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Abstract1,3‐Dioleoyl‐2‐palmitoylglycerol (OPO) is the main triacylglycerol species in human milk‐fat substitute. The production of OPO is of considerable research interest. In this study, a new strategy for producing OPO by fermentation with R. opacus is proposed. Chemically Interesterified fat (high oleic acid sunflower oil/hydrogenated palm oil 1.73:1 w/w), or a mixture of ethyl oleate/ethyl palmitate 2:1 (w/w) as a starting material. The highest biomass and oil content obtained were 3.3 g L−1 and 40.2% (dry cell weight), respectively. The yield of OPO was 0.62 g L−1. The fatty acid composition of produced OPO was 55.7–59.7% 18:1 and 28.3–29.8% 16:0, and the sn‐2 position was predominantly 16:0 (64.7–74.5%). 13C‐nuclear magnetic resonance analysis showed that the sn‐1,3 and sn‐2 positions were predominately esterified by 18:1 and 16:0, respectively. OPO (47.1%), OPL (13.9%), PPO (13.1%), and PPoO (16:0–16:1–18:1) (10.3%) were the most abundant triacylglycerol species.

Odd‐chain fatty acids (OCFA) are widely used in pharmaceutical and food industries, as well as the chemical industry. In this study, oil rich in OCFA was produced by Rhodococcus opacus PD630, and 1‐propanol in combination with glucose was used as the carbon and energy source. Oil was extracted by the subcritical extraction system. Gas chromatography (GC), ultra‐performance liquid chromatography‐mass spectrometry (UPLC‐MS), and differential scanning calorimetry (DSC) were used to evaluate the fatty acids composition, triacylglycerol (TAG) molecular species, and thermal properties, respectively. It was found that the content of OCFA reached 68.15%, where pentadecanoic acid (7.74%), heptadecanoic acid (18.20%), and heptadecenoic acid (42.71%) were the dominant OCFA. The most abundant TAG were HePaHa (15.82%), HePHa (11.42%), HePaHe (9.68%), HeHaHa (9.62%), HePaO (8.26%), and HePPa (8.00%). In terms of thermal profiles, recrystallization was detected in the oil rich in OCFA. Above all, these findings greatly extend the utilization of the microbial oil rich in OCFA, and they may have a significant impact on the future development of the microbial oil industry.