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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 (k

Peroxidation and reduction of 11S- and 13S-positions on C20 and C22 polyunsaturated fatty acids (PUFAs) by Escherichia coli expressing highly active arachidonate (ARA) 11S-lipoxygenase (11S-LOX) from Enhygromyxa salina with the reducing agent cysteine.The specific activity and catalytic efficiency of ARA 11S-LOX from E. salina were 4.1- and 91-fold higher than those of only reported ARA 11S-LOX from Myxococcus xanthus, respectively. The hydroxy fatty acids (HFAs) obtained by the biotransformation of ARA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexanoic acid (DHA) by Escherichia coli expressing 11S-LOX from E. salina in the presence of cysteine were identified as 11S-hydroxyeicosatetraenoic acid (11S-HETE), 11S-hydroxyeicosapentaenoic acid (11S-HEPE), 13S-hydroxydocosapentaenoic acid (13S-HDPA), and 13S-hydroxydocosahexaenoic acid (13S-HDHA), respectively. The recombinant cells converted 3 mM of ARA, EPA, DPA, and DHA into 2.9 mM of 11S-HETE, 2.4 mM 11S-HEPE, 1. 9 mM 13S-HDPA, and 2.2 mM 13S-HDHA in 60, 80, 120, and 120 min, corresponding to productivities of 72.5, 40.4, 18.5, and 22.4 μM minWe report the highest concentrations, conversion yields, and productivities of 11S- and 13S-hydroxy fatty acids from C20- and C22-PUFAs achieved via E. coli expressing highly active E. salina 11S-LOX.

C18 trihydroxy fatty acids (THFAs), plant oxylipins, are used as antifungal agents and vaccine adjuvants.

Platycodon grandiflorum (balloon flower) root (Platycodi radix, PR) is used as a health supplement owing to its beneficial bioactive properties. In the present study, the anti-inflammatory, antioxidant, and whitening effects of deglycosylated platycosides (saponins) from PR biotransformed by pectinase from Aspergillus aculeatus were investigated. The bioactivities of the platycosides improved when the number of sugar moieties attached to the aglycone platycosides was decreased. The deglycosylated saponins exhibited higher lipoxygenase inhibitory activities (anti-inflammatory activities) than the precursor platycosides and the anti-inflammatory compound baicalein. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pectinase-treated PR extract was higher than that of the non-treated PR extract. The trolox-equivalent antioxidant capacity (TEAC) assay showed improved values as the saponins were hydrolyzed. The tyrosinase inhibitory activities (whitening effects) of deglycosylated platycosides were higher than those of the precursor platycosides. Furthermore, 3-O-β-D-glucopyranosyl platycosides showed higher anti-inflammatory, antioxidant, and whitening activities than their precursor glycosylated platycosides. Therefore, 3-O-β-D-glucopyranosyl platycosides may improve the beneficial effects of nutritional supplements and cosmetic products.

Recombinant Escherichia coli cells expressing 8,11-linoleate diol synthase (LDS) from Penicillium chrysogenum convert oleic and palmitoleic acids to 8-hydroperoxy-9(Z)-octadecenoic acid (HPOME) and 8-hydroperoxy-9(Z)-hexadecenoic acid (HPHME) only, respectively. However, recombinant E. coli cells expressing Q889A variant 6,8-LDS from Penicillium oxalicum as an 8,11-LDS converted oleic and palmitoleic acids to 8,11-dihydroxy-9(Z)-octadecenoic acid (DiHOME) and 8,11-dihydroxy-9(Z)-hexadecenoic acid (DiHHME), respectively, which were identified using liquid chromatography-tandem mass spectrometry analysis. To select suitable variants for producing these compounds, position 889 of 6,8-LDS from P. oxalicum was substituted with other amino acids, and recombinant E. coli cells expressing Q889L and Q889A variants were chosen as the best biocatalysts for producing 8,11-DiHOME and 8,11-DiHHME, respectively. The optimal conditions for producing 8,11-DiHOME or 8,11-DiHHME using cells expressing Q889L or Q889A variant 6,8-LDS were pH 6.5 and 30 °C with 5% (v/v) dimethyl sulfoxide, 60 g L

A compound K-producing fungus was isolated from meju (fermented soybean brick) and identified as the generally recognized as safe (GRAS) strain Aspergillus tubingensis. The extracellular enzymes obtained after the cultivation of 6 days in the medium with 20 g/L citrus pectin as an inducer showed the highest compound K-producing activity among the inducers tested. Under the optimized conditions of 0.05 mM MgSO4, 55 °C, pH 4.0, 13.4 mM protopanaxadiol (PPD)-type ginsenosides, and 11 mg/mL enzymes, the extracellular enzymes from A. tubingensis completely converted PPD-type ginsenosides in the ginseng extract to 13.4 mM (8.35 mg/mL) compound K after 20 h, with the highest concentration and productivity among the results reported so far. As far as we know, this is the first GRAS enzyme to completely convert all PPD-type ginsenosides to compound K.

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

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. Th...

d-Tagatose, a low-calorie functional sweetener, is biotransformed from lactose via galactose. The discovery of an enzyme with 4-epimerization activity toward d-fructose to produce d-tagatose has be...

Platycodon grandiflorum root (Platycodi radix) saponins, platycosides, have been used as health supplements and food items for the treatment of respiratory disorders and pulmonary diseases. Deglycosylated saponins have been known to exert stronger biological effects than their glycosylated forms. In the present study, glycosylated platycosides in Platycodi radix extract were biotransformed into deglycosylated 3-O-β-D-glucopyranosyl platycosides, including 3-O-β-Dglucopyranosyl platycodigenin, 3-O-β-D-glucopyranosyl polygalacic acid, and 3-O-β-Dglucopyranosyl platyconic acid, by pectinase from Aspergillus aculeatus. This is the first report on the quantitative enzymatic production of 3-O-β-D-glucopyranosyl platycosides. The chemical structures of 3-O-β-D-glucopyranosyl platycosides were identified with LC/MS. Moreover, the biotransformation pathways of the three types of platycosides in Platycodi radix into 3-O-β-Dglucopyranosyl platycosides were established.

Medium-chain α,ω-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer-Villiger monooxygenases are used in the production of ω-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain α,ω-dicarboxylic acids from renewable long-chain fatty acids.

Enzyme cascade catalysis has critical problems in obtaining the high concentrations of products, such as the low stabilities and activities of biocatalysts and the inhibition by hydrophobic reactan...

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer–Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g−1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

One of the omega-3 essential fatty acids, docosahexaenoic acid (DHA), is a significant constituent of the cell membrane and the precursor of several potent lipid mediators. These mediators are considered to be important in preventing or treating several diseases. Resolvin D5, an oxidized lipid mediator derived from DHA, has been known to exert anti-inflammatory effects. However, the detailed mechanism underlying these effects has not yet been elucidated in human monocytic THP-1 cells. In the present study, we investigated the effects of resolvin D5 on inflammation-related signaling pathways, including the extracellular signal-regulated kinase (ERK)-nuclear factor (NF)-κB signaling pathway. Resolvin D5 downregulated the production of interleukin (IL)-6 and chemokine (C-C motif) ligand 5 (CCL5). Additionally, these inhibitory effects were found to be modulated by mitogen-activated protein kinase (MAPK) and NF-κB in lipopolysaccharide (LPS)-treated THP-1 cells. Resolvin D5 inhibited the LPS-stimulated phosphorylation of ERK and translocation of p65 and p50 into the nucleus, resulting in the inhibition of IL-6 and CCL5 production. These results revealed that resolvin D5 exerts anti-inflammatory effects in LPS-treated THP-1 cells by regulating the phosphorylation of ERK and nuclear translocation of NF-kappaB.

Double-oxygenating lipoxygenase (LOX) converted C20- and C22-polyunsaturated fatty acids (PUFAs) into C20 dihydroxy fatty acids (DiHFAs) as inflammatory mediators and C22 DiHFAs as specialized pro-resolving mediators, which are involved in the resolution of inflammation and infection in humans, and their isomers, respectively. However, the quantitative bioconversion of C20- and C22-PUFAs into 9S,15S- and 11S,17S-DiHFAs has been not attempted to date, respectively. Here, we performed the efficient quantitative production of 9S,15S- and 11S,17S-DiHFAs by Escherichia coli expressing 9S-LOX from Sphingopyxis macrogoltabida. The optimal bioconversion conditions of the C20 PUFA arachidonic acid or the C22-PUFA docosahexaenoic acid were pH 8.5, 35 °C, 6 mM substrate, and 5 g dry cells/L for C20 PUFAs or 7 g dry cells/L for C22-PUFAs, respectively. Under these conditions, E. coli expressing double-oxygenating 9S-LOX from S. macrogoltabida converted arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid

Platycosides, Platycodi radix (

11 S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid (DoPE) is a derivative of docosapentaenoic acid, a specialized pro-resolving mediator of inflammation such as lipoxins, resolvins, maresins, and protectins. PM

Lipoxygenases (LOXs) biosynthesize lipid mediators (LMs) as human signaling molecules. Among LMs, specialized pro-resolving mediators (SPMs) are involved in the resolution of inflammation and infection in humans. Here, the putative LOX from the bacterium Sphingopyxis macrogoltabida was identified as arachidonate 9S-LOX. The enzyme catalyzed oxygenation at the n-12 position of C20 and C22 polyunsaturated fatty acids (PUFAs) to form 9S- and 11S-hydroperoxy fatty acids, which were reduced to 9S- and 11S-hydroxy fatty acids (HFAs) by cysteine, respectively, and it catalyzed again oxygenation at the n-6 position of HFAs to form 9S,15S- and 11S,17S-DiHFAs, respectively. The regioselective residues of 9S-LOX were determined as lle395 and Val569 based on the amino acid alignment and homology models. The regioselectivity of the I395F variant was changed from the n-12 position on C20 PUFA to the n-6 position to form 15S-HFAs. This may be due to the reduction of the substrate-binding pocket by replacing the smaller Ile with a larger Phe. The V569W variant had a significantly lower second‑oxygenating activity compared to wild-type 9S-LOX because the insertion of the hydroxyl group of the first‑oxygenating products at the active site was seemed to be hindered by substituting a larger Trp for a smaller Val. The compounds, 11S-hydroxydocosapentaenoic acid, 9S,15S-dihydroxyeicosatetraenoic acid, 9S,15S-dihydroxyeicosapentaenoic acid, 11S,17S-hydroxydocosapentaenoic acid, and 11S,17S-dihydroxydocosahexaenoic acid, were newly identified by polarimeter, LC-MS/MS, and NMR. 11S,17S-DiHFAs as SPM isomers biosynthesized from C22 PUFAs showed anti-inflammatory activities in mouse and human cells. Our study contributes may stimulate physiological studies by providing new LMs.

To quantitatively hydroxylate 8S- and 10S-positions on polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase (8S-LOX). Hydroxylated products gained from the conversion of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z, AA), eicosapentanoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z, EPA), and (22:6Δ4Z,7Z,10Z,13Z,16Z,19Z, DHA) by recombinant E. coli cells containing 8S-LOX from mouse were identified as 8S-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetranoic acid (8S-HETE), 8S-hydroxy-5,9,11,14,17(Z,E,Z,Z,Z)-eicosapentanoic acid (8S-HEPE), and 10S-hydroxy-4,8,12,14,16,19(Z,E,Z,Z,Z,Z)-docosahexaenoic acid (10S-HDoHE), respectively. Under the optimal hydroxylation conditions of pH 7.5, 30 °C, 5% (v/v) ethanol, 15 g cells l−1, and 5 mM substrate, AA, EPA, and DHA were hydroxylated into 4.37 mM 8S-HETE, 3.77 mM 8S-HEPE, and 3.13 mM 10S-HDoHE for 60, 90, and 60 min, with 87, 75, and 63% molar conversions, respectively. To the best of our knowledge, this is the first quantitatively biotechnological production of 8S-HETE, 8S-HEPE, and 10S-HDoHE.

Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9 S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy- cis, cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy- cis, cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy- cis, cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.

This study was conducted to characterize recombinant α-l-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. The α-l-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-l-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-l-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL−1 α-l-rhamnosidase, and 30 mM rutin, α-l-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h−1. We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-l-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

The Platycodon grandiflorum root, Platycodi radix, a common vegetable, and its extract with glycosylated saponins, platycosides, have been used as food items and food health supplements for pulmonary diseases and respiratory disorders. Enzymes convert glycosylated saponins into deglycosylated saponins, which exhibit higher biological activity than glycosylated saponins. In this study, β-glucosidase from the hyperthermophilic bacterium Dictyoglomus turgidum converted platycosides in the Platycodi radix extract into deglucosylated platycosides. In addition, the enzyme completely converted platycoside E (PE), platycodin D3 (PD3), and platycodin D (PD) in Platycodi radix extract into deglucosylated platycodin D (deglu PD), which was first identified by nuclear magnetic resonance. The anti-inflammatory activities of deglu PD and deglucosylated Platycodi radix extract were higher than those of PE, PD3, PD, Platycodi radix extract, and baicalein, an anti-inflammatory agent. Therefore, deglucosylated Platycodi radix extract is expected to be used as improved functional food supplements.

Hepoxilins (HXs) and trioxilins (TrXs) are lipid mediators that regulate diverse physiological processes at trace amounts in humans. They have been synthesized by chemical methods, which have disadvantages such as low yield, byproduct formation, and use of toxic chemical reagents. Here, we reported the identification of ten types of HXs and TrXs synthesized by recombinant Escherichia coli expressing bacterial arachidonate 15-lipoxygenase and/or epoxide hydrolase using five polyunsaturated fatty acids (PUFAs). Of these, eight types were new. The discovery was due to the microbial enzymes with high activities for the same catalytic activities as those of human enzymes. We succeeded in the efficient, cost-effective, and environmentally friendly biotransformation of PUFAs into nine types of HXs and TrXs by cells, yielding >10 g L−1 of 14,15-HXB3 and 13,14,15-TrXB3, with conversion yields of >70%. Our study contributes to the environmentally friendly synthesis of medicinally important lipid mediators.

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.