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.
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| Gene/Complex | Qualifier | Gene Ontology Term | Annotation Extension | Evidence | Source | Assigned On |
|---|---|---|---|---|---|---|
| PRO1 | enables | glutamate 5-kinase activity | IDA | SGD | 2013-08-07 | |
| PRO1 | involved in | proline biosynthetic process | IDA | SGD | 2013-08-07 | |
| PRO2 | involved in | proline biosynthetic process | IDA | SGD | 2013-08-07 | |
| PRO2 | enables | glutamate-5-semialdehyde dehydrogenase activity | IDA | SGD | 2013-08-07 |
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| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| PRO1 | chemical compound accumulation: increased | classical genetics | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | L-proline | Details: increased resistance relative to wt; a high concentration of AZC was used as the parent strain contains the L-azetidine-2-carboxylic acid acetyltransferase, MPR1 |
| PRO1 | chemical compound accumulation: increased | heterozygous diploid | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | L-proline | Details: increased resistance relative to wt; a high concentration of AZC was used as the parent strain contains the L-azetidine-2-carboxylic acid acetyltransferase, MPR1 |
| PRO1 | freeze-thaw resistance: increased | classical genetics | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | Details: increased viability relative to wt after freezing at -20 deg C for 1 day | |
| PRO1 | freeze-thaw resistance: increased | heterozygous diploid | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | Details: increased viability relative to wt after freezing at -20 deg C for 1 day | |
| PRO1 | protein activity: increased Reporter: γ-glutamyl kinase | classical genetics | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | Details: ~ 25% increased activity in both the absence and presence of physiological levels of L-proline | |
| PRO1 | protein activity: increased Reporter: γ-glutamyl phosphate reductase | classical genetics | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | Details: 5 to 6-fold increased activity in both the absence and presence of physiological levels of L-proline | |
| PRO1 | resistance to chemicals: increased | classical genetics | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | 3 mg/ml (S)-azetidine-2-carboxylic acid | Details: increased resistance relative to wt; a high concentration of AZC was used as the parent strain contains the L-azetidine-2-carboxylic acid acetyltransferase, MPR1 |
| PRO1 | resistance to chemicals: increased | heterozygous diploid | activation Allele: pro1-D154N D154N (G460A); kinase domain mutated; increased catalytic activity; feedback inhibition insensitive | Other | 3 mg/ml (S)-azetidine-2-carboxylic acid | Details: increased resistance relative to wt; a high concentration of AZC was used as the parent strain contains the L-azetidine-2-carboxylic acid acetyltransferase, MPR1 |