The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol

Background Punicic acid is a conjugated fatty acid with a wide-range of nutraceutical properties naturally present in pomegranate seed oil. To meet the rising demand for pomegranate seed oil, a single-cell oil enriched in punicic acid provides a sustainable biomass-derived alternative. This study describes the production of a punicic acid-enriched single-cell oil through the engineering of the red yeast Rhodotorula toruloides grown in glucose and a low-cost substrate, crude glycerol. Results The gene for Punica granatum fatty acid conjugase, PgFADX , was randomly integrated into the genome of R. toruloides without disrupting the carotenoid synthesis. In shake flask studies, the effects of three promoters (P _PGI1 , P _NAR1 , and P _PMA1 ) on punicic acid production were evaluated. A punicic acid titer of 105.77 mg/L and 72.81 mg/L was obtained from engineered cells expressing PgFADX from the P _PMA1 promoter cultivated for 72 hours in glucose and for 168 hours in crude glycerol, respectively. Furthermore, the detailed lipid analysis revealed a high enrichment of punicic acid in the triacylglycerol lipid structures, even without substantial modifications to the metabolic pathways. Conclusions This report demonstrates the high potential of R. toruloides in the biotransformation of a low-cost substrate, crude glycerol, into a value-added products such as punicic acid. The findings support the feasibility of using engineered R. toruloides as a sustainable and efficient platform for the production of punicic acid-enriched single-cell oil.