Cis and trans conformational changes of bacterial fatty acids in comparison with analogs of animal and vegetable origin

The conditions of the formations of trans isomers of fatty acids, depending on the method of processing and storage of the raw material of microbial, plant and animal origin, were investigated. In the composition of lipids, except for the main trans-isomer elaidic acid, nonsignificant amounts of trans -2-hexen-4-ynal, trans-2-formlcyclopro-panecarboxylate, methyl octadeca-9-yn-l1-trans-enoate, trans-2, 2-dimethyl-3-(2-propenyl)-ethyl ester, trans-9-octadecenoic acid, and trans-1,5-heptadiene, and mixed isomers of methyloctadeca-9-yn-11-trans-enoate,-methyl-9-cis, 11-trans-octadecadienoate, l-[trans-4-(2-iodo-ethyl) cyclohexyl]-trans-4-pentylcyclo-hexane and cis-9, and trans 11-octadecenoic acid. The major trans elaidic acid component was detected in natural objects of different origin in quantities not exceeding 0.05–0.11%. The combination of thermal processing with other parameters, especially enzymatic treatment, led to an increased proportion of trans isomers. The content of trans isomers is usually proportional to the time of storage of materials.     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Production and identification of a novel compound, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-hexadecenoic acid from palmitoleic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.     

Intraspecific Variation of Unusual Phospholipids from Corynebacterium spp. Containing a Novel Fatty Acid

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132^T and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132^T and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.     

Identification of the components of the essential oil from Trollius europaeus flowers

The essential oil of Trollius europaeus flowers obtained by hydrodistillation was analyzed by gas chromatography coupled with mass spectrometry (GC–MS). The compounds giving fragrance of essential oils commonly used in perfumery 3,7-dimethyl-1,6-octadien-3-ol, nonanal, 3-methyl-2-pent-2-enyl-cyclopent-2-enone and oxacycloheptadec-8-en-2-one, rare in the Plant Kingdom, were tentatively identified. In the analyzed essential oil, the saturated fatty acids hexadecanoic acid (7.54 %), tetradecanoic acid (4.24 %), dodecanoic acid (3.10 %) and unsaturated fatty acids 9,12,15-octadecatrienoic acid (3.47 %), hydrocarbons, namely eicosane (20.03 %), hexadecane (8.63 %) and 1,2-benzenedicarboxylic acid (2.39 %), were also found.     

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-²H]18:1n-9, [7R-²H]18:2n-6, and [8R-²H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.     

Thiol-catalyzed cis-trans isomerization of oleic acid

Various thiols were found to catalyze the geometrical isomerization of oleic acid to trans-Δ⁹-octadecenoic acid. The reaction proceeds in neutral aqueous solution at mild temperatures and at relatively low thiol concentration, 5–20 meq/liter. Hydrogen from the medium was not incorporated into the product, and no trace of Δ⁸ or Δ¹⁰ octadecenoic acid could be detected among the products. The reaction is proposed to involve the formation of a mixed micelle of fatty acid and thiol, nucleophilic attack of the double bond by the thiol, rotation about the former double bond, and elimination of the thiol to produce the thermodynamically more stable trans isomer. The cationic reagent, 2-mercaptoethylamine, was the most efficient catalyst tested. This system should prove to be useful for the preparation of labeled trans unsaturated fatty acids.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

The absolute configuration of phaseic and dihydrophaseic acids

A sample of phaseic acid methyl ester (5 mg, isolated from tomato plants fed (±)-abscisic acid, was reduced to a mixture of the epimeric dihydrophaseates which were separated by TLC. The more polar epimer was identical with the dihydrophaseate isolated from beans by Walton et al. [14]. Comparison of the NMR and IR spectra (H-bonding) of the two epimers shows the secondary hydroxyl of the less polar epimer is cis to the oxymethylene group, which is cis to the tertiary hydroxyl group. The absolute configuration of this centre is known so the absolute configuration of phaseic acid can be deduced. Phaseic acid is (−)-3-methyl-5{8[1(R), 5(R)-dimethyl-8(S)-hydroxy-3-oxo-6-oxabicyclo-(3,2,1)-octane]} 2-cis-4-trans-pentadienoic acid and both it and the reduction products exist in chair conformations. The more polar epimer isolated by Walton et al. is (−)-3-methyl-5{8[3(S,8(S)-dihydroxy-1(R,5(R)-dimethyl-6-oxabicyclo-(3,2,1)-octane]}2-cis-4-trans-pentadienoic acid. It is suggested that the less polar epimer should be referred to as epi-dihydrophaseic acid.     

Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity

Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty acids, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.     

The fatty acid and sterol composition of two marine dinoflagellates

The fatty acid, sterol and chlorophyll pigment compositions of the marine dinoflagellates Gymnodinium wilczeki and Prorocentrum cordatum are reported. The fatty acids of both algae show a typical dinoflagellate distribution pattern with a predominance of C₁₈, C₂₀ and C₂₂ unsaturated components. The acid 18:5ω3 is present at high concentration in these two dinoflagellates. G. wilczeki contains a high proportion (93.4%) of 4-methyl-5α-stanols including 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), dinostanol and 4,23,24-trimethyl-5α-cholest-7-en-3β-ol reported for the first time in dinoflagellates. The role of this sterol in the biosynthesis of 5α-stanols in dinoflagellates is discussed. P. cordatum contains high concentrations of a number of δ ²⁴⁽²⁸⁾-sterols with dinosterol, 24-methylcholesta-5,24(28)-dien-3β-ol, 23,24-dimethylcholesta-5,22E-dien-3β-ol, 4,24-dimethyl-5α-cholest-24(28)-en-3β-ol and a sterol identified as either 4,23,24-trimethyl- or 4-methyl-24-ethyl-5α-cholest-24(28)-en-3β-ol present as the five major components. The role of marine dinoflagellates in the input of both 4-methyl- and 4-desmethyl-5α-stanols to marine sediments is discussed.     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Production of 7, 10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from triolein via lipase induction by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.     

Branched Alkanes and Other Apolar Compounds Produced by the Cyanobacterium Microcoleus vaginatusfrom the Negev Desert

Gas chromatography–mass spectrometry on serially coupled capillary columns with different polarity of stationary phases showed that the soil cyanobacterium Microcoleus vaginatusfrom the Negev desert produces an unusual mixture of 4 normal and more than 60 branched alkanes, as well as a number of fatty acids, cyclic and unsaturated hydrocarbons, aldehydes, alcohols, and ketones. The dominant compounds were heptadecane (12%), 7-methylheptadecane (7.8%), hexadecanoic acid (6.5%), (Z)-9-hexadecenoic acid (5.6%), 4-ethyl-2,2,6,6-tetramethylheptane (2.8%), (Z)-9-octadecenoic acid (2.8%), and 4-methyl-5-propylnonane (2.7%).     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Identification and characterization of a novel C20-elongase gene from the marine microalgae, Pavlova viridis, and its use for the reconstitution of two pathways of long-chain polyunsatured fatty acids biosynthesis in Saccharomyces cerevisiae

The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2

We identified [Hou CT (2003) New uses of vegetable oils: novel oxygenated fatty acids by biotransformation. SIM News 53:56–61] many novel oxygenated fatty acids produced from linoleic acid by Bacillus megaterium ALA2: 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. 12,13,17-THOA, the main product, has antiplant pathogenic fungal activity. To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products were studied. Dextrose at 5 g/l was the optimum amount for the carbon source. A combination of 15 g yeast extract and 10 g tryptone showed good results as nitrogen sources. Among the metal ions tested, the optimum concentrations for the reaction for the different ions were as follows (in mM): magnesium 2.0, iron 0.5, zinc 0.1, nickel 0.01, and cobalt 0.05. Copper ions did not affect the production of oxygenated products; however, manganese ions inhibited the reaction. Addition of these metal ions did not alter the distribution of products. The optimum temperature and pH for the production of THOAs were 30°C and pH 6.5. Time course studies showed 40–48 h is the optimum for the production of both THOAs. These data provide the basis for engineering scale-up production of these new products.