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 freezetolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding ␥-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of ␥-glutamyl kinase and ␥-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 ␥-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 ⌬ 1 -pyrroline-5-carboxylate reductase, which converts ⌬ 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 ␥-glutamyl kinase and the wild-type ␥-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not ⌬ 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.

Role of the yeast acetyltransferase Mpr1 in oxidative stress: Regulation of oxygen reactive species caused by a toxic proline catabolism intermediate

“…Proline is believed to function as a compatible solute and as a free-radical scavenger to protect macromolecular structures form damage during osmotic and oxidative stresses (46,47). We also found that intracellular proline in yeast has an important role in reducing the oxidative stress induced during freezing and thawing or during exposure to H 2 O 2 (26,(48)(49)(50). It is therefore suggested that the toxic accumulation of P5C, not proline, triggers oxidative damage in yeast cells by reacting with various cellular compounds.…”

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…For example, a mutant form of PUT1 encoding an enzyme from the proline biosynthetic pathway was isolated from yeast mutants with increased proline levels (228). In contrast to the wild-type enzyme, the mutated version of Put1p was less sensitive to feedback regulation by proline, and its overexpression led to intracellular accumulation of proline (310,347).…”

“…Although a twohybrid analysis showed a physical interaction between tQM and Put1p, it remains to be clarified how tQM increases the intracellular proline concentration and protects the PUT1-overexpressing strain from oxidative stress (54). It is worth noting that high intracellular proline also positively affects freeze tolerance (347), desiccation (340), and ethanol tolerance (339,341) in yeast (see below and "Improving tolerance to ethanol" below). If present in the medium, proline may also serve as an osmoprotectant in yeast (349).…”

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

“…Proline also has an established role in defending against various abiotic and biotic stresses with its properties as a compatible solute benefiting a broad-range of organisms [10][11][12][13][14][15][16][17][18][19][20]. Proline has been shown to diminish aggregation of the polyQ region of the huntington protein suggesting a preventive role of proline in neurodegenerative diseases [19].…”