Metabolic Engineering of <i>Yarrowia lipolytica</i> for Conversion of Waste Cooking Oil into Omega-3 Eicosapentaenoic Acid

Omega-3 polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA, C20:5), are crucial dietary fats known for their numerous health benefits. However, traditional sources of EPA, like fish oil, raise sustainability and environmental concerns, underscoring the need for alternative production methods. The engineered oleaginous yeast <i>Yarrowia lipolytica</i> has emerged as a promising candidate for sustainable production of EPA. This study explores the efficient production of EPA with an earlier engineered<i>Y. lipolytica</i> strain Y8412, utilizing waste cooking oil (WCO) as an alternative carbon source. While cofeeding WCO resulted in increased total lipid content, it also caused an increase in intracellular free fatty acid (FFA) levels, which can be toxic to cells and reduce EPA synthesis. To solve this issue, we first overexpressed <i>FAA1</i> and <i>GPD1</i> genes converting excess FFAs into triglycerides (TAGs). Additionally, we knocked out <i>TGL3</i>/<i>4</i> genes, which encode lipases linked to lipid bodies, to minimize the degradation of TAGs back into FFAs. The modified strains significantly reduced intracellular FFA levels and improved EPA production. Notably, the <i>TGL4</i> knockout strain Y8412T4^- showed 57% increase in EPA production titer and nearly 50% increase in carbon conversion yield compared to the parental strain Y8412 fed with glucose only. These findings suggest that preventing TAG degradation by knocking out <i>TGL4</i> is an effective approach for enhanced EPA production when WCO is used to partially replace glucose as the carbon source. This study offers an effective engineering strategy for low-cost, high-yield, and sustainable production of omega-3 fatty acids from waste feedstocks.