l -Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding γ-Glutamyl Kinase

Abstract

ABSTRACT 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 γ-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 γ-glutamyl kinase and γ-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 γ-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 γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-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.