Synergistic genetic module engineering for optimized eicosapentaenoic acid production in Nannochloropsis oceanica

Abstract

Eicosapentaenoic acid (EPA), an essential omega-3 polyunsaturated fatty acid, provides numerous health benefits and is a valuable resource for the biofuel and nutraceutical industries. This study aims to enhance EPA production in Nannochloropsis oceanica through synergistic genetic module engineering. Five key enzymes involved in EPA biosynthesis —Δ12 desaturase (Δ12D), Δ6 desaturase (Δ6D), Δ6 elongase (Δ6E), Δ5 desaturase (Δ5D), and ω3 desaturase (ω3D) —were co-expressed in various combinations in the heterologous expression system Saccharomyces cerevisiae to evaluate their synergistic effects on EPA production. Based on these findings, proportional combinations of Δ12D-Δ6D and Δ5D-ω3D modules were introduced into N. oceanica individually and jointly. Their impacts on lipid and EPA yield under different nutrient conditions were systematically investigated. The results demonstrated that overexpressing the Δ12D-Δ6D module, along with Δ6E significantly enhanced the ratio of the EPA precursors, C18:3 and C20:4, in total fatty acid under both nitrogen-replete and nitrogen-deplete conditions. In contrast, overexpression of the Δ5D-ω3D module, along with Δ6E, significantly increased EPA content by up to 34.96 % under nitrogen-replete conditions. Under nitrogen-deficient conditions, this genetic modification enhanced EPA content in triacylglycerol (TAG) form by up to 40.47 % and boosted overall TAG yield by 97.43 %. This study highlights the potential of gene stacking technology of Δ5D and ω3D to enhanced metabolic flux and improve EPA synthesis in microalgae. These findings offer a promising strategy for optimizing EPA production and pave the way for the development of genetically engineered strains capable of producing high-value fatty acids at an industrial scale.