title: Effect of L-proline on sake brewing and ethanol stress in Saccharomyces cerevisiae.
abstract: 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, pro1(D154N), which encodes the Asp154Asn mutant gamma-glutamyl kinase. This mutation increases the activity of gamma-glutamyl kinase and gamma-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 pro1(D154N) 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.
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. hiro@fpu.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
authors.forename: Miki
authors.initials: M
authors.lastname: Takaoka
authors.forename: Akari
authors.initials: A
authors.lastname: Kawaguchi
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kubo
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology*; D005838:Genotype; D007564:Japan; D007700:Kinetics; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000187:drug effects; D010957:Plasmids; D011392:Proline / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014920:Wine
chemical_list: D000431:Ethanol; D011392:Proline