Lipoxygenases (LOXs) are implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators involved in immune cell signaling, most of which catalyze peroxidation of polyunsaturated fatty acids by distinct regio- and stereoselectivity. Current reports suggested that conserved amino acid, Gly in R-LOXs and Ala in S-LOXs, in the catalytic domain play an important role in determining the position as well as the stereochemistry of the functional group. Recently, we have confirmed that the catalytic specificity of cyanobacterial lipoxygenase, named Osc-LOX, with alanine at 296 was 13S-type toward linoleic acid, and producing a 17S- hydroxy-docosahexaenoic acid from docosahexaenoic acid (DHA). Here, we aimed to change the catalytic property of LOX from13S-LOX to 9R-LOX by replacing Ala with Gly and to produce a lipid mediators different from the wild-type using DHA. Finally, we succeeded in generating human endogenous a 13R-hydroxy-docosahexaenoic acid and a 13R,20-dihydroxy-docosahexaenoic acid from DHA through an enzymatic reaction using the Osc-LOX-A296G. Our study could enable physiological studies and pharmaceutical research for the 13R,20-dihydroxy-docosahexaenoic acid. (C) 2020 Elsevier Inc. All rights reserved.