Abstract
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.
Highlights
Yeasts that belong to the genera of Pichia have been known for their capability of using both hexose and pentose sugars as substrates for bioethanol productions (Mussatto et al, 2012)
The intracellular proline content of P. kudriavzevii isolate R- derived mutants (RT0 and R-T1) to its wild type was not significantly different, yet resulted in essentially different phenotype in corresponding to the capability of mutants to grow in AZC stress-medium
A contrarily phenomenon was found in R-T3 which had the lowest proline content of all mutant tested (18.3nmol/ml) (Fig. 2A); the viability of this mutant strain in AZC containing medium was higher than its Wild Type (WT) (Fig. 1)
Summary
Yeasts that belong to the genera of Pichia have been known for their capability of using both hexose and pentose sugars as substrates for bioethanol productions (Mussatto et al, 2012). S. cerevisiae, the commonly used industrial yeast, is unable to use pentose as a substrate, suggesting superiority of Pichia in its application for the 2nd generation of bioethanol productions (Radecka et al, 2015). Yeasts are exposed to various environmental stresses, including an elevated temperature, osmotic pressure and high ethanol stresses, causing depletion upon ethanol production (Ansanay-Galeote et al, 2001). For more effective and valuable ethanol production, yeast strain developments are needed, to construct stress tolerance-mutant strains. Various approaches have been employed to construct mutant yeast capable of combating fermentation-related stresses. In S. cerevisiae, modifications of proline metabolism pathway results in mutant strains with enhanced stress-tolerant phenotypes against high temperature stresses, ethanol, freeze drying, dehydration, high salinity (Sekine et al, 2007; Sasano et al, 2012a)
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