Structured odd-chain PUFA TAGs in Pythium: a precursor-guided biosynthesis strategy

Structured triacylglycerols (TAGs) were biosynthetically engineered in Pythium oligandrum ATCC 38472 using a precursor-directed approach. KEGG pathway analysis confirmed that TAG assembly proceeds via the Kennedy pathway. Supplementation with valine, leucine, isoleucine, propionic acid, and pivalic acid promoted the incorporation of odd-chain polyunsaturated fatty acids (odd-PUFAs), with isoleucine, particularly in combination with TWEEN 80, showing the greatest effect. Lipidomic profiling (RP-HPLC-MS/MS) revealed enhanced odd-PUFA esterification at the sn-1 position of lysophosphatidic and phosphatidic acids, while diacylglycerols acted as key intermediates. Two-dimensional and chiral LC-MS/MS confirmed that TWEEN 80 modulates PUFA distribution within TAGs, enabling controlled molecular design. These findings demonstrate a sustainable microbial route for producing structured lipids enriched in odd-PUFAs. The resulting TAGs exhibit tunable structural and nutritional attributes, supporting their potential use as functional lipid ingredients in food formulations and nutraceutical applications.