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Abstract
The potential of lignocellulosic biomass as a sustainable substrate for bioethanol production is limited by the pretreatment process that generates inhibitory compounds impairing the growth and performance of fermenting microorganisms. Development of strains with increased tolerance to a range of inhibitors is necessary as methods of inhibitor abatement are economically and environmentally unfavorable. S. cerevisiae strain XR122N was subjected to directed evolution and adaptation in pretreated pine fermentations and resultant strains, GHP1 and GHP4, demonstrated improved fermentative ability with GHP4 exhibiting constitutive tolerance and GHP1 exhibiting conditional tolerance dependent on continuous selective pressure during preculturing. Transcriptome sequencing revealed 52 differentially expressed genes that may account for improved tolerance to multiple inhibitors simultaneously. Forty-three of the genes have yet to be investigated in pertinence to biomass-derived inhibitors. Evolved strains also demonstrated improved robustness of mitochondria that were resistant to the damaging effects of inhibitors in contrast to the parent. Whole genome sequencing revealed a diverse set of more than 300 genes having polymorphisms within the evolved strains. More than 60 were unique to each strain and majority of the polymorphisms were shared for both GHP1 and GHP4. Over 130 large structural variations and rearrangements were identified for evolved strains in comparison with parent XR122N including potential aneuploidy of chromosome 14 in GHP4. These findings have led to the identification of two novel gene targets, YNL019C and PRM9, uncharacterized membrane-associated proteins of unknown function that were identified in both comparative transcriptomics and genomics analyses. Collectively this work has contributed to overall understanding of S. cerevisiae stress tolerance and has direct implications for further development of robust yeast strains for multiple industrial applications.