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

Abstract

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.

Bacterial-Fungal Interactions: Hyphens between Agricultural, Clinical, Environmental, and Food Microbiologists

“…In wine production, S. cerevisiae and Schizosaccharomyces pombe have been engineered to express the malolactic gene from Lactococcus lactis in order to VOL. 75, 2011 BACTERIAL-FUNGAL INTERACTIONS 599 enable them to perform both the alcoholic and malolactic fermentation stages without the need for malolactic bacteria (8). E. coli strains have been engineered with a combination of genes from S. cerevisiae and from other bacterial strains to convert dihydroxyacetone phosphate into 1,3-propanediol, which can be used as building blocks for industrial polymers (271).…”

The Biochemistry of Malic Acid Metabolism by Wine Yeasts – A Review

“…The ability of a yeast strain to degrade extracellular L-malate is dependent on the efficient transport of the dicarboxylic acid, as well as the efficiency of the intracellular malic enzyme (Ansanay et al, 1996;Volschenk et al, 1997a;1997b). In addition, factors such as the availability of glucose and oxygen also influence the efficacy of malic acid degradation.…”

Characterization ofSchizosaccharomyces pombeMalate Permease by Expression inSaccharomyces cerevisiae

“…The bacterial mleS gene, encoding a bifunctional malolactic enzyme that catalyzes the conversion of L-malate into L-lactate and CO 2 , has been introduced into S. cerevisiae, to improve its ability to consume malate (1,12). The heterologous enzyme is functional in recombinant strains but increases malate degradation only slightly, and it has been shown that transport is the limiting step in L-malic acid utilization (2).…”