Biotransformation of C20- and C22-polyunsaturated fatty acids and fish oil hydrolyzates to R,R-dihydroxy fatty acids as lipid mediators using double-oxygenating 15R-lipoxygenase

C20- and C22-dihydroxy fatty acids (DiHFAs) are bioactive lipid mediators (LMs) in humans. Among them, leukotrienes are inflammatory mediators, whereas resolvins generated by M2 macrophages in humans are anti-inflammatory mediators. However, the synthesis of LMs by chemical or biological methods is inefficient. Here, we discovered a double-oxygenating arachidonate (ARA) 15R-lipoxygenase (15R-LOX) with the highest catalytic activity among the reported ARA LOXs. Cells expressing double-oxygenating 15R-LOX converted ARA, eicosapentaenoic acid, and docosahexaenoic acid (DHA) and DHA-rich fish oil hydrolyzates into 5R,15R-dihydroxyeicosatetraenoic acid, 5R,15R-dihydroxyeicosapentaenoic acid, and 7R,17R-dihydroxydocosahexaenoic acid as isomers of leukotriene B4, resolvin E4, and resolvin D5, which were identified as new compounds, within 1.5 h, with concentrations >1.5 g L⁻¹, conversions >77% (w/w), and productivity >1.0 g L⁻¹ h⁻¹, respectively. Double-oxygenating 15R-LOX was altered to single-oxygenating 15R-LOX by the L606F mutation using structure-guided engineering. Cells expressing single-oxygenating 15R-LOX produced 15R-hydroxyeicosatetraenoic acid, 15R-hydroxyeicosapentaenoic acid, and 17R-hydroxydocosahexaenoic acid, respectively. These results demonstrated that the six medicinally important LMs could be prepared from C20- and C22-polyunsaturated fatty acids and inexpensive DHA-rich fish oil hydrolyzates by cost-effective, eco-friendly, and efficient biosynthesis using the discovered double-oxygenating and engineered single-oxygenating LOXs.