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 <i>Rhodotorula toruloides</i> grown in glucose and a low-cost substrate, crude glycerol. The gene for <i>Punica granatum</i> fatty acid conjugase, <i>PgFADX</i>, was randomly integrated into the genome of <i>R. toruloides</i> without disrupting the carotenoid synthesis. In shake flask studies, the effects of three promoters (P<i>_PGI1</i>, P<i>_NAR1</i>, and P<i>_PMA1</i>) on PuA production were evaluated. PuA titers of 105.77 mg/L and 72.81 mg/L were obtained from engineered cells expressing <i>PgFADX</i> from the P<i>_PMA1</i> 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 <i>R. toruloides</i> 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 <i>R. toruloides</i> for sustainable production of PuA-enriched single-cell oil.