Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (kcat/Km) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25 °C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2 g/l cells, and 7 mM ARA as substrate in a 500 ml-Erlenmeyer baffled flask with 50 ml reaction solution with agitation at 200 rpm in the presence of 10 mM cysteine as a reduction agent, respectively. Under these conditions, 6.4 mM 9S-hydroxyeicosatetraenoic acid, 6.2 mM 9S-hydroxyeicosapentaenoic acid, and 5.9 mM 11S-hydroxydocosahexaenoic acid were produced in 30 min, 40 min, and 60 min with specific productivities of 1067 µmol/min/g, 775 µmol/min/g, and 492 µmol/min/g, volumetric productivities of 213 µM/min, 155 µM/min, and 98 µM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.

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

Leukotrienes (LTs) and maresins (MaRs) are human lipid mediators (LMs) involved in immune response and anti-inflammation, respectively. These compounds and their isomers are generated in trace amounts by lipoxygenases (LOXs) in human macrophages and neutrophils. These LMs have been synthesized using nonenvironmentally benign synthetic protocols, which are expensive. 8S- and 15S-LOXs with double dioxygenating activities have previously been reported, whereas 12S-LOX with double dioxygenating activity have not been reported to date. Here, we discovered a wild-type 12S-LOX with double dioxygenating activity from the bacterium Endozoicomonas numazuensis, which produced dihydroxy fatty acids (DiHFAs) as LMs from polyunsaturated fatty acids via double dioxygenation. The enzyme activity for producing DiHFA was approximately 550-fold higher than that of mammalian LOX with double dioxygenating activity. The microbial 12S-LOX converted 3.00 mM of arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid to 2.37 mM (797 mg/L) 6-trans-8-cis-12S-epimer of LTB4, 1.59 mM (532 mg/L) 6-trans-8-cis-12S-epimer of LTB5, 1.35 mM (498 mg/L) 10-cis-12-trans-7S-epimer of MaR1n-3 DPA , and 1.54 mM (555 mg/L) 10-cis-12-trans-7S-epimer of MaR1 within 2 h, which were 5.3-, 7.6-, 3.1-, and 5.5-fold higher than those biosynthesized by the previously reported microbial engineered 12S-LOX with double dioxygenating activity, respectively. These findings contribute to the efficient and environmentally friendly biosynthesis of LMs and stimulate physiological study on LMs.

Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells.

We evaluated the antioxidant activity and anti-melanogenic effects of Oenothera laciniata methanol extract (OLME) in vitro by using melan-a cells. The total polyphenol and flavonoid content of OLME was 66.3 and 19.0 mg/g, respectively. The electron-donating ability, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity, and superoxide dismutase (SOD)-like activity of OLME (500 µg/mL) were 94.5%, 95.6%, and 63.6%, respectively. OLME and arbutin treatment at 50 µg/mL significantly decreased melanin content by 35.5% and 14.2%, respectively, compared to control (p < 0.05). OLME and arbutin treatment at 50 µg/mL significantly inhibited intra-cellular tyrosinase activity by 22.6% and 12.6%, respectively, compared to control (p < 0.05). OLME (50 µg/mL) significantly decreased tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor-M (MITF-M) mRNA expression by 57.1%, 67.3%, 99.0%, and 77.0%, respectively, compared to control (p < 0.05). Arbutin (50 µg/mL) significantly decreased tyrosinase, TRP-1, and TRP-2 mRNA expression by 24.2%, 42.9%, and 48.5%, respectively, compared to control (p < 0.05). However, arbutin (50 µg/mL) did not affect MITF-M mRNA expression. Taken together, OLME showed a good antioxidant activity and anti-melanogenic effect in melan-a cells that was superior to that of arbutin, a well-known skin-whitening agent. The potential mechanism underlying the anti-melanogenic effect of OLME was inhibition of tyrosinase activity and down-regulation of tyrosinase, TRP-1, TRP-2, and MITF-M mRNA expression.

Action Mechanism of Chamaecyparis obtusa Oil on Hair Growth.

This study was carried out to examine the action mechanism of Chamaecyparis obtusa oil (CO) on hair growth in C57BL/6 mice. For alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT) activities in the skin tissue, at week 4, the 3% minoxidil (MXD) and 3% CO treatment groups showed an ALP activity that was significantly higher by 85% (p < 0.001) and 48% (p < 0.05) and an γ-GT activity that was significantly higher by 294% (p < 0.01) and 254% (p < 0.05) respectively, as compared to the saline (SA) treatment group. For insulin-like growth factor-1 (IGF-1) mRNA expression in the skin tissue, at week 4, the MXD and CO groups showed a significantly higher expression by 204% (p < 0.05) and 426% (p < 0.01) respectively, as compared to the SA group. At week 4, vascular endothelial growth factor (VEGF) expression in the MXD and CO groups showed a significantly higher expression by 74% and 96% (p < 0.05) respectively, however, epidermal growth factor (EGF) expression in the MXD and CO groups showed a significantly lower expression by 66% and 61% (p < 0.05) respectively, as compared to the SA group. Stem cell factor (SCF) expression in the MXD and CO groups was observed by immunohistochemistry as significant in a part of the bulge around the hair follicle and in a part of the basal layer of the epidermis. Taking all the results together, on the basis of effects on ALP and γ-GT activity, and the expression of IGF-1, VEGF and SCF, which are related to the promotion of hair growth, it can be concluded that CO induced a proliferation and division of hair follicle cells and maintained the anagen phase. Because EGF expression was decreased significantly, CO could delay the transition to the catagen phase.