Integrated metabolic and process engineering of Rhodotorula toruloides for light-driven hydrocarbon production

Long-chain hydrocarbons are bulky chemicals traditionally produced via petroleum refining. Microbial production of these hydrocarbons from renewable resources has attracted much attention, but is limited by precursor accessibility and catalysis efficiency. Here, we combined genetic engineering and photobioprocess optimization for blue-light-driven hydrocarbon production by the oleaginous yeast Rhodotorula toruloides . Cytosolic expression of the Chlorella variabilis fatty acid photodecarboxylase ( Cv FAP) in R . toruloides conferred cells producing 15.0 mg/L hydrocarbons, while targeting Cv FAP to lipid droplets (LDs) via fusion with the LD-associated protein Ldp1 enhanced titer by more than 3-fold. Systematic optimization of blue-light fermentation further doubled hydrocarbon titers. Metabolic rewiring of the pathways related to fatty acid activation, lipid turnover, and carotenoid biosynthesis showed that CRT1 deletion had the most pronounced effect, increasing hydrocarbon titer by 2.5-fold. Additional improvements were achieved by reinforcing free fatty acid supply and increasing CvFAP copy number. The final strain R. toruloides NF72D produced 557.7 mg/L hydrocarbons in flask culture, over 37-fold higher than that by the initial cytosolic expression strain. Collectively, this study demonstrates the importance of proper localization of key enzymes for improved biosynthesis of fatty acid-derived metabolites and a great potential for light-driven production of renewable hydrocarbons by oleaginous yeasts.