Production of hydroxy fatty acids by microbial fatty acid-hydroxylation enzymes

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.     

Characterization of the hydroxy fatty acid content ofBasidiomycotina

Fatty acids (FA) of nine fungal species belonging to the subphylumBasidiomycotina were identified by using capillary GC-MS, MS and HPLC. The identified fatty acids included 45 saturated (iso-, anteiso-, and 19 hydroxy acids) and 42 monoenoic acids (including 14 hydroxy acids); dienes and polyenes were represented by 13 fatty acids. The proportion of hydroxy acids in the total fatty acids in the fungal species ranged from 4.3 to 10.2%. Very long-chain fatty acids (C₂₄-C₃₀) were also determined. Four fatty acids 16:0 (8.8–14.3%), 18:1(11) (3.9–14.9%), 18:1(9) (7.7–19.0%) and 18:2(6) (7.6–19.4%), were found as major acids. Of the identified acids, 17 were detected inBasidiomycotina for the first time.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

Production of long-chain hydroxy fatty acids by microbial conversion

Hydroxy fatty acids (HFAs) are very important chemicals for versatile applications in biodegradable polymer materials and cosmetic and pharmaceutical industries. They are difficult to be synthesized via chemical routes due to the inertness of the fatty acyl chain. In contrast, these fatty acids make up a major class of natural products widespread among bacteria, yeasts, and fungi. A number of microorganisms capable of producing HFAs from fatty acids or vegetable oils have been reported. Therefore, HFAs could be produced by biotechnological strategies, especially by microbial conversion processes. Microorganisms could oxidize fatty acids either at the terminal carbon or inside the acyl chain to produce various HFAs, including α-HFAs, β-HFAs, mid-position HFAs, ω-HFAs, di-HFAs, and tri-HFAs. The enzymes and their encoded genes responsible for the hydroxylation of the carbon chain have been identified and characterized during the past few years. The involved microbes and catalytic mechanisms for the production of different types of HFAs are systematically demonstrated in this review. It provides a better view of HFA biosynthesis and lays the foundation for further industrial production.     

Growth inhibition of plant pathogenic fungi by hydroxy fatty acids

Hydroxy fatty acids are plant self-defense substances (Masui et al, Phytochemistry1989). Three types of hydroxy fatty acids: 10-hydroxystearic acid (HSA), 7S,10S-dihydroxy-8(E)-octadecenoic acid (DOD), and 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA) were tested against the following plant pathogenic fungi: Erysiphe graminis f sp tritici (common disease name, wheat powdery mildew); Puccinia recondita (wheat leaf rust); Pseudocercosporella herpotrichoides (wheat foot rot); Septoria nodorum (wheat glume blotch); Pyricularia grisea (rice blast); Rhizoctonia solani (rice sheath blight); Phytophthora infestans (potato late blight); and Botrytis cinerea (cucumber botrytis). At a concentration of 200 ppm, both HSA and DOD showed no fungal disease control activity. However, THOA at the same concentration showed weak activity and provided disease control (percent) of the following plant pathogenic fungi: Erysiphe graminis 77%; Puccinia recondita 86%; Phytophthora infestans 56%; and Botrytis cinerea 63%. The position of the hydroxy groups on the fatty acids seems to play an important role in activity against specific fungi. Journal of Industrial Microbiology & Biotechnology (2000) 24, 275–276. Received 12 August 1999/ Accepted in revised form 06 January 2000     

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.     

Novel membrane target proteins for lipoxygenase-derived mono(S)hydroxy fatty acids.

Hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) are major bioactive lipids formed via the lipoxygenase oxygenation of arachidonic and linoleic acid, respectively. These metabolites appear to be involved in various cellular actions including cell proliferation, migration and regulation of enzyme activities such as phospholipases and kinases. In view of the diversity of biological effects of these hydroxy fatty acids, it seems likely that multiple mechanisms are involved. Previous reports showed that 15(S)-HETE inhibited the 5-lipoxygenase in rat basophilic leukemia (RBL-1) cell homogenates and established the presence of specific cellular HETE binding sites in these and other cells. The present study used 15(S)-HETE biotin hydrazide and 15(S)-HETE biotin pentyl amide as probes to identify membrane target proteins present in RBL-1 cells that specifically interact with HETEs and HODEs. Two membrane-associated proteins, with apparent molecular weights of 43 and 58 kDa, were identified that specifically interact with these probes and competition experiments indicated that 13(S)-HODE and 15(S)-HETE were the most effective competitors for the hydrazide probe, followed in decreasing effectiveness by 5(S)-HETE, arachidonic acid, 15(R)-HETE, stearic acid and 12(S)-HHT, a cyclooxygenase product. The two proteins were isolated and microsequencing analysis established their identities as actin and the alpha-subunit of mitochondrial ATP synthase, respectively. In vitro binding studies confirmed that purified actin is a potential 15-HETE binding protein. Subcellular cytosolic fractions exhibited fewer protein-probe complexes than membrane fractions. The association of HETEs and HODEs with these cytoskeletal and mitochondrial proteins, respectively, represents a new development in the potential actions of these hydroxy fatty acids.     

Characteristic lipids of Bordetella pertussis: simple fatty acid composition, hydroxy fatty acids, and an ornithine-containing lipid.

The lipids and fatty acids of Bordetella pertussis (phases I to IV) were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry and compared with those of B. parapertussis and B. bronchiseptica. The major lipid components of the three species were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, lysophosphatidylethanolamine, and an ornithine-containing lipid. The ornithine-containing lipid was characteristic of the genus Bordetella. The fatty acid composition of the total extractable cellular lipids of B. pertussis was mostly hexadecanoic and hexadecenoic acids (90%) in a ratio of about 1:1. The hexadecenoic acid of B. pertussis was in the cis-9 form. The fatty acid composition of the residual bound lipids was distinctly different from that of the extractable lipids, and residual bound lipids being mainly 3-hydroxytetradecanoic, tetradecanoic, and 3-hydroxydecanoic acids, with 3-hydroxydodecanoic acid occurring in some strains. It was determined that the 3-hydroxy fatty acids were derived from lipid A. The fatty acid composition of the total extractable cellular lipids of B. parapertussis and B. bronchiseptica, mainly composed of hexadecanoic and heptadecacyclopropanoic acid, differed from that of B. pertussis. Although the fatty acid composition of the residual bound lipids of B. parapertussis was similar to that of the residual bound lipids of B. pertussis, 2-hydroxydodecanoic acid was detected only in the bound lipids of B. bronchiseptica.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

New hydroxy fatty acid from seed oil of Baliospermum axillare

A fatty acid, found as a minor component in the seed oil of Baliospermum axillare, is shown to be the hitherto unknown 11,13-dihydroxytetracos-trans-9-enoic acid (axillarenic acid).     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

A new method for the analysis of amide-linked hydroxy fatty acids in lipid-As from gram-negative bacteria.

Lipid-A represents the ubiquitous, covalently bound hydrophobic component of bacterial lipopolysaccharides (endotoxins). Lipid-As isolated and characterized from rhizobial species have large variations in their backbone sugars, as well as in their hydroxy fatty acid substituents. The sugar backbones consist of either glucosamine and galacturonic acid or glucosamine and 2,3-diaminoglucose. The published procedures for characterizing amide-linked fatty acids do not release all these fatty acids, hence a new method was developed to characterize the amide-linked hydroxy fatty acids. This method involves a mild methanolysis procedure to release glucosamine methyl glycosides which still contain the amide-bound hydroxy fatty acids. The products were analysed by fast atom bombardment mass spectrometry (FAB-MS) and, after trimethylsilylation, by electron impact (E.I.) and chemical ionization (C.I.) gas chromatography-mass spectrometry (GC-MS). The procedure was applied to lipid-A preparations from several gram-negative bacteria. This method allows the unequivocal identification of amide-linked hydroxy fatty acids and also allows determination of the microheterogeneity of the N-acyl substituents in lipid-As from gram-negative bacteria.     

Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species

An analysis of the phospholipid ester-linked and the lipopolysaccharide (LPS) fatty acids and hydroxy fatty acids of six lactate-utilizing Desulfovibrio-type sulfate-reducing bacteria (SRB) has been performed using capillary gas-liquid chromatography-mass spectrometry (GLC-MS). The concentrations of normal fatty acids were essentially similar, with the possible exception of a high content of normal fatty acids in the LPS of Desulfovibrio gigas. Determination of monounsaturated acid double bond configuration was performed by GLC-MS analysis of the derivatized fatty acids. A total of nine branched chain and eight straight chain monounsaturated fatty acids was detected in the Desulfovibrio species analyzed. The major component detected in five Desulfovibrio was the 17-carbon iso-branched monoenoic acid which showed cis unsaturation [i17:1(n-7)c] seven carbons from the terminal methyl group of the fatty acid chain. D. gigas, in contrast, contained almost no unsaturated fatty acids and was greatly enriched in iso-branched 15:0. Major differences between strains were found in the phospholipid and LPS hydroxy fatty acids. These components, in addition to the i17:1(n-7)c and other characteristic branched chain unsaturated acids, can possibly be utilized as signatures of the lactate-utilizing SRB.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pK_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.