Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk.

The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.