L-proline accumulation and freeze tolerance of Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding gamma-glutamyl kinase.
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 degrees 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.
Morita
Yuko
Y
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Fukui 910-1195, Japan.
Nakamori
Shigeru
S
Takagi
Hiroshi
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005615:Freezing; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development* / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins
D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins; D011392:Proline; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase