Discovery and Engineering of a Microbial Double-Oxygenating Lipoxygenase for Synthesis of Dihydroxy Fatty Acids as Specialized Proresolving Mediators

Abstract

Specialized proresolving mediators (SPMs), such as resolvins and maresins, resolve inflammation and protect against infection at trace amounts generated by neutrophils and macrophages in humans. Thus, SPMs have been leading compounds in treatment of inflammation and infection. These mediators have been synthesized using chemical methods, which have disadvantages such as use of toxic chemical reagents, many (20−30) step reactions, and expensive processes. Here, we first discovered a human SPM-producing microorganism and found a 15-lipoxygenase (15-LOX) from the microorganism that produces SPMs via double oxygenation from polyunsaturated fatty acids. The 15-LOX was altered to 12-LOX using structure-based engineering. We performed the quantitative biocatalytic synthesis of SPMs by wild-type 15-LOX and engineered 12-LOX. Owing to the high double-oxygenating activities of LOXs, we succeeded in the one-enzyme biotransformation of docosahexaenoic acid and docosapentaenoic acid into the SPM resolvin D5 (RvD5, 7S,17S-dihydroxydocosahexaenoic acid) and RvD5 n−3 DPA (7S,17Sdihydroxydocosapentaenoic acid) (>40% conversion), respectively, by Escherichia coli expressing wild-type 15-LOX and into the 7Sepimer of maresin (MaR) 1 (7S,14S-dihydroxydocosahexaenoic acid) and 7S-epimer of MaR1 n−3 DPA (7S,14S-dihydroxydocosapentaenoic acid) (>20% conversion), respectively, by E. coli expressing engineered 12-LOX. Our study contributes to the environmentally friendly synthesis of medicinally important SPMs.

A New E-Series Resolvin: RvE4 Stereochemistry and Function in Efferocytosis of Inflammation-Resolution

“…using recombinant human 5- and 15-LOX co-incubations in vitro ( 18 ). Recently, EPA was shown to be converted by wild type and recombinant engineered lipoxygenases to double dioxygenation products 5 S ,12 S -diHEPE and RvE4 on large-scale studies ( 30 ). In addition, it is possible that the lipoxygenation of EPA is initiated via hydrogen abstraction at carbon position C7 by 5-LOX ( Figure 5 ) to biosynthesize RvE4 in human neutrophils or by transcellular biosynthesis ( 31 ).…”

Biocatalytic synthesis of dihydroxy fatty acids as lipid mediators from polyunsaturated fatty acids by double dioxygenation of the microbial 12S‐lipoxygenase

“…The chiral column was eluted at 30°C with solvent mixtures of n-hexane, isopropyl alcohol, and acetic acid (92:8:0.1%, v/v/v) at a flow rate of 1 mL/min for 35 min. (Lee et al, 2020), respectively. Although the amino acid sequence of the putative protein showed 10%-30% identity with those of other LOXs, five metal-binding residues, which acted as catalytic residues, and a determinant residue involved in stereoselectivity (Coffa site) were completely conserved (Figure S2), suggesting that the putative protein is a LOX.…”

“…Among LMs, leukotrienes (LTs) as EpFAs or DiHFAs are inflammatory mediators (Peters‐Golden et al, 2005), the EpHFA hepoxilins (HXs) are involved in calcium migration and insulin secretion (Munoz‐Garcia et al, 2014), and the DiHFA maresins (MaRs) are pro‐resolving inflammatory mediators (Abdulnour et al, 2014). MaR types can be distinguished by the chirality and position of the hydroxyl group on the carbon of DHA as MaR1 (7 R ,14 S ‐dihydroxy docosahexaenoic acid, DiHDHA), MaR2 (13 R ,14 S ‐DiHDHA), 7 S ‐epimer‐MaR1 (7 S ,14 S ‐DiHDHA), and 10‐ cis ‐12‐ trans ‐7 S ‐epimer‐MaR1 (10‐ cis ‐12‐ trans ‐7 S ,14 S ‐DiHDHA) (Lee et al, 2020; Serhan et al, 2012). The biological functions of the original human LMs, such as LTB4 (5 S ,12 R ‐dihydroxy eicosatetraenoic acid, DiHETE) and MaR1 (Abdulnour et al, 2014; Peters‐Golden et al, 2005) have been intensively studied, whereas those of their isomers, such as 6‐ trans ‐8‐ cis ‐12 S ‐epimer of LTB4 and 10‐ cis ‐12‐ trans ‐7 S ‐epimer of MaR1 have not.…”

Highly efficient oxidation of plant oils to C18 trihydroxy fatty acids by Escherichia coli co-expressing lipoxygenase and epoxide hydrolase

“…Plant oxylipins, including hydroxy fatty acids (HFAs), HpFAs, EHFAs, and THFAs, were prepared via a three-step process, including whole-cell reactions, prep-HPLC, and resin treatment. First, the whole-cell reactions of E. coli expressing A. violaceum LA 13 S -LOX, previously reported as ARA 15 S -LOX, 21 were performed at 30 °C in 50 mM 4-(2-hydroxyethyl)piperazinyl-1-propanesulfonic acid (HEPPS) buffer (pH 8.0) containing 2 mM PUFA ( 6 or 11 ), 5 g L −1 cells, and 5% (v/v) methanol with 10 mM cysteine as a reducing agent, with shaking at 200 rpm for 3 h to prepare the HFA standards 13 S -hydroxy-9 Z ,11 E ,15 Z -octadecatrienoic acid (compound 10 ; 13 S -HOTrE) and 13 S -hydroxy-6 Z ,9 Z ,11 E -octadecatrienoic acid (compound 15 ; 13 S -HOTrE(γ)). The cell reactions were carried out with 5 mM PUFA ( e.g.…”