Diol Synthase Research Articles

A novel integrated membrane bioreactor-stirred tank bioreactor system for simultaneous biodegradation of bisphenol A contaminated wastewater and biopolyol production: An approach towards circular bioeconomy

The present work examined the biodegradation of bisphenol A (BPA) under batch and continuous modes using hydrocarbonoclastic bacterium, Pseudomonas aeruginosa PR3. The strain showed great biodegradation potential under batch shake flask and stirred tank reactor. Bacterial growth was considerably inhibited at initial BPA concentrations > 50 mg/L. Further, membrane bioreactor (MBR) for BPA biodegradation was also evaluated under different inlet BPA concentration and hydraulic retention time (HRT) and the maximum BPA removal efficiency of 94.4 % was attained with an influent BPA concentration of 25 mg/L at 5 d of HRT. MBR also showed better performance under shock loading conditions. Identification of BPA biodegraded pathways was also performed using liquid chromatography-mass spectrometry, elucidating the different metabolites and end products formed. A subsequent enhancement in BPA removal efficieny was also observed owing to the microfiltration system of MBR that was applied to separate bacterial biomass from effluent. The maximum removal efficiency was further enhanced to 98.5 % under the same operational schedule. As the strain has the ability to produce extracellular diol synthase enzyme which transforms oleic acid into the biopolyol 7,10-dihydroxy-8(E)-octadecenoic acid, the effluent containing the enzymes was supplemented with 1 % (v/v) oleic acid under whole cell and cell-free approaches. Cell-free technique showed an enhanced bioconversion efficiency in comparison to the traditional whole cell approach. Further, the produced biopolyol was characterized using Fourier transform infrared spectroscopy, thermogravimetry, differential scanning calorimetry, and 1H nuclear magnetic resonance analyses and also depicted the antibacterial activity.

The Biosynthesis of 1-octene-3-ol by a Multifunctional Fatty Acid Dioxygenase and Hydroperoxide Lyase in Agaricus bisporus.

The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and 10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.

Characterisation of the enzymes involved in the diol synthase metabolic pathway in Pseudomonas aeruginosa

This study is the first attempt to report the biochemical characterisation of the 10S-dioxygenase (10S-DOX) and 7S, 10S-diol synthase (7,10-DS). Both enzymes showed similar pH profiles with 10S-DOX presenting the highest activity at 30 °C whereas 7,10-DS did not show a clear preferred temperature. These differences were reflected in the thermostability assay, the Km values were 0.89 ± 0.22 mM and 3.26 ± 0.31 mM for 10S-DOX and 7,10-DS, respectively. Inductively coupled plasma mass spectrometry indicated that both enzymes contained bound to haem group Fe2+ as a prosthetic group: 10S-DOX (0.95 mol Fe2+/mol of protein) and 7,10-DS (1.18 mol Fe2+/mol of protein), respectively. Assays using metal cations as cofactors revealed that Mg2+ and Ni2+ enhance 7,10-DS activity, whereas Hg2+ decrease it up to 50 %. The activity of 10S-DOX in the presence of Mn2+ and Fe2+ was reduced to 51.6 % and 61.8. Aggregated proteins producing 10S-DOX and 7,10-DS were characterised as inclusion bodies: IBs-77 and IBs-78 respectively, was performed by Fourier Transform spectroscopy (FT-IR), Atomic Force Microscopy, dye binding, and proteolysis. The specific activity was 1.55 IU/mg for IBs-77 and 1.05 IU/mg for IBs-78. The presence of the oleate-diol synthase pathway in proteobacteria other than Pseudomonas aeruginosa was detected.

Pseudomonas aeruginosa Secretes the Oxylipin Autoinducer Synthases OdsA and OdsB via the Xcp Type 2 Secretion System.

The oxylipin-dependent quorum-sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two extracellular oxylipins, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, we implemented a genetic screen of P. aeruginosa strain PAO1 aimed to identify genes required for 10-HOME and 7,10-DiHOME production. Among the 14 genes identified, four encoded previously known components of the ODS and 10 encoded parts of the Xcp type II secretion system (T2SS). We subsequently created a clean xcpQ deletion mutant, which encodes the necessary outer membrane component of Xcp, and found it recapitulated the impaired functionality of the T2SS transposon mutants. Further studies showed that the ΔxcpQ mutant was unable to secrete the oxylipin synthase enzymes across the outer membrane. Specifically, immunoblotting for OdsA, which is responsible for the generation of 10-HOME from oleic acid, detected the enzyme in supernatants from wild-type PAO1 but not ΔxcpQ cultures. Likewise, chromatography of supernatants found that 10-HOME was not in supernatants collected from the ΔxcpQ mutant. Accordingly, diol synthase activity was increased in the periplasm of ΔxcpQ mutant consistent with a stoppage in its transport. Importantly, after exposure of the ΔxcpQ mutant to exogenous 10-HOME and 7,10-DiHOME, the ODS effector genes become active; thus, the sensing component of the ODS does not involve the T2SS. Finally, we observed that Xcp contributed to robust in vitro and in vivo biofilm formation in oleic acid availability- and ODS-dependent manner. Thus, T2SS-mediated transport of the oxylipin synthase enzymes to outside the bacterial cell is required for ODS functionality. IMPORTANCE We previously showed that the ODS of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Here, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp type 2 secretion system (T2SS) of P. aeruginosa, and so the biosynthesis of oxylipins occurs extracellularly. T2SS promoted biofilm formation in the presence of oleic acid as a result of ODS activation. Our results identify two new T2SS secreted proteins in P. aeruginosa and reveal a new way by which this important opportunistic pathogen interacts with the host environment.

Production of 8,11-dihydroxy fatty acids from oleic and palmitoleic acids by Escherichia coli cells expressing variant 6,8-linoleate diol synthases from Penicillium oxalicum.

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 pH6.5 and 30 °C with 5% (v/v) dimethyl sulfoxide, 60 g L-1 cells, and 10g L-1 oleic acid or 7.5g L-1 palmitoleic acid, respectively. Under these conditions, 10.7g L-1 8,11-DiHOME and 8.1g L-1 8,11-DiHHME were produced for 1.5h with molar yields of 96.4% and 96.2% and productivities of 7.1 and 5.4g L-1 h-1 , respectively. The molar yields and concentrations of 8,11-DiHOME and 8,11-DiHHME were highest among those of other reported DiHOMEs and DiHHMEs. To the best of our knowledge, this is the first quantitative production of 8,11-DiHOME and 8,11-DiHHME.

T-cell epitope-based vaccine prediction against Aspergillus fumigatus: a harmful causative agent of aspergillosis

BackgroundAmong the most common causes of invasive aspergillosis and acute bronchopulmonary aspergillosis is Aspergillus fumigatus. Transmission with A. fumigatus produces aggressive aspergillosis in allogeneic haematopoietic stem cell transplant recipients, HIV patients, and cancer patients. Asthmatics and cystic fibrosis patients are allergic to A. fumigatus. MHC class-II binding epitopes can initiate immunogenic responses in patients. In this study, we deployed immunoinformatic study to reveal epitopes from fungal proteins. ResultsIn modern research, we found multiple epitopes ITLKLLHRYSYKLAG, KLVLRAFPNHFRGDS, RYSYKLAGVNQVDVV, GKSFELNQAARAVTQ, and LHRYSYKLAGVNQVD from crucial proteins of A. fumigatus 5,8-linoleate diol synthase (ACO55067.2) and ChainB-chitinase A1 (2XVN_B). RYSYKLAGVNQVDVV, GKSFELNQAARAVTQ, and LHRYSYKLAGVNQVD epitopes interact with HLA-DRB01_0101, while ITLKLLHRYSYKLAG and KLVLRAFPNHFRGDS epitopes interact with HLA-DRB01_1501. Molecular docking analysis reveals atomic contact energy (ACE) value for these five epitopes shown below −5 Kcal/mol in docked state. ConclusionsThe invasive aspergillosis and acute bronchopulmonary aspergillosis are caused by harmful fungal pathogen Aspergillus fumigatus. Our modern immunoinformatic research shows ITLKLLHRYSYKLAG, KLVLRAFPNHFRGDS, RYSYKLAGVNQVDVV, GKSFELNQAARAVTQ, and LHRYSYKLAGVNQVD epitopes could bind to MHC-II HLA allelic determinants and can initiate immunogenic response in patients affected by Aspergillus fumigatus.

Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis

Histoplasma capsulatum is an ascomyceteous fungus and a human lung pathogen, which is present in river valleys of the Americas and other continents. H. capsulatum and two related human pathogens, Blasmomyces dermatitidis and Paracoccidioides brasiliensis, belongs to the Ajellomycetaceae family. The genomes of all three species code for three homologous and tentative enzymes of the linoleate diol synthase (LDS) family of fusion enzymes with dioxygenase (DOX) and cytochrome P450 domains. One group aligned closely with 8R-DOX-5,8-LDS of Aspergilli, which oxidizes linoleic acid to 5S,8R-dihydroxylinoleic acid; this group was not further investigated. The second group aligned with 10R-DOX-epoxy alcohol synthase (EAS) of plant pathogens. Expression of this enzyme from B. dermatitidis revealed only 10R-DOX activities, i.e., oxidation of linoleic acid to 10R-hydroperoxy-8E,12Z-octadecadienoic acid. The third group aligned in a separate entity. Expression of these enzymes of H. capsulatum and B. dermatitidis revealed no DOX activities, but both enzymes transformed 13S-hydroperoxy-9Z,11E-octadecadienoic acid efficiently to 12(13S)epoxy-11-hydroperoxy-9Z-octadecenoic acid. Other 13-hydroperoxides of linoleic and α-linolenic acids were transformed with less efficiency and the 9-hydroperoxides of linoleic acid were not transformed. In conclusion, a novel EAS has been found in H. capsulatum and B. dermititidis with 13S-hydroperoxy-9Z,11E-octadecadienoic acid as the likely physiological substrate.

Production of 6,8‐Dihydroxy Fatty Acids by Recombinant Escherichia coli Expressing T879A Variant 6,8‐Linoleate Diol Synthase from Penicillium oxalicum

AbstractRecombinant Escherichia coli expressing T879A variant 6,8‐linoleate diol synthase (LDS) from Penicillium oxalicum showed 2.1‐fold higher activity than recombinant E. coli expressing wild‐type 6,8‐LDS for the production of 6,8‐dihydroxy fatty acids (DiHFA) from linoleic acid. The optimal conditions for the production of 6,8‐DiHFA by recombinant E. coli expressing T879A variant 6,8‐LDS were pH 6.5, 35°C, 50 g L−1 cells, 10 g L−1 (35.7 mM) substrate, and 5% (v/v) dimethyl sulfoxide. Under these optimized conditions, 6.6 g L−1 (22.1 mM) 6,8‐dihydroxy‐9,12(Z,Z)‐octadecadienoic acid (DiHODE) and 7.1 g L−1 (22.6 mM) 6,8‐dihydroxy‐9(Z)‐octadecenoic acid (DiHOME) were produced from linoleic acid and oleic acid in 40 min, respectively. The volumetric productivities of 6,8‐DiHODE and 6,8‐DiHOME under these conditions were 9.9 and 10.7 mg L−1 h−1, respectively. The volumetric productivities of 6,8‐DiHODE and 6,8‐DiHOME were the highest values among those of all reported regiospecific DiHODE and DiHOME, respectively. To the best of our knowledge, this is the first quantitative biotechnological production of 6,8‐DiHFA.

Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn886, Arg707, and Arg934, are catalytic importance and belong to the catalytic groove. Phe794 and Gln889 were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C18 fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.

Oxylipins from both pathogen and host antagonize jasmonic acid‐mediated defence via the 9‐lipoxygenase pathway in Fusarium verticillioides infection of maize

Oxylipins are a newly emerging group of signals that serve defence roles or promote virulence. To identify specific host and fungal genes and oxylipins governing the interactions between maize and Fusarium verticillioides, maize wild-type and lipoxygenase3 (lox3) mutant were inoculated with either F.verticillioides wild-type or linoleate-diol-synthase 1-deleted mutant (ΔFvlds1D). The results showed that lox3 mutants were more resistant to F.verticillioides. The reduced colonization on lox3 was associated with reduced fumonisin production and with a stronger and earlier induction of ZmLOX4, ZmLOX5 and ZmLOX12. In addition to the reported defence function of ZmLOX12, we showed that lox4 and lox5 mutants were more susceptible to F.verticillioides and possessed decreased jasmonate levels during infection, suggesting that these genes are essential for jasmonic acid (JA)-mediated defence. Oxylipin profiling revealed a dramatic reduction in fungal linoleate diol synthase 1 (LDS1)-derived oxylipins, especially 8-HpODE (8-hydroperoxyoctadecenoic acid), in infected lox3 kernels, indicating the importance of this molecule in virulence. Collectively, we make the following conclusions: (1) LOX3 is a major susceptibility factor induced by fungal LDS1-derived oxylipins to suppress JA-stimulating 9-LOXs; (2) LOX3-mediated signalling promotes the biosynthesis of virulence-promoting oxylipins in the fungus; and (3) both fungal LDS1- and host LOX3-produced oxylipins are essential for the normal infection and colonization processes of maize seed by F.verticillioides.

Use of palmitoleic acid and its oxidation products for monitoring the degradation of ice algae in Arctic waters and bottom sediments

Degradation of palmitoleic acid (C16:1ω7), the main fatty acid component of sea ice-associated (sympagic) diatoms, was monitored in Arctic sea ice at the beginning of ice melting and in the underlying sinking particles and superficial bottom sediments. In sea ice, degradation of sympagic algae involved biotic oxidation induced by 10S-DOX-like lipoxygenase of unknown salinity-stressed attached bacteria, while photo- and autoxidation were limited. In the water column, strong hydratase and Z/E isomerase activity were observed. Hydration of unsaturated fatty acids seems to be a detoxification strategy, which is essential for bacterial survival when associated with free fatty acid-rich environments such as ice algae. In contrast, Z/E isomerisation of palmitoleic acid was attributed to the release of Fe2+ ions during radical-induced damage of the active site of the bacterial 10S-DOX-like lipoxygenase and Z/E isomerases. Due to the poor physiological state of their attached bacteria resulting from salinity stress in brine channels or toxicity of free ice algae fatty acids, sympagic algae appeared to be only very weakly biotically degraded within the water column. In bottom sediments, free radicals resulting from 10S-DOX-like lipoxygenase activity induced a strong autoxidation of the ice algal material. The presence in bottom sediments of a significant proportion of oxidation products resulting from 10S-DOX-like lipoxygenase activity attested to the strong contribution of sea ice-derived OM released during the early stages of ice melt prior to deposition in the sediments. However, on the basis of the highest fatty acid photooxidation state observed in these sediments, an additional contribution of highly photooxidized material (ice algal material released at the end of ice melting or open water phytoplankton) seems likely. The degradation of hydroperoxides, resulting from biotic and abiotic degradation of palmitoleic acid, appeared to involve: (i) homolytic cleavage of the peroxyl group affording the corresponding hydroxy- and oxoacids, (ii) reduction to the corresponding hydroxyacids by peroxygenases, (iii) heterolytic proton-catalysed cleavage and (iv) conversion to allylic 1,4-diols by diol synthases and hydroperoxide isomerases.

Biosynthesis of Oxylipins by Rhizoctonia solani with Allene Oxide and Oleate 8S,9S-Diol Synthase Activities.

Oxylipin biosynthesis by fungi is catalyzed by both the lipoxygenase (LOX) family and the linoleate diol synthase (LDS) family of the peroxidase-cyclooxygenase superfamily. Rhizoctonia solani, a pathogenic fungus, infects staple crops such as potato and rice. The genome predicts three genes with 9-13 introns, which code for tentative dioxygenase (DOX)-cytochrome P450 fusion enzymes of the LDS family, and one gene, which might code for a 13-LOX. The objective was to determine whether mycelia or nitrogen powder of mycelia oxidized unsaturated C18 fatty acids to LDS- or LOX-related metabolites. Mycelia converted 18:2n-6 to 8R-hydroxy-9Z,12Z-octadecadienoic acid and to an α-ketol, 9S-hydroxy-10-oxo-12Z-octadecenoic acid. In addition to these metabolites, nitrogen powder of mycelia oxidized 18:2n-6 to 9S-hydroperoxy-10E, 12Z-octadecadienoic, and 13S-hydroperoxy-9Z,11E-octadecadienoic acids; the latter was likely formed by the predicted 13-LOX. 18:1n-9 was transformed into 8S-hydroperoxy-9Z-octadecenoic and into 8S,9S-dihydroxy-10E-octadecenoic acids, indicating the expression of 8,9-diol synthase. The allene oxide, 9S(10)epoxy-10,12Z-octadecadienoic acid, is unstable and decomposes rapidly to the α-ketol above, indicating biosynthesis by 9S-DOX-allene oxide synthase. This allene oxide and α-ketol are also formed by potato stolons, which illustrates catalytic similarities between the plant host and fungal pathogen.

Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism

Plants and fungi form jasmonic acid from α-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-lipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8- and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of α-linolenic and linoleic acids to allene oxides/α-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues.

Production of 8,11-dihydroxy and 8-hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11-linoleate diol synthase from Penicillium chrysogenum.

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11-Linoleate diol synthase (8,11-LDS) catalyzes the conversion of unsaturated fatty acid to 8-hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11-dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11-LDS for the production of 8,11-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8,11-DiHODE), 8,11-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid (8,11-DiHOTrE), 8-hydroxy-9(Z)-hexadecenoic acid (8-HHME), and 8-hydroxy-9(Z)-octadecenoic acid (8-HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α-linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11-DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11-DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8-HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8-HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11-DiHODE, 8,11-DiHOTrE, 8-HHME, and 8-HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390-396, 2017.

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence.

The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria.

Production of 7,8-Dihydroxy Unsaturated Fatty Acids from Plant Oils by Whole Recombinant Cells Expressing 7,8-Linoleate Diol Synthase from Glomerella cingulata

The reaction conditions for the production of 7S,8S-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant Escherichia coli expressing 7,8-linoleate diol synthase from Glomerella cingulata were optimized using response surface methodology. The optimal reaction conditions were pH 7.0, 18.6 °C, 10.8% (v/v) dimethyl sulfoxide, 44.9 g/L cells, and 14.3 g/L linoleic acid, with agitation at 256 rpm. Under these conditions, recombinant cells produced 7,8-dihydroxy unsaturated fatty acids in the range of 7.0-9.8 g/L from 14.3 g/L linoleic acid, 14.3 g/L oleic acid, and plant oil hydrolysates such as waste oil and olive oil containing 14.3 g/L linoleic acid or oleic acid. To the best of the authors' knowledge, this is the first report on the biotechnological production of 7,8-dihydroxy unsaturated fatty acids.

Genome sequence of the filamentous soil fungus Chaetomium cochliodes reveals abundance of genes for heme enzymes from all peroxidase and catalase superfamilies.

BackgroundThe ascomycetous family Chaetomiaceae (class Sordariomycetes) includes numerous soilborn, saprophytic, endophytic and pathogenic fungi which can adapt to various growth conditions and living niches by providing a broad armory of oxidative and antioxidant enzymes.ResultsWe release the 34.7 Mbp draft genome of Chaetomium cochliodes CCM F-232 consisting of 6036 contigs with an average size of 5756 bp and reconstructed its phylogeny. We show that this filamentous fungus is closely related but not identical to Chaetomium globosum and Chaetomium elatum. We screened and critically analysed this genome for open reading frames coding for essential antioxidant enzymes. It is demonstrated that the genome of C. cochliodes contains genes encoding putative enzymes from all four known heme peroxidase superfamilies including bifunctional catalase-peroxidase (KatG), cytochrome c peroxidase (CcP), manganese peroxidase, two paralogs of hybrid B peroxidases (HyBpox), cyclooxygenase, linoleate diol synthase, dye-decolorizing peroxidase (DyP) of type B and three paralogs of heme thiolate peroxidases. Both KatG and DyP-type B are shown to be introduced into ascomycetes genomes by horizontal gene transfer from various bacteria. In addition, two putative large subunit secretory and two small-subunit typical catalases are found in C. cochliodes. We support our genomic findings with quantitative transcription analysis of nine peroxidase & catalase genes.ConclusionsWe delineate molecular phylogeny of five distinct gene superfamilies coding for essential heme oxidoreductases in Chaetomia and from the transcription analysis the role of this antioxidant enzymatic armory for the survival of a peculiar soil ascomycete in various harsh environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3111-6) contains supplementary material, which is available to authorized users.

Characterization of a novel 8R,11S-linoleate diol synthase from Penicillium chrysogenum by identification of its enzymatic products

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus.

Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate.

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.