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1,3-Unsaturated fatty acid-2-palmitic acid triacylglycerol (UPU) is a key component of human milk fat substitutes. In this present study, a novel approach was employed to produce UPU by cell factory of Rhodococcus opacus. The resulting UPU displayed a remarkable fatty acid composition, with 37.6% docosahexaenoic acid (DHA), 26.3% palmitic acid (P) and 15.6% docosapentaenoic acid (DPA). Notably, palmitic acid dominated the sn-2 position with a content of 51.1%. Furthermore, 13C nuclear magnetic resonance analysis revealed the predominant presence of DHA at the sn-1,3 position and palmitic acid at the sn-2 position. Among the triacylglycerol species, β-DHA P DHA (6.6%) and β-DPA P DHA (6.6%) exhibited the highest levels. The fermentation process achieved a biomass of 2.1 g L-1and an oil content of 39.9% (DCW). The yield of UPU reached 0.89 g L-1. This study presented a novel method for manufacturing 1,3-unsaturated fatty acid-2-palmitic acid triacylglycerol enriched with DHA.