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Engineering pathways for malate degradation in Saccharomyces cerevisiae

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dc.contributor.author Volschenk H en
dc.date.accessioned 2016-09-22T08:39:49Z
dc.date.available 2016-09-22T08:39:49Z
dc.date.submitted 1996 en
dc.identifier.uri http://hdl.handle.net/20.500.11892/38563
dc.description.abstract L-Malic acid is one of the most important organic acids in grapes and wine. Together with tartaric acid, it contributes to 90% of the total acidity in grapes. The level of acidity in grapes greatly influences the quality of wine. In the cold viticultural regions, the level of acidity in grapes is usually too high and excess amounts of malate has to be reduced in order to create well-balanced wines. The yeast Saccharomyces cerevisiae is unable to degrade L-malic acid during alcoholic fermentation. Uptake of malate relies on simple diffusion, since S. cerevisiae lacks an active transport system for malate. Furthermore, the malic enzyme of S. cerevisiae has a low substrate affinity for malate which contributes to the yeast's weak ability to degrade malate. Malolactic fermentation, spontaneous or induced, is often employed by winemakers to reduce excess acidity. During malolactic fermentation, certain strains of lactic acid bacteria, especially strains of Leuconostoc oenos, proliferate in the must during or soon after alcoholic fermentation and results in deacidification, increased bacteriological stability, as well as enhanced sensory characteristics of the wine. The malolactic enzyme, encoded by the mleS gene, catalyses the direct decarboxylation of malate to lactate and carbon dioxide. The use of other yeasts that can degrade malate during alcoholic fermentation has also been investigated. The fission yeast Schizosaccharomyces pombe actively degrades malate under fermentative conditions through a malo-alcoholic pathway. The malic enzyme of S. pombe, encoded by the mae2 gene, catalyses the oxidative decarboxylation of malate to pyruvate and carbon dioxide. The malic enzyme of S. pombe has a high substrate affinity for malate and malate is actively transported by a malate permease, encoded by the mael gene. However, this yeast is unsuitable for the production of wine since it produces atypical wine flavours. The genes responsible for the malo-alcoholic fermentation (MAF) in S. pombe, mae1 and mae2, were subcloned into expression cassettes containing the alcohol dehydrogenase I (ADH1) and/or the phosphoglycerate kinase (PGK1) promoter and terminator sequences. A laboratory strain of S. cerevisiae was transformed with these cassettes. Expression of the mael gene of S. pombe in S. cerevisiae resulted in active transport of radioactively-labeled malate by the recombinant strain of S. cerevisiae. Recombinant strains of S. cerevisiae containing both the mael and mae2 genes efficiently degraded malate in seven days under respiratory conditions, and in 19 days in fermentative conditions. Co-expression of the mael gene of S. pombe and the malolactic enzyme gene (mleS) of Lactococcus lactis in S. cerevisiae, also led to efficient malate degradation via the malolactic pathway in Shiraz, Cabernet Sauvignon and Chardonnay grape must. In Cabernet Sauvignon and Shiraz grape must complete degradation of malate was obtained in less than three days, whereas in Chardonnay must, complete malate degradation was established in seven days. en
dc.language English en
dc.subject Microbiology en
dc.title Engineering pathways for malate degradation in Saccharomyces cerevisiae en
dc.type Masters degree en
dc.description.degree MSc en

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