Phenotypic characterization and comparative transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic derived inhibitors

Lignocellulosic biomass continues to be investigated as a viable source for bioethanol production. However, the pretreatment process generates inhibitory compounds that impair the growth and fermentation performance of microorganisms such as Saccharomyces cerevisiae. Pinewood specifically has been shown to be challenging in obtaining industrially relevant ethanol titers. An industrial S. cerevisiae strain was subjected to directed evolution and adaptation in pretreated pine biomass and resultant strains, GHP1 and GHP4, exhibited improved growth and fermentative ability on pretreated pine in the presence of related inhibitory compounds. A comparative transcriptomic approach was applied to identify and characterize differences in phenotypic stability of evolved strains. Evolved strains displayed different fermentative capabilities with pretreated pine that appear to be influenced by the addition or absence of 13 inhibitory compounds during pre-culturing. GHP4 performance was consistent independent of culturing conditions, while GHP1 performance was dependent on culturing with inhibitors. Comparative transcriptomics revealed 52 genes potentially associated with stress responses to multiple inhibitors simultaneously. Fluorescence microscopy revealed improved cellular integrity of both strains with mitochondria exhibiting resistance to the damaging effects of inhibitors in contrast to the parent. Multiple potentially novel genetic targets have been discovered for understanding stress tolerance through the characterization of our evolved strains. This study specifically examines the synergistic effects of multiple inhibitors and identified targets will guide future studies in remediating effects of inhibitors and further development of robust yeast strains for multiple industrial applications.