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Metabolic engineering and characterisation of the malolactice wine yeast ML01 Husnik, John Ivan

Abstract

Malolactic fermentation (MLF) is essential for deacidification o f high acid grape must and the production of well-balanced wines. The bacterial MLF is unreliable and stuck MLFs often lead to spoilage of wines and the production of biogenic amines. A genetically stable industrial strain of Saccharomyces cerevisiae was constructed by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mael) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast strain. The malolactic yeast strain, ML01, completes the MLF during the alcoholic fermentation in a variety of musts including a high acid Chardonnay must containing 9.2 g/L of malate. ML01 cannot appreciably decarboxylate L-malic acid to Llactic acid when present at levels below 1% of the total inoculum. ML01 contains no antibiotic resistance marker genes or vector DNA sequences. Global gene expression patterns and analysis of the proteome showed that no metabolic pathway was affected by the introduction of the malolactic cassette. The presence of the malolactic cassette in the genome does not affect growth, ethanol production, fermentation kinetics or metabolism of ML01. Wines produced by the ML01 yeast have lower volatile acidity and improved color properties compared to wines produced with the parental yeast and a bacterial MLF. GC/MS analysis of volatile compounds revealed that wine produced by ML01 did not contain any compounds that were not detected in wine produced with the parental strain S92 or with S92 and malolactic bacteria. Moreover, ML01 reduces the processing time after alcoholic fermentation and produces wine that is judged highest in overall quality by trained tasters. Analyses of the phenotype, DNA, RNA, and proteins demonstrate that the recombinant yeast ML01 is substantially equivalent to the parental strain S92. ML01 has been approved for use in Canada and has 'Generally Regarded As Safe' status with the US FDA. It is the first metabolically engineered yeast to be commercialised by the wine industry and is currently available in Canada, the USA and Moldova.

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