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Applied and Environmental Microbiology, January 2003, p. 212-219, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.212-219.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding {gamma}-Glutamyl Kinase

Yuko Morita, Shigeru Nakamori, and Hiroshi Takagi*

Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Fukui 910-1195, Japan

Received 1 August 2002/ Accepted 8 October 2002

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular L-proline derived from L-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and L-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-L-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding {gamma}-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of {gamma}-glutamyl kinase and {gamma}-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis from L-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated {gamma}-glutamyl kinase were found to accumulate intracellular L-proline and showed a prominent increase in cell viability after freezing at -20°C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered {gamma}-glutamyl kinase results in stabilization of the complex or has an indirect effect on {gamma}-glutamyl phosphate reductase activity, which leads to an increase in L-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.

* 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.

Applied and Environmental Microbiology, January 2003, p. 212-219, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.212-219.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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