CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate

Abstract

Background Baker’s yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity. Results Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate. Conclusion Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes.

A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

“…In previous studies where CRISPRi technology was applied for massive genotype-phenotype mapping in S. cerevisiae ( 9 , 11 – 13 ), the strains were pooled and screened for competitive growth. Although competitive growth assays have the advantage of throughput, they come with a major weakness: the nutrient-specific advantage for cells/strains with a shorter lag phase is amplified.…”

“…Moreover, the characterization of a population enriched after a specific time provides merely an endpoint observation. In the previously described competitive growth assays of whole-genome CRISPRi libraries ( 11 , 13 ), the genes identified to give beneficial phenotypes when repressed have not been essential. This is likely due to the phenotypes of strains with altered expression of essential genes not being as pronounced as the phenotypes of the strains becoming enriched or due to the alteration in expression being detrimental.…”

“…Furthermore, as the strength of the expression alteration is greatly dependent on the efficiency and positioning of the gRNA, one can study a gradient of repression by testing multiple gRNA sequences for each target gene ( 8 , 9 ). Based on this technology, several CRISPRi strain libraries were constructed for many species, including Saccharomyces cerevisiae ( 9 – 13 ).…”

“…In this strain collection of roughly 9,000 strains, nearly 99% of the essential and 98% of the respiratory growth-essential genes have been targeted with up to 17 gRNAs per target gene ( 12 ). Recently, the construction and phenotypic screening of CRISPR technology-based S. cerevisiae libraries have been demonstrated to be very efficient to identify bioengineering genetic candidates to increase the production of β-carotene or endoglucanase ( 15 ), regulate polyketide synthesis ( 16 ), or improve tolerance to furfural ( 11 ) or lignocellulose hydrolysates ( 13 ).…”

Recent Advances in Directed Yeast Genome Evolution

“…Simple phenotypes, such as carbon catabolism and chemicals biosynthesis, are easy to focus on for genes in relevant pathways [ 38 ]. However, when it comes to more complicated phenotypes, such as cell growth and resistance to environmental stresses, more genes such as transcription factors and protein kinases are selected as targets for perturbation, because these proteins control global genes spanning from synthetic pathways to cellular signaling [ 40 ]. While it is impossible to precisely predict genes involved in some phenotypes due to a limited knowledge of cellular networks, whole-genome coverage by an sgRNA library bypassed this requirement by targeting all genes within the genome.…”