Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing greater than 50 weight percent of eicosapentaenoic acid [“EPA”], an ω-3 polyunsaturated fatty acid, in the total oil fraction are described. These strains over-express heterologous Δ9 elongases, Δ8 desaturases, Δ5 desaturases, Δ17 desaturases, Δ12 desaturases and C16/18 elongases, and optionally over-express diacylglycerol cholinephosphotransferases. Preferred gene knockouts are described, as are methods for producing EPA within the host cells and products comprising EPA from the optimized Yarrowia lipolytica strains.