Relieving an elongation bottleneck unlocks eicosapentaenoic acid biosynthesis in the industrial microalga Nannochloropsis oceanica.

Nannochloropsis oceanica is an industrially relevant marine microalga that accumulates high levels of lipids and synthesizes the health-beneficial ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). However, EPA biosynthesis in this alga is constrained by inefficient conversion of abundant C16 fatty acids into the C18 precursors required for downstream desaturation and elongation reactions. Here, we identified NoELO2, a low-abundance fatty acid elongase, as a key regulatory enzyme controlling early carbon flux into EPA biosynthesis in N. oceanica. Heterologous expression in yeast demonstrated that NoELO2 catalyzed elongation of C16 to C18 fatty acids. Subcellular localization revealed that NoELO2 resided in the chloroplast-associated endoplasmic reticulum, positioning it at a strategic interface between plastidial fatty acid synthesis and extraplastidial lipid metabolism. Boosting NoELO2 expression in N. oceanica substantially enhanced EPA accumulation without compromising growth. EPA content reached up to 66.1 mg/g dry weight (49.2% increase)-the highest value reported in transgenic N. oceanica achieved through manipulation of a single endogenous enzyme. Conversely, knockout of NoELO2 reduced EPA levels and triggered compensatory transcriptional changes in other elongase genes. Together, our results identify NoELO2 as a critical metabolic gatekeeper that alleviates a previously underappreciated C16 bottleneck in EPA biosynthesis. This work provides both mechanistic insight into fatty acid flux control in microalgae and a practical metabolic engineering strategy for improving ω-3 fatty acid production.