isobutyric acid exhibited greater vari-
ations than the production of isovaleric acid. Moreover,
it is noteworthy that the response to changes in nutrient
availability with regard to the production of acids dif-
fered from that of the synthesis of higher alcohols, as
described above. Regarding the isotopic enrichments,
the labelling incorporation found in acids presented the
same pattern as was observed in the case of the
higher alcohols: the isotopic enrichments remained low
and tended to increase together with the nitrogen
content (from 5% to 11% in SM70 and SM425, respec-
tively), but we did not observe a significant impact of
the lipid dose.

Together, these results are consistent with the fact
that both acids and their corresponding higher alcohols
were produced from the same ketoacids (Hazelwood
et al., 2008), with a further distribution of these alcohol
precursors towards acids depending on the nutrient com-
position of the medium.

Finally, we observed an enrichment of isovaleric acid
in fermentations conducted with ¹³C-valine (Figs 3 and
5), confirming the conversion of a part of the consumed
valine into the α-ketoisocaproate intermediate.

Discussion

In yeast, α-ketoacids play a key metabolic role at the
interface between the CCM and the nitrogen metabolism.
In fact, these intermediates may both originate from the
CCM and be directly derived from the amino acid cata-
bolism via transamination. Furthermore, they have been
reported to be the main precursors in the anabolism of
amino acids (Albers et al., 1996) and the starting point in
the synthesis of branched acids, higher alcohols and
their acetate derivatives through the Ehrlich pathway
(Hazelwood et al., 2008). Under defined conditions
(180 mg l⁻¹ assimilable nitrogen and 2 mg l⁻¹ ergos-
terol), a recent study has elucidated the fluxes dis-
tributed around the α-ketoacids that are the metabolic
origin of these compounds – relative to the contribution
of CCM and amino acids catabolism – and their alloca-
tion (Crépin et al., 2017). In this work, we demonstrated
how the partitioning around two α-ketoacids nodes,
α-ketoisovalerate and α-ketoisocaproate, varies depend-
ing on two environmental factors identified as modulators
of biomass formation and fermentative aroma production:
nitrogen and phytosterol availability.

First, our data underlined that the important contribu-
tion of catabolism to the fate of consumed amino acids –
a feature that was unexpected until recently (Crépin
et al., 2017) – was found regardless of the nitrogen and
phytosterol content in the medium. However, we
observed that only a small fraction of higher alcohols
were synthetized using the carbon skeletons of amino
acids. This could be explained by the fact that the quan-
tity of amino acids consumed is often much lower than
the amount of the corresponding higher alcohol pro-
duced. Consequently, most of these volatile compounds
originated from the backbones of compounds produced
through the CCM.

Concerning the influence of the nitrogen content, part
of the catabolism of N-containing compounds increased
with the decrease in nitrogen availability. This finding is
particularly relevant for leucine, one of the most

abundant amino acids in biomass (Lange and Heijnen,
2001). In media containing moderate to high concentra-
tions of nitrogen, direct incorporation into biomass
accounted for approximately 70% of the consumed leu-
cine. This behaviour, which is specific to this branched
amino acid, may be explained by (i) its poor ability to
support yeast growth as the sole nitrogen source (God-
ard et al., 2007), and (ii) its early importation into cells
through the SPS-controlled transporters Bap2p and
Bap3p (Iraqui et al., 1999; Gaber et al., 2003), which
occurs before the initiation of the redistribution of nitro-
gen from consumed amino acids to the de novo synthe-
sis of N-containing compounds. Conversely, in nitrogen-
poor medium, the direct incorporation of exogenous leu-
cine into biomass was severely limited (30%). It has
recently been demonstrated that during growth in a com-
plex nitrogen resource, yeasts catabolized a large por-
tion of the consumed amino acids to provide an
intracellular nitrogen pool that was further used in combi-
nation with CCM precursors for the de novo synthesis of
proteinogenic amino acids corresponding to anabolic
requirements (Crépin et al., 2017). Our latter observa-
tions suggested that to overcome nitrogen deficiency,
yeast strongly favours the catabolism of all the available
amino acids to provide this intracellular pool of nitrogen
for improved management of a limited nitrogen resource.

Surprisingly, the total production of higher alcohols
changed differently depending on nitrogen availability, as
it exhibited a maximal level at the moderate dose of
nitrogen (250 mg l⁻¹), in agreement with previous stud-
ies (Jiménez-Martí et al., 2007; Vilanova et al., 2012;
Mouret et al., 2014; Rollero et al., 2015). From the inte-
grated analysis of carbon mass balances and labelling
patterns, an explanatory model elucidating the effect of
the nitrogen availability on the redistribution of fluxes
around the α-ketoisovalerate and the α-ketoisocaproate
nodes could be proposed. When the nitrogen resource is
limited (70 mg l⁻¹, Fig. 7A), a substantial part of the
consumed amino acids is catabolized to provide intracel-
lular nitrogen; the subsequent release of α-ketoacids
combined with the low anabolic requirements for leucine
and valine results in a limited formation of the α-ketoiso-
valerate and α-ketoisocaproate intermediates from CCM
and a moderate production of higher alcohols, which
removes the surplus of α-ketoacids precursors. When
the nitrogen content increases from 70 to 250 mg l⁻¹
(250 mg l⁻¹, Fig. 7B), the synthesis of α-ketoacids
through the CCM increases to fulfil the greater require-
ments of these precursors for anabolism (three times
higher). This response caused an excess of α-ketoacids,
which results in a marked increase in the flux towards
the formation of higher alcohols. Finally, for a greater
increase in nitrogen content, and thus a greater increase
in the anabolic demand (425 mg l⁻¹, Fig. 7C), the
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Distribution of fluxes leading to the formation of higher alcohols on the basis of the initial nitrogen content: SM70 (A), SM250 (B) and SM425 (C).</figcaption></figure>

intracellular α-ketoacids were to a large extent directed towards the synthesis of amino acids at the expense of higher alcohol formation, which results in a decreased flux in the formation of aromas compared with the previous conditions. Further analyses of changes in the intracellular α-ketoacids content according to assimilable nitrogen availability should help to support and strengthen our hypothesis. Nevertheless, in any case, this view is in agreement with the nitrogen-dependent regulation of the biosynthetic pathways of amino acids mediated by the derepressor Gcn4: the accumulation of uncharged tRNAs, which especially occurs in nitrogen-starved cells, results in the activation of *GCN2* that mediates the expression of *GCN4* (Hinnebusch, 1997; Tate et al., 2017). In our context, the increase in protein formation to sustain yeast growth with nitrogen availability could modify the balance between synthesis and use of tRNAs, and thus activate the *GCN4*-mediated derepression of genes responsible for the biosynthesis of amino acids, providing an excess of α-ketoacid intermediates.

It is noteworthy that the isotopic enrichment profiles were similar between higher alcohols and their corresponding branched acids. This is consistent with the existence of shared precursors for the synthesis of these compounds. However, nitrogen availability affected the total production of acids and higher alcohols differently. The redox state of cells may explain this differential response, as the formation of both these compounds involves many dehydrogenases (Schoondermark-Stolk et al., 2005). The activity of these enzymes depends on redox cofactor availability, which in turn depends on the amount of biomass produced, which requires the consumption of NADPH and NAD⁺. In nitrogen-poor medium, the growth is limited, resulting in an excess of NAD⁺ that can be regenerated through the synthesis of acids via the Ehrlich pathway (Hazelwood et al., 2008). In agreement with this, Bloem et al. (2015) have shown that a perturbation in the availability of redox cofactors resulted in a decrease in the production of these volatile compounds.

The impact of phytosterols on the biosynthetic pathways of higher alcohols and acids was more complex: more alcohols were produced overall at high phytosterol content, but no real impact on acids was detected. Our results also highlighted the interaction between nitrogen and lipids having a more pronounced effect on isobutanol than on isoamyl alcohol. Moreover, the quantitative analysis of flux distributions in the metabolic network revealed that α-ketoisovalerate was largely directed towards the synthesis of α-ketoisocaproate, with its conversion into isobutanol and isobutyric acid accounting for less than one-third of the total flux. As a consequence, the different responses of the formation of isobutyl derivatives and isoamyl compounds to nitrogen and lipid availability may be explained by the regulating effect of the synthesis of α-ketoisocaproate from α-ketoisovalerate. This synthesis involves *LEU4*, *LEU2* and *LEU1*, which are controlled by the complex Leu3p-α-isopropylmalate, which in turn is sensitive to the level of nitrogen (Kohlhaw, 2003). Thus, it can first act as an inhibitor in leucine-rich medium or as an activator when α-isopropylmalate (an intermediate in the biosynthesis of leucine) accumulates in medium deficient in leucine (Sze et al., 1992). In addition, the expression of *LEU3* is regulated by the general amino acid control system mediated by the transcription factor Gcn4 (Zhou et al., 1987), which also controls the expression of genes involved in the biosynthetic pathway of leucine (*ILV3*, *LEU4*, *BAT1* and *BAT2*) (Hsu et al., 1982; Hu and Kohlhaw, 1995). Finally, differences in the responses of isoamyl alcohol and isobutanol production have previously been reported in the case of redox perturbation, where the availability of acetyl-CoA was modified according to the redox status of the cell (Celton et al., 2012). These differences were explained by the modulation of the conversion of α-ketoisovalerate and α-ketoisocaproate by the intracellular content of acetyl-CoA, which is required for the first step of this metabolic pathway (Roeder and Kohlhaw, 1980). Thus, a change in the availability of acetyl-CoA triggered by modifications in the phytosterol content
could explain why the response to change was more sig-
nificant for the formation of isobutyl compound than for
the production of isoamyl derivatives.

Overall, this study highlights the potential of isotopic
filiation approaches to provide new insight into the meta-
bolism of nitrogen-containing and aroma compounds. A
future challenge will be to predict and to adjust the for-
mation of aroma compounds, which remains a complex
task due to the fact that their production depends not
only on the amount and nature of nutrients but also on
the yeast strain used to perform the fermentation. The
combination of the knowledge gained at the intracellular
level of kinetics data, such as specific rates and dynam-
ics of production, will pave the way for the development
of an explanatory, dynamic and predictive model of the
production of fermentative aromas.

**Experimental procedure**

Yeast strains, fermentation conditions and sampling

All experiments in this study were performed with the commercial wine strain *Saccharomyces cerevisiae* Lalvin EC1118® (Lallemand SA, Montreal, Canada). Fermentation flasks were inoculated with 10 g l⁻¹ active dry yeast that had been previously rehydrated for 30 min at 37°C in a 50 g l⁻¹ glucose solution (1 g of dry yeast diluted in 10 ml of this solution).

Fermentations were carried out in 330 ml reactors equipped with fermentation locks to maintain anaerobiosis, obtained by bubbling argon into the medium at 24°C with continuous magnetic stirring (150 rpm). Cultures were performed in a synthetic medium (SM) that simulates standard grape juice (Bely et al., 1990) and is characterized by a low pH (3.3) and high sugar content (100 g l⁻¹ glucose and 100 g l⁻¹ fructose). The nitrogen source was composed of ammonium chloride and amino acids (Table SD3). SM was initially supplemented with two different concentrations of phytosterols (85451, Sigma-Aldrich, Saint-Louis, MO, USA): 2 and 8 mg l⁻¹, to satisfy the lipid requirements of yeast cells during anaerobic growth. The stock solution was composed of 15 g l⁻¹ phytosterols in a mixture of Tween 80 and ethanol (1:1, v/v). The labelled nitrogen sources used were obtained from Euriso-top® (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA): L-valine (U-¹³C₅, 97-98%, CLM-2249) and L-leucine (U-¹³C₆, 97-99%, CLM-2261).

Each condition was tested in duplicate. By measuring
the change in weight throughout the process, the
amount of CO₂ released was determined and the fer-
mentation progress was monitored (Rollero *et al*., 2015).
Samples (2 aliquots of 6 ml) were taken throughout the
fermentation process at stages corresponding to partial
(¹/₂ and ¾) or complete consumption of the nitrogen

resource and at the end of fermentation, which were
referred to as N₁/₂, N₃/₄, N_T and EF respectively. Cells
were pelleted by centrifugation (2000 × g, 5 min and
4°C) and washed twice with distilled water. Supernatants
and cell pellets were stored at -20°C until analysis.

Quantification of consumed and proteinogenic amino
acids

Dry weights were determined by weight difference. Culture (10 ml) was filtered through preweighed nitrocellulose filters (pore size 0.45 µm, Millipore, Molsheim, France). The filters were twice washed with 20 ml of distilled water and dried at 105°C for 48 h before weighing (i.e. until no further change in weight was observed).

Residual ammonium ions in the supernatant were
assayed spectrophotometrically using an Enzytec™ kit
(5380, Enzytec™ Grosseron SAS, Coueron, France)
according to the manufacturer's instructions. Before
quantifying the residual amino acids, molecules with high
molecular weights were removed from the supernatants
by the addition of one volume of 25% (w/v) sulfosalicylic
acid solution to four volumes of sample, followed by
incubation at 4°C for 1 h. After centrifugation (4°C,
10 min, 3000 × g), the sample was filtered through a
0.22-µm pore-size Millipore nitrocellulose membrane.
The amino acid concentrations were determined using a
specific amino acid analyser (Biochrom 30, Biochrom,
Holliston, MA, USA) combining ion-exchange chromatog-
raphy and spectrophotometric detection after reaction
with ninhydrin, as previously described (Crepin *et al.*,
2012).

We used the protein fraction of the biomass and the
relative concentrations of amino acids within proteins
previously determined by Crépin *et al.* (2017). The per-
centage of each amino acid in the proteins was further
calculated from these data by dividing the measured
amount of each amino acid (mg l⁻¹) by the total amount
of amino acids measured in the protein extract (sum in
mg l⁻¹). To assess the concentration of each proteino-
genic amino acid in the culture (mg l⁻¹), these percent-
ages were then multiplied by the concentration of total
protein in the culture (mg l⁻¹), which was the product of
the protein content of the biomass and the dry weight.

Measurement of the isotopic enrichment of intracellular
amino acids

Biomass hydrolysis. The determination of the isotopic
enrichment of proteinogenic amino acids was performed
using 1–2 mg dried biomass prehydrolysed in 1200 µl of
6 M HCl for 16 h at 105°C. The sample, after the
addition of 800 µl of distilled water, was centrifuged at
3000 × g for 5 min to remove cellular debris. The
supernatant was distributed into four 400 µl fractions, which were further dried at 105°C until they reached a syrup-like state (4–5 h). These fractions were then utilized for amino acid derivatization using two different agents.

The *ethylchloroformate (ECF) derivatization* procedure was modified from Christensen and Nielsen (1999). The dried hydrolysate was dissolved in 200 µl of 20 mM HCl and 133 µl of a pyridine–ethanol mixture (1:4). The amino acids in this mixture were derivatized by adding 50 µl of ECF. The derivatives were extracted into 500 µl of dichloromethane. For analysis, the upper organic phase was collected after 4 min of centrifugation at 9000 × g and injected directly into a GC/MS system.

The *N,N-dimethylformamide dimethyl acetal (DMF/DMA)* derivatization procedure was modified from Christensen and Nielsen (1999). The dried hydrolysate was dissolved in 50 µl of methanol and 200 µl of acetonitrile, and the mixture was derivatized by adding 300 µl of DMF/DMA. The extract was directly injected into the GC/MS.

**GC-MS analysis.** Samples were then analysed with a Hewlett Packard 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a CTC Combi PAL Autosampler AOC-5000 (Shimadzu, Columbia, SC, USA) and coupled to an HP 5973 mass spectrometer. The instrument was controlled and the data were analysed using HP G1701DA ChemStation software. The gas chromatograph was fitted with a 30 m × 0.25 mm DB-17 ms column with a film thickness of 0.15 µm (Agilent Technologies). Compounds obtained from the ECF and DMF/DMA derivatizations were separated using the analytical conditions described by Crépin *et al.* (2017).

The MS was operated in selected ion monitoring (SIM) mode with positive ion electron impact at 70 eV using the characteristic ions of amino acid fragments reported in Crépin *et al.*, (2017). For each amino acid fragment, the outcome of the analysis was a cluster of intensities corresponding to its different mass isotopomers. These data were subsequently processed using the IsoCor software recently developed by Millard *et al.* (2012) to correct for natural labelling and to assess the isotopic enrichment of each amino acid, which is defined as the fraction of labelled compound with respect to its total amount in proteins (expressed in percentage).

## Quantification and isotopic enrichment of volatile compounds

The labelled volatile compounds were extracted according to the method described by Rollero *et al.* (2015), using dichloromethane after the addition of deuterated

internal standards. The extracted volatile molecules were separated using a Hewlett Packard 6890 gas chromatograph (Agilent Technologies) equipped with a 30 m × 0.25 mm Phenomenex ZB-WAX-fused silica capillary column with a 0.25 µm film thickness (Agilent Technologies) and helium as the carrier gas using the procedure previously described by Rollero *et al.* (2015). Compounds were detected using an HP 5973 mass spectrometer in SIM mode with positive ion electron impact at 70 eV. For the quantification and the determination of the labelling patterns of volatile compounds we used the ion clusters reported in Crépin *et al.* (2017). These ion clusters were selected on the basis of their high signal-to-noise ratio and low interference from other compounds. The concentration of each volatile molecule was quantified from the sum of the intensities of the corresponding ion cluster. In parallel, for each ion clusters, the intensities were corrected for natural labelling using *isocor* software (Millard *et al.*, 2012) and processed to assess to the isotopic enrichment of volatile compounds, which is defined as the fraction of labelled molecule with respect to its total production (expressed in percentage).

## *Outline of calculations for flux quantification*

The labelled fraction of a proteinogenic amino acid or a volatile compound was calculated by multiplying its concentration in mM by its isotopic enrichment. The difference from the total amount of the compound corresponded to the unlabelled part. Fluxes in the metabolic reactions involved in the synthesis of a target compound (proteinogenic amino acid or volatile molecule) from a labelled nitrogen source were quantified by dividing the labelled fraction of the compound by the total amount of consumed labelled molecule in mM (Tables SD1 and SD2).

## Conflict of Interest

None declared.

## Acknowledgments

This work was supported by the French Ministry of Higher Education and Research (grant CIFRe 2012/0086).

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**Supporting information**

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<caption>**Table S1.** Summary of the data set obtained during fermentation with ¹³C valine.</caption>

**Table S2.** Summary of the data set obtained during fermentation with ¹³C leucine.

**Table S3.** Percentage of yeast assimilable nitrogen provided by each nitrogen sources.