(86f) Metabolic Engineering of Non-Model Yeast Cutaneotrichosporon Oleaginosus for Valorization of Lignin and Lignin-Derived Aromatics

Cutaneotrichosporon oleaginosus,previously known as Cryptococcus curvatus, is a non-model oleaginous yeast that is known for its ability to metabolize many alternative sugars, including xylose, and toxic lignocellulosic hydrolysate inhibitors such as 5-hydroxymethylfurfural (5-HMF) and furfural. We discovered C. oleaginosusalso tolerates and metabolizes lignin-derived phenolics, highlighting this organisms’ potential to utilize all components of lignocellulosic biomass. C. oleaginosusis able to fully metabolize phenol, 4-hydroxybenzoic acid (pHBA), and resorcinol as sole carbon sources, as well as co-utilization with glucose and xylose. We exploited feeding strategies to overcome aromatic toxicity and increased lipid accumulation to over 69% of biomass by weight. Furthermore, we have measured the depolymerization of lignin hydrolysates. Since yeast aromatic metabolism is poorly characterized, we used transcriptomic analyses to elucidate aromatic metabolic pathways in C. oleaginosus and improved the existing genome annotation significantly. Biochemical analysis suggests ortho ring cleavage. With such an exceptionally desirable natural phenotype, this yeast could become a preferred host for oleochemical production if novel synthetic biology tools are developed. We identified both strong constitutive and phenolic-regulated promoters using our transcriptomic data and used these to drive expression of Claviseps purpurea delta-12 hydroxylase gene required for ricinoleic acid production. In parallel, we are establishing higher efficiency transformation methods for increasing the pace of engineering efforts. Overall, our work establishes C. oleaginosusas a promising platform to robustly convert all components of lignocellulosic biomass into novel high-value oleochemicals.