Background Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. Results The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. Conclusion This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.

Cutaneotrichosporon oleaginosus is a highly efficient single cell oil producer, which in addition to hexoses and pentoses can metabolize organic acids. In this study, fed-batch cultivation with consumption-based acetic acid feeding was further developed to integrate the transformation of an industrial paper mill lignocellulosic hydrolysate (LCH) into yeast oil. Employing pentose-rich LCH as a carbon source instead of glucose significantly improved both biomass formation and lipid titer, reaching 55.73 ± 5.20 g/L and 42.1 ± 1.7 g/L (75.5% lipid per biomass), respectively. This hybrid approach of using acetic acid and LCH in one process was further optimized to increase the share of bioavailable carbon from LCH using a combination of consumption-based and continuous feeding. Finally, the techno-economic analysis revealed a 26% cost reduction when using LCH instead of commercial glucose. In summary, we developed a process leading to a holistic approach to valorizing a pentose-rich industrial waste by converting it into oleochemicals.