During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the l-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of l-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for l-proline utilization, and replacing the wild-type PRO1 allele with the pro1D154N allele. The resultant strain accumulated l-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate l-proline to brew sake containing five times more l-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles.
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