UC Riverside

Yarrowia lipolytica is a versatile oleaginous yeast used for bioprocessing and bioproduction with uses ranging from supplement production to bioremediation. Its ability to metabolize a wide range of carbon sources, generate and store surplus Acetyl-CoA and lipids, and grow in a wide range of environmental conditions make it a promising candidate for a wide range of novel bioprocessing applications. Engineering optimized strains of Y. lipolytica remains a challenge due to an incomplete understanding of its genome and biological function under industrially-relevant conditions. An efficient approach to address these knowledge gaps and design optimized strains is a genome-wide CRISPR knockout screen. Here, we use optimized whole-genome gRNA libraries and innovative bioinformatic pipelines to carry out functional knockout screens and identify genes to target for strain engineering. First, we conducted a qualitative screen to identify knockouts that abolish hyphal formation, multicellular filaments that can interfere with industrial bioreactor function. We identified a benign null-hyphal knockout ΔRAS2 with equivalent growth and production titer characteristics of existing strains. We also developed a pipeline to identify potential gene knockouts that may optimize the usage of Y. lipolytica for solid state fermentation, a growing need in industrial bioprocessing. We used GO-term analysis of the screen results to elucidate putative differences in the relative importance of different cellular systems between solid and liquid environments. We identified genes responsible for endomembrane system and mitochondrial function as more essential under solid conditions, and genes responsible for biosynthesis, metal ion/redox, and ribosomal function as more essential under liquid conditions. Finally, we screened for gene knockouts with improved metabolism of non-glucose carbon sources and identified several with improved acetate catabolism and growth with the knockouts ΔE37234g, ΔE01193g, ΔC02904g, and ΔD21022g exhibiting the best growth and acetate catabolism characteristics. We also identified gene knockouts with preliminary improvements to hydrocarbon and fatty acid catabolism including ΔE36308g, ΔE03584g, ΔA18344g and ΔD20202g. These discoveries validate our genome wide knockout libraries and screen pipelines while providing novel gene targets for industrial Y. lipolytica strain engineering.