ABSTRACT
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 γ-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of γ-glutamyl kinase and γ-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 γ-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 Δ1-pyrroline-5-carboxylate reductase, which converts Δ1-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 γ-glutamyl kinase and the wild-type γ-glutamyl phosphate reductase was effective for l-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Δ1-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.
Genes that cause overproduction of isoamyl alcohol by increased gene-dosage effect in Saccharomyces cerevisiae.
