Comparative Expression of Diacylglycerol Acyltransferases for Enhanced Accumulation of Punicic Acid-Enriched Triacylglycerols in Yarrowia lipolytica

Punicic acid is an uncommon ω-5 conjugated fatty acid with significant biological activity, mainly found in pomegranate seed oil. Due to limited natural availability, heterologous production of punicic acid in oleaginous yeasts offers a sustainable alternative. In this study, Yarrowia lipolytica was engineered for punicic acid biosynthesis by expressing the PgFADX gene from Punica granatum and subsequently modified to evaluate the influence of distinct diacylglycerol acyltransferases on punicic acid accumulation. The effects of seven acyltransferases, originating from P. granatum or Y. lipolytica, were compared under various cultivation conditions. The PgDGAT1 enzyme demonstrated the most favorable balance between total lipid content and punicic acid accumulation. Medium containing crude glycerol as a low-cost carbon source was initially tested in flask experiments with punicic acid accumulation in yeast cells of 129 mg/L. Further optimization of crude glycerol medium and subsequent scale-up experiments confirmed the potential of crude glycerol as an effective substrate, yielding up to 147.8 mg/L of punicic acid. Overall, this work identifies key enzymatic determinants for efficient punicic acid biosynthesis and supports Y. lipolytica as a robust host for the sustainable production of conjugated fatty acids from waste substrates.