Expanding toolkits of EPA/DHA enrichment from natural sources is essential for better satisfying increasing demands for them. Lipase K80, from Proteus vulgaris K80, showed an application potential in EPA/DHA enrichment, whereas no desired heterologous expression in generally regarded as safe (GRAS) hosts restricted its relevant applications. In this study, expression of lipase K80 in a well-reputed GRAS host, Pichia pastoris, was achieved and further enhanced via combining disruption of its C-terminal KKL motif with co-expression of N-Acetyltransferase Mpr1, with a cumulative increment of nearly 200% in the secretion level and the volumetric activity. Its application in EPA/DHA enrichment from fish oil was thereafter obtained with merits of low temperature and much less time, yielding an increase of ~31% in their total percentage. To gain mechanistic insights into its substrate chain-length specificity, we performed molecular dynamics simulation and revealed the substrate-dependent significant yet divergent conformational shifts of predominantly distal surface-exposed regions, suggesting a predominant long-range modulation mechanism. Together, this work provided in-depth insights into substrate specificity of lipase K80 and an alternate engineering site, the C-terminal KKL motif, for its expression optimization in P. pastoris, as well as extended toolboxes of EPA/DHA enrichment and application scopes of lipase K80.