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Applied and Environmental Microbiology, December 2005, p. 8656-8662, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8656-8662.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of L-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae

Hiroshi Takagi,^1* Miki Takaoka,¹ Akari Kawaguchi,¹ and Yoshito Kubo²

Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan,¹ Fukui Prefectural Food Processing Research Institute, 1-1-1 Tsubonouchi, Maruoka-cho, Fukui 910-0343, Japan²

Received 29 May 2005/ Accepted 14 September 2005

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 -glutamyl kinase. This mutation increases the activity of -glutamyl kinase and -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.

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* Corresponding author. Mailing address: Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. Phone: 81-776-61-6000. Fax: 81-776-61-6015. E-mail: hiro{at}fpu.ac.jp.

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Applied and Environmental Microbiology, December 2005, p. 8656-8662, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8656-8662.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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