ISOLATION OF FREEZE-TOLERANT LABORATORY STRAINS OF SACCHAROMYCES-CEREVISIAE FROM PROLINE-ANALOG-RESISTANT MUTANTS

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contr ibute to enhancing freeze tolerance in yeast cells. In fact, proline a nd charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces ce revisiae. To investigate the cryoprotective effect of proline on the f reezing stress of yeast, we isolated proline-analogue-resistant mutant s derived from a proline-non-utilizing strain of S. cerevisiae. When c ultured in liquid minimal medium, many mutants showed a prominent incr ease, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at -20 degrees C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amou nt of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic str ess. These results suggest that the increased flux in the metabolic pa thway of specific amino acids such as proline is effective for breedin g novel freeze-tolerant yeasts.