Human milk fat substitutes (HMFSs) are the structured lipids intended for infant formula. It provides energy and essential fatty acid for infant. HMFSs are mainly prepared by enzymatic method. In this study, we aim to explore the potential for producing HMFSs by fermentation using R. opacus. The results indicated that different compounds with chain length from 12 to 18, used as carbon source, could be incorporated into triacylglycerols directly. Polyunsaturated fatty acids in term of ARA, EPA, DHA could enter the kennedy pathway directly and involved in the biosynthesis of triacylglycerols. GC, UPLC-MS and C-NMR analysis demonstrated that typical structured lipids β-OPL (40.09%) was synthesized in R. opacus. Transcriptome analysis revealed that β-oxidation, fatty acid elongation and kennedy pathways existed in R. opacus. It was concluded that fatty acid supplied as carbon source could enter the kennedy pathways directly or via the de novo fatty acid biosynthesis pathway depending on the chain length, thus, affect the triacylglycerol species formed in the Rhodococcus opacus.
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