(250c) Using CRISPR Libraries to Identify Engineering Targets for Improved Growth of Yarrowia Lipolytica on Lignocellulosic Hydrolysate

Lignocellulosic hydrolysate is an abundant source of carbon, and a promising feedstock for use in microbial production of specialty chemicals traditionally derived from petroleum. Pretreatment of the biomass prior to hydrolysis of the lignocellulose results in the formation of inhibitory compounds detrimental to microbial growth. Yarrowia lipolytica has demonstrated some innate tolerance to these inhibitory compounds in addition to a robust ability to direct carbon towards the production of oleochemicals. Using CRISPR libraries as part of a systems biology approach to direct engineering efforts, we are developing a strain of Y. lipolytica with improved growth on lignocellulosic hydrolysate for oleochemical production. Collaborators in Prof. Ian Wheeldon’s lab (University of California, Riverside) have developed a CRISPR knockout (CRISPR KO) library for Y. lipolytica with full genome coverage. We used this library to run enrichment screens and to identify genes involved in tolerance to the inhibitory compounds found in lignocellulosic hydrolysate. Genes conferring both growth benefits and detriments were identified. While many of these genes have known functions related to stress response, genes of seemingly unrelated or of unknown function have also been identified. To be used in conjunction with this CRISPR KO library, we have developed a CRISPR activation (CRISPRa) library for Y. lipolytica. Each member of this library contains a guide RNA targeting upstream of a different gene, with multiple guide RNAs for each gene. These guides work with a deactivated cas9 tied to an activator domain to upregulate transcription of individual genes. By upregulating gene expression instead of knocking genes out, this library can identify additional genes of interest that are either essential or under cryptic expression. Ultimately, we are using information from these CRISPR screens to direct engineering efforts of Y. lipolytica to develop a strain with more robust growth on lignocellulosic hydrolysate to be used as a chassis for specialty oleochemical production.