Ethanol Production by Novel Proline Accumulating Pichia kudriavzevii Mutants Strains Tolerant to High Temperature and Ethanol Stresses

Accumulation of osmoprotectant molecule, proline, has been reported to induce a stress tolerance phenotype in yeast cells. In this study, two ethanologenic isolates of yeast, Pichia kudriavzevii (R and T), both capable of using both five and six sugar compounds to produce ethanol, were mutated via ethyl methanesulfonate (EMS) treatment. Prolineaccumulating mutant strains were selected by using proline-analogue (L-azetidine-2-carboxylic acid). Mutant strains were confirmed to accumulate proline in various level ranging from 11% to 154% compared to the wild type cells. Selected mutant strains were more resistant to high ethanol concentration (15%) and high temperature stress (45°C) compared to their Wild Type (WT) cells and industrial bioethanol yeast, Saccharomyces cerevisiae. Interestingly, the fermentation rate of isolate R-T1 and T-T2 was higher than its WT, based on quantitation of reducing sugar and ethanol content in both glucose and mixed glucose-xylose fermentations. The highest ethanol production was shown by strain R-T1 (3.3g/100 mL) that produced 7% and 20% higher ethanol compared to its WT in glucose and glucose-xilose as fermentation substrate, respectively. It is worth noting that ethanol production activity of T-T2 was 22% and 172% higher than its WT and industrial yeast S. cerevisiae, respectively. Our study indicates that proline accumulation in yeast P. kudriavzevii may promote ethanol production, especially in mixed substrate fermentations.