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

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 Δ ¹ -pyrroline-5-carboxylate reductase, which converts Δ 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 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 Δ 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.