Pierre Barre

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe.

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of l‐lactate during fermentation on glucose‐rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the l‐lactate produced originates from endogenous l‐malate and 25% from exogenous l‐malate. Moreover, although a small amount of exogenous l‐malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded l‐malate was converted into l‐lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades l‐malate. Total malolactic fermentation was obtained in this strain, with most of the l‐malate converted into l‐lactate and CO2. Moreover, l‐malate was used preferentially by the malolactic enzyme in this strain also.

Examination of the transcriptional specificity of an enological yeast. A pilot experiment on the chromosome-III right arm

Abstract The adaptation of yeasts to industrial environments is thought to be largely dependent on gene-expression specificity. To assess the transcriptional specificity of an enological strain, we performed a pilot experiment and examined the transcript level of 99 ORFs of the chromosome-III right arm with two strains, an enological-derived strain and a laboratory strain, grown under three different physiological conditions: respiration, standard alcoholic fermentation and enological alcoholic fermentation. The use of 99 single ORF-derived probes led to the detection of 49 transcripts, most of which were present at low levels and were not regulated. Ethanol respiration induced transcripts, in a similar manner with both strains. While standard alcoholic fermentation led to only minor regulations, the enological fermentation conditions triggered the expression of different genes. In addition, a specific transcriptional response to these conditions was observed with the enological-derived strain. The known or predicted functions of several genes induced under enological conditions is related to either alcoholic fermentation or stress, suggesting that their specific induction could reflect adaptation of the strain to the enological environment. Our data suggest that systematic transcriptional studies are an effective way to assess the molecular basis of yeast adaptation to industrial environments.