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 bett er 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) pr omoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisia e expressing the malolactic enzyme produces significant amounts of L-l actate during fermentation on glucose-rich medium in the presence of m alic acid. Isotopic filiation was used to demonstrate that 75% of the L-lactate produced originates from endogenous L-malate and 25% from ex ogenous L-malate. Moreover, although a small amount of exogenous L-mal ate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded L-malate was converted into L-lactate via a malolac tic reaction in the recombinant strain, providing evidence for very ef ficient competition of malolactic enzyme with the endogenous malic aci d 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 efficien tly degrades L-malate. Total malolactic fermentation was obtained in t his 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.