Efficient One‐Step Production of 7<i>S</i>,17<i>S</i>‐ and 10<i>S</i>,17<i>S</i>‐Dihydroxydocosahexaenoic Acids by a Double‐Oxygenating 15<i>S</i>‐Lipoxygenase From <i>Chlamydomonas incerta</i>

Specialized pro-resolving mediators (SPMs), such as resolvin D5 (7S,17S-dihydroxydocosahexaenoic acid, 7S,17S-DiHDHA; RvD5) and protectin DX (10S,17S-DiHDHA; PDX), are critical in resolving inflammation in humans. In this study, a unique double-oxygenating 15S-lipoxygenase (15S-LOX) from the alga Chlamydomonas incerta was identified and characterized for its ability to simultaneously produce RvD5 and PDX from docosahexaenoic acid (DHA). Recombinant Escherichia coli expressing the C. incerta 15S-LOX demonstrated enhanced RvD5 and PDX production under the following optimized reaction conditions: pH 8.0, 25°C, 0.5 g dry cells/L, 7.0 mM DHA, 2.0% (w/v) PVP, 2.0% (v/v) DMSO, and 200 mM cysteine used as a reductant. This one-step biocatalytic process produced 2.91 mM (1.05 g/L) RvD5 and 2.18 mM (0.78 g/L) PDX in 90 min, with a total of 5.09 mM (1.83 g/L) and a total conversion yield of 79.6% (w/w). Compared to previously reported two-step biocatalytic processes, this one-step process significantly enhanced the production of particular PDX with improved productivity and simplicity. Structural analysis identified residues Phe667, Ile705, and Leu713 as regioselectivity modulators for the second oxygenation step. This study demonstrates the efficiency and industrial potential of the double-oxygenating LOX as a biocatalyst for simultaneously producing RvD5 and PDX.