A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg(-1) was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k cat/K m) were the highest for linoleic acid (k cat of 93.7 s(-1) and K m of 41.5 μM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l(-1) linoleic acid, 2.5 g l(-1) enzyme, 0.1 mM Cu(2+), and 6% (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l(-1) 13-HODE (70.2 mM) from 20 g l(-1) linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5% and productivity of 10.4 g l(-1) h(-1). The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158% higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.

A new biotransformation process for the production of the flavor lactone was developed by using permeabilized Waltomyces lipofer, which was selected as an efficient ␥-dodecalactone-producing yeast among 10 oleaginous yeast strains. The optimal reaction conditions for ␥-dodecalactone production by permeabilized W. lipofer cells were pH 6.5, 35°C, 200 rpm, 0.7 M Tris, 60 g/liter of 10-hydroxystearic acid, and 30 g/liter of cells. Under these conditions, nonpermeabilized cells produced 12 g/liter of ␥-dodecalactone after 30 h, with a conversion yield of 21% (wt/wt) and a productivity of 0.4 g/liter/h, whereas permeabilized cells obtained after sequential treatments with 50% ethanol and 0.5% Triton X-100 produced 46 g/liter of ␥-dodecalactone after 30 h, with a conversion yield of 76% (wt/wt) and a productivity of 1.5 g/liter/h. These values were 3.7-and 3.8-fold higher than those obtained using nonpermeabilized cells. These are the highest reported concentration, conversion yield, and productivity for the production of the bioflavor lactone.

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.

No abstract

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l(-1) yeast extract, 10 g l(-1) peptone, 5 g l(-1) oleic acid, 1 g l(-1) glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l(-1) hydroxy fatty acid, and 20 g l(-1) cells. Non-induced cells produced 38 g l(-1) γ-dodecalactone from 60 g l(-1) 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l(-1) h(-1) under the optimized conditions, whereas induced cells produced 51 g l(-1) γ-dodecalactone from 60 g l(-1) 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l(-1) h(-1). The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l(-1) γ-decalactone and 12 g l(-1) γ-butyrolactone from 60 g l(-1) 12-hydroxystearic acid and 60 g l(-1) 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

Candida boidinii was selected as a γ-dodecelactone producer because of the highest production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid among the 11 yeast strains tested. Under the reaction conditions of pH 5.5 and 25 °C with 5 g/L 10-hydroxy-12(Z)-octadecenoic acid and 30 g/L cells, whole C. boidinii cells produced 2.1 g/L γ-dodecelactone from 5 g/L 10-hydroxy-12(Z)-octadecenoic acid after 6 h, with a conversion yield of 64% (mol/mol) and a volumetric productivity of 350 mg/L/h. The production of γ-dodecelactone from safflower oil was performed by lipase hydrolysis reaction and two-step whole-cell biotransformation using Stenotrophomonas nitritireducens and C. boidinii. γ-Dodecelactone at 1.88 g/L was produced from 7.5 g/L safflower oil via 5 g/L 10-hydroxy-12(Z)-octadecenoic acid intermediate by these reactions after 8 h of reaction time, with a volumetric productivity of 235 mg/L/h and a conversion yield of 25% (w/w). To the best of the authors' knowledge, this is the highest volumetric productivity and conversion yield reported to date for the production of γ-lactone from natural oils.

Hepoxilins (HXs) and trioxilins (TrXs) are lipid mediators that regulate diverse physiological processes at trace amounts in humans.