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Applied and Environmental Microbiology, November 2003, p. 6527-6532, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6527-6532.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Gene Dosage Effect of L-Proline Biosynthetic Enzymes on L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae

Yukiyasu Terao, Shigeru Nakamori, and Hiroshi Takagi*

Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan

Received 16 June 2003/ Accepted 2 September 2003

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding {gamma}-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of {gamma}-glutamyl kinase and {gamma}-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant {gamma}-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including {Delta}1-pyrroline-5-carboxylate reductase, which converts {Delta}1-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant {gamma}-glutamyl kinase and the wild-type {gamma}-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not {Delta}1-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.

* 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, November 2003, p. 6527-6532, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6527-6532.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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