Altering 15‐Lipoxygenases to 18‐Lipoxygenases and Their Application to the Production of 5,18‐Dihydroxyeicosapentaenoic Acids

TL;DR

Scientists engineered enzymes to efficiently produce anti-inflammatory compounds from eicosapentaenoic acid, achieving much higher concentrations than before.

Contribution

First identification of 18-LOXs and first qualitative production of RvE2 and 18S-RvE2 using engineered enzymes.

Findings

Engineered 18R-LOX and 18S-LOX achieved >105-fold higher concentrations of 18R-HEPE and 18S-HEPE than previously reported.

5S-LOX converted 18R- and 18S-HEPE into RvE2 and 18S-RvE2 with measurable yields.

E. coli expressing the engineered enzymes produced 641 mg/L of 18R-HEPE and 577 mg/L of 18S-HEPE in 20 minutes.

Abstract

Resolvin E2 (RvE2), 5S,18R‐dihydroxyeicosapentaenoic acid (5S,18R‐DiHEPE), and 18S‐RvE2 (5S,18S‐DiHEPE) are specialized pro‐resolving mediators that function in the resolution of inflammation. These SPMs have been produced in trace amounts from eicosapentaenoic acid (EPA) using acetylated cyclooxygenase‐2 or cytochrome P450 and 5‐lipoxygenase (5‐LOX) via 18R‐ and 18S‐hydroxyeicosapentaenoic acid (18R‐ and 18S‐HEPE) intermediates. In this study, we engineered 15R‐LOX from Sorangium cellulosum and 15S‐LOX from Archangium violaceum into 18R‐LOX (L423W/L424M/L568M variant of 15R‐LOX) and 18S‐LOX (L429W/L430M/L575M variant of 15S‐LOX), respectively, via structure‐guided enzyme engineering. The engineered 18R‐LOX converted EPA into 72.5% 18R‐HEPE and 27.5% 15R‐HEPE, while the engineered 18S‐LOX formed 81.8% 18S‐HEPE and 18.2% 15S‐HEPE. Escherichia coli expressing the engineered 18R‐ or 18S‐LOX converted 4.0 or 3.0 mM EPA into 2.0 mM (641 mg/L) 18R‐HEPE or 1.8 mM (577 mg/L) 18S‐HEPE in 20 min, respectively, achieving concentrations that were > 105‐fold higher than those reported previously. Furthermore, 5S‐LOX from Danio rerio (zebrafish) converted a concentration of 0.5 mM of the prepared 18R‐ or 18S‐HEPE into 0.24 mM (81 mg/L) RvE2 or 0.22 mM (74 mg/L) 18S‐RvE2 in 30 min, respectively. To the best of our knowledge, this represents the first identification of 18‐LOXs and first qualitative production of RvE2 and 18S‐RvE2.

15R‐ and 15S‐lipoxygenases (15R‐ and 15S‐LOXs) were engineered into 18R‐ and 18S‐LOXs, respectively. Escherichia coli expressing these enzymes converted eicosapentaenoic acid into 18R‐ and 18S‐hydroxyeicosapentaenoic acids (18R‐ and 18S‐HEPEs), respectively. 5S‐LOX converted 18R‐ and 18S‐HEPEs into resolvin E2 (RvE2), 5S,18R‐dihydroxyeicosapentaenoic acid (5S,18R‐DiHEPE), and 18S‐RvE2 (5S,18S‐DiHEPE), respectively.

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