Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology

Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society.  However, the substitution of fossil based biochemical with its renewable counterpart requires the production to be significantly more efficient and price competitive. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulose hydrolysate. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. Numerous attempts have been reported to improve the stress tolerance of Saccharomyces cerevisiae by different bioengineering strategies such as deletion/overexpression of genes. However, the inability to achieve a fine balance of the transcriptional expression of the target and the ancillary gene(s) is one of the major factors that impedes the efficiency of many of these strategies. In this project, we apply CRISPR interference (CRISPRi) technology to investigate the potential of fine-tuning the expression of genes that are related to the stress regulation. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using a sgRNA. Strains with altered regulation will be screened for inhibitor tolerance. Furthermore, transcriptomics analysis of tolerant mutants will be conducted to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate. Here, we will show data on a methodology that we have developed for studying hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates.