Complex interactions between organisms occur when fermentations are conducted with different yeasts (Fleet, 2003; Alexandre et al., 2004; Liu et al., 2015; Albergaria and Arneborg, 2016; Ciani et al., 2016). Considerable differences have been shown in the metabolism of Saccharomyces cerevisiae in single and in co-culture with non-Saccharomyces yeasts. Moreira et al. (2005) reported an increase in the quantity of desirable compounds, such as higher alcohols and esters, when S. cerevisiae was co-fermented with Hanseniaspora uvarum. A previous study (Sadoudi et al., 2012) based on the analysis of 48 volatile compounds belonging to different chemical families, highlighted the existence of different types of interactions independent of biomass production between non-Saccharomyces yeasts co-cultured with S. cerevisiae. More precisely, a positive interaction (synergistic effect) between Metschnikowia pulcherrima and S. cerevisiae resulted in a higher level of aromatic compounds than the sum of the aromatic compounds present in each monoculture. In addition, in a sequential M. pulcherrima/S. cerevisiae culture, acetic acid production was significantly lower compared to that obtained with a S. cerevisiae monoculture. Different studies reported low acetic acid production for certain non-Saccharomyces yeasts (M. pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris) and their capacity in culture with S. cerevisiae to produce lower acetic acid concentrations in comparison to S. cerevisiae monoculture (Bely et al., 2008; Comitini et al., 2011; Milanovic et al., 2012; Rantsiou et al., 2012). These studies suggest that the acetic acid metabolic pathway can be affected by interactions occurring between yeasts, leading to a decrease in the amount of acetic acid. However, little is known as yet of the impact of sequential non-Saccharomyces/S. cerevisiae culture on the genes involved in the acetic acid metabolic pathway of S. cerevisiae.

Acetic acid is the principal volatile acid of wine. It has a negative impact on yeast fermentative performance and affects the quality of some wines when present above a given concentration (Rasmussen et al., 1995). The OIV (2010) states that the maximum acceptable limit for volatile acidity for most wines is 1.2 g l^-1 of acetic acid. Unfortunately, higher levels are sometimes produced, depending on the strain (Erasmus et al., 2004; Orlić et al., 2010), on grape or must composition (Delfini and Costa, 1993) and on the winemaking process (Barbosa et al., 2009). Therefore, strains with reduced acetate production would have a high enological value. Studies on the production of volatile acidity by S. cerevisiae in winemaking conditions showed that this acid is mainly formed at the beginning of alcoholic fermentation (Alexandre et al., 2004; Bely et al., 2008). Acetic acid is formed rapidly during the fermentation of the first 50–100 g l^-1 of sugar, but part of it is metabolized by S. cerevisiae (Ribéreau-Gayon et al., 2006). This yeast can also assimilate acetic acid added at the beginning of alcoholic fermentation (Vasserot et al., 2010).

Acetic acid is a by-product of alcoholic fermentation produced via the pyruvate dehydrogenase (PDH) bypass (Figure 1). It is produced at the onset of anaerobic growth conditions, as a reducing equivalents regeneration mechanism (NADH and NADPH) essential for maintaining the redox balance (Remize et al., 2000). Enzymes involved in the PDH bypass include pyruvate decarboxylase (Pdc), acetaldehyde dehydrogenase (Ald), and acetyl-CoA synthetase (Acs) (Figure 1). The PDH complex leads to the formation of acetyl-CoA in the mitochondria through the oxidative decarboxylation of pyruvate. However, S. cerevisiae is unable to transport acetyl-CoA out of the mitochondria. Moreover, cytosolic NADP⁺-dependent Ald is active during alcoholic fermentation, while PDH activity is limited under anaerobic conditions (Remize et al., 2000). Therefore, the PDH bypass is necessary for providing acetyl-CoA in the cytosolic compartment which is used, inter alia, in lipid synthesis (for a review, see Pronk et al., 1996).

Pdc catalyzes the decarboxylation of pyruvate to acetaldehyde and carbon dioxide. In S. cerevisiae, Pdc is encoded by three structural genes, PDC1, PDC5, and PDC6 which encode Pdc1, Pdc5, and Pdc6 isoforms, respectively (Hohmann, 1991; Pronk et al., 1996). Pdc1 and Pdc5 are 88% identical (Hohmann and Cederberg, 1990). Pdc1 is the predominant isoenzyme form, performing 80–90% of the activity in cells. The Pdc6p is an active Pdc (Hohmann, 1991; Zeng et al., 1993; Baburina et al., 1994) but is not apparently involved in glucose fermentation and its role remains unclear (Hohmann, 1991). The regulatory genes PDC2, PDC3, and PCD4 encode probably positive transcriptional regulators required for high-level expression of structural PDC1 and PDC5 genes (Milanovic et al., 2012).

Ald is responsible for the conversion of acetaldehyde to acetate. The S. cerevisiae Ald family counts five isoenzymes localized in the mitochondria or the cytosol. Ald6 and Ald4 have been shown to be the main cytosolic and mitochondrial Ald, respectively. Cytosolic Ald is encoded by ALD2, ALD3, and ALD6 (occasionally named ALD1) genes and the mitochondrial enzymes are encoded by ALD4 (occasionally named ALD7) and ALD5 genes (Navarro-Aviño et al., 1999). Ald6 uses the NADP⁺ co-enzyme, activated by Mg²⁺, and is not glucose-repressed (Dickinson, 1996; Meaden et al., 1997). Ald4 uses both the NAD⁺ and NADP⁺ co-enzymes activated by K⁺ and thiols, and it is highly glucose-repressed (Jacobson and Bernofsky, 1974). Numerous studies stated that cytosolic Ald is responsible for the formation of acetate from glucose and that mitochondrial enzymes are involved during growth on ethanol or glycerol as carbon sources (Saigal et al., 1991; Wang et al., 1998). Remize et al. (2000) showed that a strain deleted in the ALD6 gene led to a considerable decrease in acetate yield. The absence of Ald6p was compensated by mitochondrial isoforms, involving the transcriptional activation of the ALD4 gene (Saint-Prix et al., 2004). More recently, it was demonstrated that the fermentation stress response gene AAF1 regulates acid acetic production under standard laboratory conditions. This gene encodes a probable transcription factor, containing a C2-H2 zinc finger domain at the N-terminus. Indeed, AAF1 regulates the expression of ALD4 and ALD6 (Walkey et al., 2012). The deletion of this gene significantly reduced acetic acid levels without increasing the acetaldehyde concentration in wine (Luo et al., 2013).

Acs catalyzes the formation of acetyl-CoA from acetate. S. cerevisiae contains two structural genes ACS1 and ACS2, each encoding an active Acs (Van den Berg et al., 1996). It has been shown that Acs is an essential enzyme in S. cerevisiae. A disruption of both ACS1 and ACS2 genes is lethal (Van den Berg and Steensma, 1995).

An imbalance of reduction equivalents at the beginning of S. cerevisiae growth in must, due to the initial lack of alcohol dehydrogenase, triggers another mechanism: glycerol production (Gancedo and Serrano, 1989) (Figure 1). Dihydroxyacetone phosphate, the substrate for the glycerol formation pathway, can be provided either by the glycolytic degradation of sugar or by gluconeogenic flux when non-fermentable carbon sources are used (Nevoigt and Stahl, 1997). Dihydroxyacetone phosphate is converted to glycerol-3-phosphate, which is an intermediate for glycerol formation. Two homologous genes GPD1 and GPD2 encode the isoenzymes glycerol-3-phosphate dehydrogenase (Gpd). GPD1 expression is induced by osmotic stress. The repressor/activator Rap1p was demonstrated to be an important determinant of induced transcriptional activities of the GPD1 promoter (Eriksson et al., 2000). Expression of GPD2 is not affected by changes in external osmolarity, but it is stimulated by anoxic conditions (Ansell et al., 1997). A recent study by Pérez-Torrado et al. (2016) showed the induction of GPD1 after the first hour of growth in wine fermentation conditions for different Saccharomyces species. For the GPD2 gene, the time and the level of induction seem to be species- or strain-dependent. Moreover, some strains do not seem to activate this gene which presents very low mRNA levels.

In the present study, we performed sequential fermentations, combining M. pulcherrima and S. cerevisiae strains, in order to evaluate the effect of the presence of M. pulcherrima on the production of acetic acid and glycerol during alcoholic fermentation. Moreover, the impact of this sequential culture on the expression of genes in S. cerevisiae encoding enzymes involved in acetic acid and glycerol pathways during alcoholic fermentation was investigated.

The early inoculation of M. pulcherrima MCR 24 did not compromise the growth of S. cerevisiae PB2023, preventing the risk of a sluggish or a stuck alcoholic fermentation. Moreover, the M. pulcherrima population dropped after the inoculation of S. cerevisiae and no viable cells were detected after 8 days (Figure 2B). Such an antagonistic effect has been reported previously (Jolly et al., 2003; Rodríguez et al., 2010; Comitini et al., 2011; Sadoudi et al., 2012). This result could not be linked to intolerance to ethanol concentration, since we previously demonstrated that the MCR 24 strain can produce approximately 10% v/v ethanol (Sadoudi et al., 2012). According to Nguyen and Panon (1998), the antagonistic effect could be attributed to killer toxins. Another explanation is the interaction occurring between both yeasts, mediated by the cell–cell contact mechanism (Nissen and Arneborg, 2003) or competition between yeasts for the nutrients available in the must. S. cerevisiae PB2023 grew faster than M. pulcherrima MCR 24 and thus it could impoverish the medium. Sequential inoculation did not affect the ethanol level in the wine despite the death of M. pulcherrima. On the other hand, it induced a significant increase in glycerol content and a decrease in acetic acid concentration (Figure 3). These data confirm the benefits of using M. pulcherrima prior the inoculation of the S. cerevisiae starter, in accordance with previous results (Bely et al., 2008; Comitini et al., 2011), but they do not explain the positive impact of M. pulcherrima on S. cerevisiae metabolism.

All previous analytical data suggest that the metabolic pathways could be rerouted by interactions occurring between both yeasts during alcoholic fermentation. During the latter, acetic acid is produced via the cytosolic PDH bypass. In aerobic conditions, the PDH complex leads to the formation of acetyl-CoA in the mitochondria by oxidative decarboxylation of pyruvate. However, in fermentative conditions, the conversion of pyruvate to acetyl-CoA can occur via an indirect route, involving Pdc (which is also a key enzyme in alcoholic fermentation), Ald and Acs. This bypass route is the source in the cytosolic compartment of acetyl-CoA, which is used for lipid synthesis and acetate which can be precursor of volatile esters.

The production of glycerol involves the reduction of dihydroxyacetone phosphate derived from the glycolytic degradation of sugar. The NAD⁺-dependent Gpd catalyzes the first step in glycerol production. This metabolism also permits the regeneration of reducing equivalents (NADH), more particularly at the beginning of S. cerevisiae growth in fermentative conditions.

The over-expression of PDC1 and PDC5 encoding two isoforms of Pdc and the ALD6 gene encoding cytosolic aldehyde dehydrogenase (Figures 4B,C) leads to the assumption of an over production of acetic acid by-product, which appears inconsistent with the analytical data which shows that acetate was reduced in mixed culture condition. One explanation could be due to the conversion of acetate into acetyl-CoA used in other metabolic pathways such as lipid synthesis or esterification related to the production of esters. Indeed, we previously observed higher levels of acetate esters in Sauvignon wine from a M. pulcherrima/S. cerevisiae sequential culture (Sadoudi et al., 2012). However, it is clear that acetyl-CoA was not transported into mitochondria since the CAT2 gene encoding carnitine acetyltransferase was under-expressed in sequential culture condition (Figure 4E).

The lower acetate production could not be due ethanol production since the ethanol contents are comparable under the two fermentation conditions. Another hypothesis that could explain our analytical data is that a part of dihydroxyacetone phosphate is used for glycerol production at the beginning of fermentation. Glycerol can be produced mostly at the beginning of fermentation in response to hyper osmotic conditions (high concentration in sugars). Moreover, anaerobic conditions require the production of endogenous electron acceptors and glycerol production can serve as a redox valve to eliminate excess reducing power in S. cerevisiae (Ansell et al., 1997). The M. pulcherrima strain MCR 24 may have depleted oxygen in the must during sequential culture, since it was inoculated 48 h before S. cerevisiae. Oxygen depletion (anaerobiotic conditions) could explain the modulation of glyceropyruvic fermentation and the orientation of metabolism to the PDH bypass, leading to the production of acetate and glycerol. These metabolism orientations are necessary to maintain the redox balance by regenerating NAD and NADH co-enzymes. Furthermore, increased glycerol formation requires an equimolar amount of cytoplasmic NADH. This requirement could be satisfied by a lower reduction of acetaldehyde to ethanol on the one hand and an increase in oxidation to acetate on the other (Blomberg and Adler, 1989; Nevoigt and Stahl, 1997). Therefore an increase in acetate production is usually accompanied by an increase in glycerol formation; however, a high levels of glycerol is not necessarily accompanied by high levels of acetic acid or acetaldehyde (Remize et al., 2000).

Independently of the expression of genes involved in acetate and glycerol production pathways, we hypothesized the possible consumption by M. pulcherrima MCR 24 of part of the acetate produced by S. cerevisiae in sequential culture fermentation. We performed a mono-culture with the M. pulcherrima MCR 24 strain using standardized grape juice supplemented with 1.5 g l^-1 of acid acetic and observed the consumption of 0.57 g l^-1 of the initial acetic acid during 8 days of fermentation (data not shown).

This work is the first attempt to investigate M. pulcherrima and S. cerevisiae yeast–yeast metabolic interaction, reflected by gene expression in the acetic acid and glycerol production pathway in S. cerevisiae during controlled sequential fermentation in winemaking. The environmental changes in must induced by the presence of M. pulcherrima induced the alteration of the entire acetic acid and glycerol metabolic pathway of S. cerevisiae.

Future accession to the M. pulcherrima genome may provide very interesting investigative leads on the nature of interactions occurring in sequential fermentations at the transcriptomic level.

MS designed the experiments, analyzed the data, and wrote the manuscript. SR analyzed the data and wrote the manuscript. VD analyzed the data. HA and RT-M supervised the study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.