Exploring Saccharomyces cerevisiae’s responses to acetic acid and other inhibitors found in lignocellulosic hydrolysates

Abstract: The limited tolerance of the budding yeast Saccharomyces cerevisiae to lignocellulosic hydrolysate inhibitors is a key challenge to its use in biorefinery cell factories. Considerable resources have been invested in the isolation of yeast strains with better tolerance towards the inhibitors released during lignocellulose hydrolysis, such as acetic acid. The goal of this thesis was twofold: characterize the transcriptional response of S. cerevisiae to wheat straw hydrolysate and explore the role of essential S. cerevisiae genes in acetic and formic acid tolerance, using a new biosensor and competitive growth assays. The transcriptomes of one laboratory strain, two industrial strains, and two wild-type isolates grown in wheat straw hydrolysate were profiled. Despite similar growth, the isolates showed different expression of genes encoding proteins involved in oxidative stress response, lipid accumulation, and transport, suggesting different genetic strategies for tolerance. The new acetic acid biosensor was based on two transcription factors, Haa1 from S. cerevisiae and BM3R1 from Bacillus megaterium. Biosensor and competitive growth were used in parallel to screen a S. cerevisiae CRISPR interference strain library. While fluorescence-activated cell sorting led to the isolation of cells with higher acetic acid retention and sensitivity, competitive growth assays allowed the identification of cells with higher acid tolerance. The results confirmed the role in acid stress response of genes involved in glycogen accumulation, chromatin modification, and mitochondrial or proteasomal functions. Two novel targets for improving tolerance were also identified: PAP1 and HIP1. Altogether, this thesis provides mechanistic insight into the stress response to lignocellulosic hydrolysates or weak acid inhibitors that limit yeast-mediated conversion of lignocellulosic biomass into biochemicals. Additionally, it offers new tools for the identification of strains with altered acetic acid tolerance.