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, <i>Yarrowia lipolytica</i> was engineered for punicic acid biosynthesis by expressing the <i>PgFADX</i> gene from <i>Punica granatum</i> and subsequently modified to evaluate the influence of distinct diacylglycerol acyltransferases on punicic acid accumulation. The effects of seven acyltransferases, originating from <i>P. granatum</i> or <i>Y. lipolytica</i>, 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 <i>Y. lipolytica</i> as a robust host for the sustainable production of conjugated fatty acids from waste substrates.