Genetic construction of malolactic wine yeasts (Schizosaccharomyces pombe, Oenococcus oeni)

The Schizosaccharomyces pombe mae1 and Oenococcus oeni mleA genes were subcloned into expression cassettes containing the 3-phosphoglycerate kinase promoter and terminator sequences and linked together to create a single malolactic cassette. Different yeast transformation protocols were evaluated and modified to obtain efficient transformation of DNA into industrial wine yeast strains. A novel screening method for the detection of malolactic yeast was also developed. The malolactic cassette on a centromeric-based plasmid was transformed into five different industrial wine yeasts. The recombinant malolactic wine yeasts completed the malolactic fermentation in approximately five days in synthetic grape juice. Fermentation efficiencies were evaluated and the best three recombinant strains, L1001, L1002, and L1004, were produced as active dry yeast strains. The recombinant active dry yeast completed the malolactic fermentation in three days in synthetic grape juice.{09}Enzymatic analyses of fermented synthetic grape juice showed that L-malate was completely decarboxylated to L-lactate. The recombinant malolactic yeasts utilized glucose and fructose and produced ethanol as efficiently as did the wild type strains.