Construction of ethanol-tolerant yeast strains with combinatorial library-selected peptides

Abstract

Combinatorial yeast libraries were constructed by transformation of expression plasmids containing artificially synthesized random sequences into Saccharomyces cerevisiae MT8-1 and IFO10150. Approximately 200 yeast strains with enhanced ethanol tolerance were obtained from yeast libraries by incubation in 10% ethanol for 24 h. Following separate evaluation of their ethanol tolerance, the 10 clones with the highest values were selected. After 3 h incubation in 12.5% ethanol, whereas most of the control cells died, the clone with the highest tolerance from the MT8-1 library, M-1, showed approximately 40% cell viability, and the corresponding clone from the IFO10150 library, I-12, 48% viability. The half-life of M-1 cells was 20 times greater than that of control cells. Three of the library-selected peptides endowing with ethanol tolerance were identified as Gly-Thr-Arg-Leu-His pentapeptides. Four seemed to be extremely hydrophobic, and three of these were predicted to be transmembrane peptides. The three other peptides seemed to be more hydrophilic than standard yeast proteins. The results of the study show that yeast strains with fairly high ethanol tolerance can be successfully constructed by directed selection from yeast libraries expressing combinatorial peptides.