Combining metabolic engineering and fermentation optimization to achieve cost-effective oil production by...

Microbial oils, produced by oleaginous microorganisms, offer a sustainable alternative to plant-derived oils. Among these microorganisms, Cutaneotrichosporon oleaginosus is a promising microbial cell factory for sustainable oil production due to its capacity to accumulate lipids with a similar composition to palm oil. Although C. oleaginosus can reach higher lipid contents than other oleaginous yeasts, enhanced productivity is still necessary to have a feasible microbial oil production process via C. oleaginosus. In this study, we followed a combinatorial approach for strain design and bioprocess development to improve the lipid content and lipid yield. Initially, we deployed a full factorial design with genetic factors (ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS) and carbon-to-nitrogen ratio (C/N) in the medium. The C/N ratio appeared to have the most impact on oil accumulation. Combined with genetic modifications, lipid content and lipid yield increased by 1.6-fold. In a two-stage fermentation approach at a 2 L scale, the triple transformant overexpressing ACL, ACC, and TS outperformed the wild-type by achieving a lipid content of 75.4 % (w/w) with lipid productivity of 0.40 g L^-1 h^-1 and around 0.30 g lipids/g glycerol. In all, we established a cultivation strategy and strain that reached almost the theoretical maximum yield, and highest lipid content reported for a medium containing glycerol as a carbon source. These results strengthen the basis of using C. oleaginous as a platform for microbial oil production, thereby facilitating the development of processes substituting palm oil with a sustainable alternative.