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

Area of Science:

• Enology and Fermentation Science
• Microbial Biotechnology
• Yeast Genetics

Background:

• Malolactic fermentation (MLF) is crucial for wine quality, traditionally performed by lactic acid bacteria.
• Controlling MLF in winemaking is challenging, prompting research into alternative microbial systems.
• Engineering yeast for MLF could offer better control and integration with alcoholic fermentation.

Purpose of the Study:

• To investigate the feasibility of enabling yeast strains to perform malolactic fermentation.
• To express the malolactic gene (mleS) from Lactococcus lactis in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
• To assess the efficiency and control of heterologous malolactic enzyme activity in yeast.

Main Methods:

• Genetic engineering: Expression of Lactococcus lactis mleS gene in S. cerevisiae and S. pombe using the ADH1 promoter on a multicopy plasmid.
• Enzyme activity assays: Measurement of malolactic enzyme specific activity in cell extracts.
• Fermentation studies: Analysis of L-lactate production using glucose-rich medium and isotopic labeling to trace malate origins.

Main Results:

• High-level expression and significantly enhanced malolactic enzyme activity observed in S. cerevisiae compared to L. lactis.
• S. cerevisiae engineered with mleS produced substantial L-lactate from both endogenous and exogenous L-malate.
• S. pombe demonstrated efficient L-malate degradation and complete malolactic fermentation, indicating malate transport as a key limitation in S. cerevisiae.

Conclusions:

• Yeast strains can be successfully engineered to perform malolactic fermentation.
• Malate transport into the yeast cell is the primary limiting factor for malolactic fermentation in S. cerevisiae.
• S. pombe shows high potential for complete malolactic fermentation, offering a promising avenue for winemaking applications.