The Plasma Membrane H+-ATPase Promoter Driving the Expression of FADX Enables Highly Efficient Production of Punicic Acid in Rhodotorula toruloides Cultivated on Glucose and Crude Glycerol

Punicic acid (PuA) 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 PuA provides a sustainable biomass-derived alternative. This study describes the production of a PuA-enriched single-cell oil through the engineering of the red yeast Rhodotorula toruloides grown in glucose and a low-cost substrate, crude glycerol. 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 (PPGI1, PNAR1, and PPMA1) on PuA production were evaluated. PuA titers of 105.77 mg/L and 72.81 mg/L were obtained from engineered cells expressing PgFADX from the PPMA1 promoter cultivated for 72 h in glucose and for 168 h in crude glycerol, respectively. Furthermore, the detailed lipid analysis revealed a high enrichment PuA in the triacylglycerol lipid structures, even without substantial modifications to the metabolic pathways. This report demonstrates the high potential of R. toruloides in the upcycling of a low-cost substrate, crude glycerol, into a value-added product such as PuA. The findings support the feasibility of using engineered R. toruloides for sustainable production of PuA-enriched single-cell oil.