Jean-Michel Salmon

Determination of the relative ploidy in different Saccharomyces cerevisiae strains used for fermentation and 'flor' film ageing of dry sherry-type wines.

The full chromosomal karyotype of six enological Saccharomyces cerevisiae strains used for fermentation and biological ageing of sherry‐type wines was studied. A genetic method based on the analysis of segregation frequencies of auxotrophic markers, among random spore progeny of hybrids, constructed between laboratory and industrial wine strains (Bakalinsky and Snow, 1990) was used. This method was combined with the analysis of strains by pulsed‐field gel electrophoresis. The results obtained clearly indicate the presence of two, three or four copies of a chromosome in the industrial strains examined, and thus confirm that aneuploidy/polyploidy is not uncommon in these strains. In all strains examined, chromosome XIII polysomy is observed. This chromosome contains the ADH2 and ADH3 loci, that code for the ADHII and ADHIII isoenzymes of alcohol dehydrogenase, which are involved in ethanol oxidative utilization during biological ageing of wines. Tetrad analysis for the ‘flor formation’ character suggests two possibilities: this character is either regulated by at least a digenic system, or by only one gene present on a chromosome which is, at least, disomic.© 1997 John Wiley & Sons, Ltd.

Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics

ABSTRACT The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.

DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

SummaryA gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol

Investigations into physiological aspects of glycerol conversion to dihydroxyacetone (DHA) by Gluconobacter oxydans ATCC 621 were made. The activity levels of the enzymes involved in the three catabolic pathways previously known and the effects of specific inhibitors and uncoupling agents on cellular development, DHA synthesis, and cellular respiratory activity were determined. It was established that only two catabolic pathways are involved in glycerol dissimilation by this micro-organism. The only enzyme responsible for DHA production is membrane-bound glycerol dehydrogenase, which employs oxygen as the final acceptor of reduced equivalents without NADH mediation. The ketone is directly released into the culture broth. As the glycolytic and carboxylic acid pathways are absent, the pathway provided by the membrane-bound enzyme is indispensable for the energy requirements of G. oxydans. The cytoplasmic pathway, which begins by phosphorylation of glycerol followed by a dehydrogenation to dihydroxyacetone phosphate, allows growth of the bacterium. At the same time, the substrate transport mode was characterized as facilitated diffusion using radioactive [1(3)-3H]-glycerol. Concerning the DHA inhibition of microbial activity, the enzymatic study of the membrane-bound glycerol dehydrogenase showed the enzymatic origin of this phenomenon: a 50% decrease of the enzyme activity was observed in the presence of 576 mm DHA. The decrease in the rate of penetration of glycerol into cells in the presence of DHA indicates that growth inhibition is essentially due to the high inhibition exerted by the ketone on the substrate transport system.

Oxygen addition and sterol synthesis in Saccharomyces cerevisiae during enological fermentation.

Under anaerobic conditions, yeast growth normally requires oxygen in order to favour the synthesis of sterols and unsaturated fatty acids. However, in such conditions, superfluous oxygen consumption by yeast cells is observed. The superfluous oxygen consumed by the yeast cells appears to be not related to classical respiration, but mainly to the operation of several alternative oxygen consumption pathways. In this study, the potential relationship between this superfluous oxygen consumption and the yeast sterol synthesis pathway was investigated during enological fermentation. Additions of small (7 mg l(-1)) and excess (37 mg l(-1)) amounts of oxygen at the end of cell growth phase were used as a method of comparing oxygen consumption by normal synthetic pathways with that by alternative respiration pathways. The superfluous oxygen consumption by yeast cells during fermentation seemed not to alter and strongly favoured fermentation kinetics and cell biomass formation. However, a marked decrease of the orderliness of the membrane phospholipids is observed, which is not related to the drop of cell viability. After oxygen additions, squalene contents of the cells decreased, while the relative proportions of ergosterol or its precursors in the total sterol fraction did not correlatively increase. It was further found that an oxygen-dependent sterol degradation occurred when oxygen was added in excess amounts with respect to the cellular requirements for sterol synthesis. At present, this modification of the sterol contents of yeast membranes has not been related to any physiological parameters.

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

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 gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.

Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions

ABSTRACT Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.

Enological fermentation kinetics of an isogenic ploidy series derived from an industrial Saccharomyces cerevisiae strain

Abstract We used a heat-induced endomitotic polyploidization procedure to construct an isogenic ploidy series (2N to 4N) from an industrial strain of Saccharomyces cerevisiae . The nature of the yeast ploidy series observed in this work indicates that the physical and metabolic differences observed among the strains in the yeast ploidy series must be due to differences in gene dosage alone and not to heterosis. We then investigated the fermentation kinetics of the yeast ploidy series on simulated standard grape juice under enological conditions. Cell specific CO 2 production rates appeared to be as high as cell ploidy increases. Cell specific CO 2 production rates relative to the cytoplasmic volume appeared to be surprisingly different among strains within the ploidy series. We further related this phenomenon to the fact that cell specific CO 2 production rates relative to cell plasma membrane surface areas were equal for all the strains tested. The results obtained indicate that, although the normal balance of gene copy numbers was maintained for all enzyme-driven metabolic processes of strains within the ploidy series, a metabolic bottleneck seemed to occur at the plasma membrane level.

Early thiamin assimilation by yeasts under enological conditions: Impact on alcoholic fermentation kinetics

The effect of early must thiamin depletion by wild yeast strains (i.e. Kloeckera and Saccharomyces species) on alcoholic fermentation kinetics was studied. Experimental conditions affecting thiamin assimilation by yeasts were first determined, using factorial designs. Then sequential and/or mixed cultures simulating wild yeast contamination, as observed during winemaking, were carried out in order to study the influence of early thiamin depletion on fermentation kinetics. The obtained results indicate that biological thiamin depletion leads to slow fermentation and may lead to sluggish or stuck fermentation. Moreover this phenomenon is amplified in media with high assimilable nitrogen contents. No release of thiamin from contaminant yeast and subsequent assimilation by fermentative yeast was observed, indicating that some enological practices (such as centrifugation of musts contaminated early) may lead to stuck fermentation.

New Precursor of 3‑Mercaptohexan-1-ol in Grape Juice: Thiol- Forming Potential and Kinetics during Early Stages of Must Fermentation

Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are key aroma impact compounds in many young white wines, especially of the variety Sauvignon blanc (SB). Although great effort has been invested to identify their precursors in recent years, the origin of the majority of 3MH and 3MHA generated during wine fermentation still cannot be explained. Here we demonstrate that supplying an external source of hydrogen sulfide to grape juice hugely increases its thiol-forming potential. We further describe the discovery of (E)-2-hexen-1-ol as an additional new thiol precursor and demonstrate that it possesses, together with (E)-2-hexenal, an immense thiol-forming potential during fermentation. Both C6-compounds are extremely rapidly metabolized by yeast during the first hours after inoculation, even under commercial conditions, and can be interconverted during this phase depending on their initial concentration in the grape juice. Spiking grape juice with additional acetaldehyde greatly enhanced the (E)-2-hexen-1-ol to (E)-2-hexenal conversion rate. Delaying the metabolization of the two unsaturated C6-thiol precursors by yeast, at the same time as increasing hydrogen sulfide production early in fermentation, opens up a great opportunity to tap into this enormous potential 3MH and 3MHA source in grape juice and extends the possibility of thiol production to other non-grape-based alcoholic beverages as well.