(231i) Comparative Evaluation of 13 Yeast Species in the Yarrowia Clade on Lignocellulosic Biomass Hydrolysate and Genetic Engineering of Inhibitor Tolerant Strains for Lipid and Biofuel Production

Yarrowia lipolytica is an oleaginous yeast that has garnered interest for commercial production of single cell oil and other fatty acid-derived chemicals because of its GRAS status and genetic tractability. Three recent peer-reviewed studies have highlighted the possibility of lipid production by this yeast using lignocellulosic biomass hydrolysates, which provide an inexpensive and renewable feedstock for bioprocessing. Prior research on Saccharomyces cerevisiae suggests that yeast strains can vary widely in their tolerance to common inhibitors in cellulosic hydrolysates. In the present study, we investigated growth and lipid production by 57 yeast isolates, representing 13 species in the Yarrowia clade, on a dilute acid-pretreated switchgrass hydrolysate with Y. lipolytica strain W29 as a control. Phenotypic traits, such as inhibitor tolerance, substrate utilization, cell growth, and oleaginicity, varied considerably within the clade. Furthermore, we identified a lesser-known Yarrowia species that was superior to W29 in terms of lipid content (49.7% of dry weight) and lipid titer (2.9-fold improvement). The top performing strains, including members of a novel species, were subsequently evaluated for amenability to genetic engineering. An integration vector was constructed with a selectable antibiotic resistance marker and successful transformations were demonstrated by modifying a protocol developed for Y. lipolytica. Then, endogenous Yarrowia promoters were characterized in the new species by monitoring specific expression of a fluorescent reporter protein in relevant conditions. Using our newly developed genetic tools, we were able to manipulate a set of candidate target genes in a non-model host species and improve promising strains for enhanced conversion of cellulosic sugars to triacylglycerols. In summary, this work describes identification, characterization, and engineering of novel oleaginous strains in the Yarrowia clade for lipid and biofuel production from renewable biomass.