(325c) CRISPR-Cas9 Based Genome-Wide Functional Screening Yarrowia Lipolytica genes contributing to Improved Lipid Accumulation.

With global concerns about environmental problems and the depletion of fossil resources, the demand for renewable feedstocks to produce fuels and chemicals has increased. In this aspect, the utilization of microbes to produce lipids instead of relying on petroleum-derived oleochemicals has become significant. Nonconventional yeast Yarrowia lipolytica has gained prominence as a host for the bioproduction of lipids since it has a high acetyl-CoA pool. With metabolic engineering, Yarrowia has shown the ability to accumulate lipids comprising over 90% of its dry cell weight and achieving productivity levels close to 1 g/L/hr. These attributes position Yarrowia as a primary candidate for microbial oil production. However, there remains a lack of comprehensive understanding regarding the genetic mechanisms underlying its lipid metabolism and accumulation. In this study, we employ an optimized CRISPR-Cas9 genome-wide library to conduct a functional genetic screening, particularly in the presence of the fatty acid synthesis inhibitor cerulenin. Such screens in other species have identified mutants that enhance lipid production. In this work, we coupled growth screens and cell sorting to screen a genome-wide mutational pool to identify loss of function hits that enhance lipid accumulation. From the analysis of the gene hits resulting from this screen include novel genes of unknown function, along with other genes whose knockout leads to increased lipid production (such as peroxisome formation and lipase). To validate our screen, single gene knockouts were made using CRISPR-Cas9 gene editing. significant improvement for lipid production was shown in most of the loss of function strains as demonstrated by lipid staining and analysis of extracted lipids. Collectively, this work demonstrates the functionality of our optimized CRISPR-Cas9 genome-wide library. The application of this new synthetic biology tool in a classic genetic screen for lipid biosynthesis can rapidly identify genetic hits for metabolic engineering. This screen will help advance Yarrowia as a production host for the renewable production of lipids, oleochemicals, and biofuels.