Lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids.Notably, most microbial lipoxygenases including the lipoxygenase from Pseudomonas aeruginosa (Pa-LOX) catalyze oxygenation of linoleic acid and arachidonic acid into 13S-hydroperoxyoctadecenoic acid (13S-HPODE) and 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE), resp.Therefore, this study has focused on modification of positional specificity or regioselectivity of Pa-LOX.The linoleic acid oxygenations and substrate-docking simulations suggested that the regioselectivity of Pa-LOX might depend on the geometry of the hydrocarbon tail-binding cavity.Therefore, the interior end of the substrate-binding cavity was enlarged to make C10 instead of C13 face the iron active site.Remarkably, the M434G mutation led to alteration of the oxygenation products from 15S-hydroxyeicosatetraenoic acid (15S-HPETE) to 11S-HPETE as the major product from arachidonic acid.On the other hand, the Y609G substitution allowed the formation of 8S-HPETE from arachidonic acid. 8S-HPETE was recovered after reduction by tris(2-carboxyethyl)phosphine hydrochloride with an isolated yield of 62% with a purity of 94% via Escherichia coli-based whole-cell biocatalysis, solvent extraction, and silica gel chromatog.This is the first report of the production of 11S-HPETE and 8S-HPETE from arachidonic acid at high conversions.Therefore, this study contributes to the preparation of biol. active oxylipins from renewable fatty acids in a sustainable way.