CRISPRi/a for investigating yeast tolerance to inhibitors in lignocellulosic hydrolysates

Sammanfattning: Saccharomyces cerevisiae has immense potential as a cell factory in various biotechnological processes where biomass from agricultural industry residues is used as feedstock. Nonetheless, the inhibitors released during the pretreatment of the biomass makes lignocellulosic hydrolysates a challenging substrate for microorganisms. In this thesis, the CRISPR interference/activation (CRISPRi/a) technology was used in combination with high-throughput screening methods to improve tolerance of S. cerevisiae towards inhibitors found in lignocellulosic hydrolysates. The focus was on understanding the genetics behind formic and acetic acid tolerance, two abundant inhibitory compounds.  The aims were to compare the responses to either acid and to explore how the results could be extrapolated to understand hydrolysate tolerance. The CRISPRi/a technology was used to improve the hydrolysate tolerance of an industrial strain and to alter the expression of the transcription factor encoding genes YAP1 and PDR1 , leading to strains with altered tolerance to acetic acid. We performed ChIP-exo experiments, which demonstrated that both transcription factors showed increased binding of target genes in the presence of acetic acid. Notably, genes related to amino acid synthesis and cell membrane transporters were highly bound to Yap1 and Pdr1 in the presence of acetic acid. Furthermore, A CRISPRi strain library targeting the essential and respiratory essential genes in S. cerevisiae was studied for acetic and formic acid tolerance. The strains were screened by using various high-throughput methods such as competitive growth assays, fluorescence-activated cell sorting and screening for growth on solid media. Systematic analysis of the data highlighted genes encoding proteins with functions in intracellular vesicle transport, glycogen accumulation or chromatin regulation as important for tolerance towards acetic and formic acid. Interesting strains were further characterized individually in the presence of acetic or formic acid, in a synthetic hydrolysate medium or in the presence of oxidative stress causing agents. To conclude, this research advances our knowledge on how the regulation of genes such as the ones related to chromatin remodeling can influence tolerance to weak acids as well as other inhibitors found in lignocellulosic hydrolysates. The results demonstrate the potential of CRISPRi/a technology to accelerate the development of more tolerant industrial yeast strains.