The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3‐phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol‐3‐phosphatase were found when low and high glycerol‐forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose‐rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by‐products such as pyruvate, acetate, acetoin, 2,3 butane‐diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate bySaccharomyces cerevisiaeduring alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with thePDC1gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressingACS2and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies ofALD6, encoding the cytosolic Mg²⁺-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion ofALD4, encoding the mitochondrial K⁺-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, theald6 ald4double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of theALDfamily.

Among other fermentation processes, lactic acid fermentation is a valuable process which enhances the safety, nutritional and sensory properties of food. The use of starters is recommended compared to spontaneous fermentation, from a safety point of view but also to ensure a better control of product functional and sensory properties. Starters are used for dairy products, sourdough, wine, meat, sauerkraut and homemade foods and beverages from dairy or vegetal origin. Among lactic acid bacteria, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Pediococcus are the majors genera used as starters whereas Weissella is not. Weissella spp. are frequently isolated from spontaneous fermented foods and participate to the characteristics of the fermented product. They possess a large set of functional and technological properties, which can enhance safety, nutritional and sensory characteristics of food. Particularly, Weissella cibaria and Weissella confusa have been described as high producers of exo-polysaccharides, which exhibit texturizing properties. Numerous bacteriocins have been purified from Weissella hellenica strains and may be used as bio-preservative. Some Weissella strains are able to decarboxylate polymeric phenolic compounds resulting in a better bioavailability. Other Weissella strains showed resistance to low pH and bile salts and were isolated from healthy human feces, suggesting their potential as probiotics. Despite all these features, the use of Weissella spp. as commercial starters remained non-investigated. Potential biogenic amine production, antibiotic resistance pattern or infection hazard partly explains this neglecting. Besides, Weissella spp. are not recognized as GRAS (Generally Recognized As Safe). However, Weissella spp. are potential powerful starters for food fermentation as well as Lactococcus, Leuconostoc or Lactobacillus species.

A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway‐dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p–Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE‐binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co‐repressor complex Tup1p–Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Δ and sko1Δ mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.