Oleaginous microalgae represent a valuable resource for the production of high-value molecules. Considering the importance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high-value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand; however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA.Recently, we have engineered the marine diatom to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG, we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native was taken forward for detailed characterisation. Overexpression of the endogenous was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment.This study provides further insight into lipid metabolism in and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.