Enzymatic Formation of Protectin Dx and Its Production by Whole-Cell Reaction Using Recombinant Lipoxygenases

Abstract

In the human body, docosahexaenoic acid (DHA) contained in fish oil is converted to trace amounts of specialized pro-resolving mediators (SPMs) as the principal bioactive metabolites for their pharmacological effects. Protectin Dx (PDX), an SPM, is an important medicinal compound with biological activities such as modulation of endogenous antioxidant systems, inflammation pro-resolving action, and inhibition of influenza virus replication. Although it can be biotechnologically synthesized from DHA, it has not yet been produced quantitatively. Here, we found that 15S-lipoxygenase from Burkholderia thailandensis (BT 15SLOX) converted 10S-hydroxydocosahexaenoic acid (10S-HDHA) to PDX using enzymatic reactions, which was confirmed by LC-MS/MS and NMR analyses. Thus, whole-cell reactions of Escherichia coli cells expressing BT 15SLOX were performed in flasks to produce PDX from lipase-treated DHA-enriched fish oil along with E. coli cells expressing Mus musculus (mouse) 8S-lipoxygenase (MO 8SLOX) that converted DHA to 10S-HDHA. First, 1 mM DHA (DHA-enriched fish oil hydrolysate, DFOH) was obtained from 455 mg/L DHA-enriched fish oil by lipase for 1 h. Second, E. coli cells expressing MO 8SLOX converted 1 mM DHA in DFOH to 0.43 mM 10S-HDHA for 6 h. Finally, E. coli cells expressing BT 15SLOX converted 0.43 mM 10S-HDHA in MO 8SLOX-treated DFOH to 0.30 mM (108 mg/L) PDX for 5 h. Consequently, DHA-enriched fish oil at 455 mg/L was converted to 108 mg/L PDX after a total of 12 h (conversion yield: 24% (w/w); productivity: 4.5 mg/L/h). This study is the first report on the quantitative production of PDX via biotechnological approaches.

Biotransformation of docosahexaenoic acid into 10R,17S- dihydroxydocosahexaenoic acid as protectin Dx 10-epimer by serial reactions of arachidonate 8R- and 15S-lipoxygenases

“…that of E. coli expressing B. thailandensis 15S-LOX (Table1), which was used for the biotransformation of 10S-HDHA into PDX(Shin et al, 2022). These results suggest that E. coli expressing A. violaceum 15S-LOX to produce 10-epi PDX is a more e cient biocatalyst than E. coli expressing B. thailandensis 15S-LOX to produce PDX.…”

“…16,17-Hepoxilin B 5 (15R-hydroxy-16S,17S-epoxy-docosahexaenoic acid) was prepared as previously reported (Lee et al, 2019). 10R-HDHA and 10-epi PDX, obtained from the conversion of DHA and 10R-HDHA by P. homomalla 8R-LOX and A. violaceum 15S-LOX, respectively, were puri ed using a preparative HPLC system as described previously (Kim et al, 2021;Lee et al, 2020;Oh et al, 2022;Shin et al, 2022). The puri ed 10R-HDHA was used as both a standard and substrate to produce 10-epi PDX, and the puri ed 10-epi PDX was used as a standard.…”

“…The retention times of PDX and 10-epi PDX also differed in the LC/MS chromatograms (Serhan et al, 2006). 15S-LOX converted 10S-HDHA into PDX (10S,17S-DiHDHA) (Shin et al, 2022), indicating that the product obtained from the conversion of 10R-HDHA by A. violaceum 15S-LOX was 10epi PDX (10R,17S-DiHDHA).…”

“…For instance, bacterial double-oxygenating ARA 9S-, 12S-, and 15S-LOXs convert PUFAs into 170 − 2120 mg/L of SPM and SPM isomers, including 10S,17Sdihydroxydocosahexaenoic acid (11S,17S-DiHDHA) and 11S,17S-dihydroxydocosapentaenoic acid (11S,17S-DiHDPA) as the 11-isomers of protectin Dx (PDX, 10S,17S-DiHDHA) and PDX n−3 (10S,17S-DiHDPA) (Oh et al, 2022), 7S,14S-DiHDHA and 7S,14S-DiHDPA as the 10-cis-12-trans-7S-epimers of maresin 1 (MaR1) (7R,14S-DiHDHA) and MaR1 n − 3 (7R,14S-DiHDPA) (Kim et al, 2021), and resolvin E4 (RvE4, 5S,15S-dihydroxyeicosapentaenoic acid, 5S,15S-DiHEPA), resolvin D5 (RvD5, 7S,17S-DiHDHA), and RvD5 n − 3 (7S,17S-DiHDPA) (Lee et al, 2020), respectively. In contrast, the SPM PDX is synthesized by the combined reactions of 8S-and 15S-LOXs (Shin et al, 2022) because mouse double-oxygenating 8S-LOX, the only reported 8S-LOX, shows 310 − 680-fold lower activity in the second oxygenation than the bacterial double-oxygenating LOXs (Kim et al, 2021;Lee et al, 2020;Oh et al, 2022). Protectin Dx 10epimer (10-epi PDX, 10R,17S-DiHDHA) can only be produced by the combined reactions of singleoxygenating 8R-and 15S-LOXs because there are no double-oxygenating LOXs that perform both Rand S-form oxygenation.…”

“…The biosynthetic pathways from DHA to PD1 and PDX are constructed by utilizing different mechanisms of 15S-LOX(An et al, 2021). The epoxidation activity of 15S-LOX converts DHA into the intermediate 16S,17S-epoxy-DHA, which is hydrolyzed to PD1 (10R,17S-11E,13E,15Z-triene) by epoxide hydrolase(Aursnes et al, 2015), whereas the oxygenation activity of 15S-LOX converts DHA into the intermediate 17S-HDHA under reduction conditions, which is converted into PDX (10S,17S-11E,13Z,15E-triene) by its double oxygenation activity or 8S-LOX(Shin et al, 2022) (Figure…”