α-Linolenic acid-derived metabolites from gut lactic acid bacteria induce differentiation of anti-inflammatory M2 macrophages through G protein-coupled receptor 40.

Among dietary fatty acids with immunologic effects, ω-3 polyunsaturated fatty acids, such as α-linolenic acid (ALA), have been considered as factors that contribute to the differentiation of M2-type macrophages (M2 macrophages). In this study, we examined the effect of ALA and its gut lactic acid bacteria metabolites 13-hydroxy-9(Z),15(Z)-octadecadienoic acid (13-OH) and 13-oxo-9(Z),15(Z)-octadecadienoic acid (13-oxo) on the differentiation of M2 macrophages from bone marrow-derived cells (BMDCs) and investigated the underlying mechanisms. BMDCs were stimulated with ALA, 13-OH, or 13-oxo in the presence of IL-4 or IL-13 for 24 h, and significant increases in M2 macrophage markers CD206 and Arginase-1 (Arg1) were observed. In addition, M2 macrophage phenotypes were less prevalent following cotreatment with GPCR40 antagonists or inhibitors of PLC-ß and MEK under these conditions, suggesting that GPCR40 signaling is involved in the regulation of M2 macrophage differentiation. In further experiments, remarkable M2 macrophage accumulation was observed in the lamina propria of the small intestine of C57BL/6 mice after intragastric treatments with ALA, 13-OH, or 13-oxo at 1 g/kg of body weight per day for 3 d. These findings suggest a novel mechanism of M2 macrophage differentiation involving fatty acids from gut lactic acid bacteria and GPCR40 signaling.-Ohue-Kitano, R., Yasuoka, Y., Goto, T., Kitamura, N., Park, S.-B., Kishino, S., Kimura, I., Kasubuchi, M., Takahashi, H., Li, Y., Yeh, Y.-S., Jheng, H.-F., Iwase, M., Tanaka, M., Masuda, S., Inoue, T., Yamakage, H., Kusakabe, T., Tani, F., Shimatsu, A., Takahashi, N., Ogawa, J., Satoh-Asahara, N., Kawada, T. α-Linolenic acid-derived metabolites from gut lactic acid bacteria induce differentiation of anti-inflammatory M2 macrophages through G protein-coupled receptor 40.

Supplemental feeding of a gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, alleviates spontaneous atopic dermatitis and modulates intestinal microbiota in NC/nga mice.

The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer's patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16 S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.

A gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, ameliorates intestinal epithelial barrier impairment partially via GPR40-MEK-ERK pathway.

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress.

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H: quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,ß-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress.

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage.

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon-carbon double bond were cleaved at the carbon-carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.

Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition.

In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus.

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis.

Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increased adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism.

Estimating the catechin concentrations of new shoots in green tea fields using ground-based hyperspectral imagery.

Hyperspectral imagery was applied to estimating non-galloyl (EC, EGC) and galloyl (ECG, EGCG) types of catechins in new shoots of green tea. Partial least squares regression models were developed to consider the effects of commercial fertilizer (CF) and organic fertilizer (OF). The models could explain each type of catechin with a precision of more than 0.79, with a few exceptions. When the CF model was applied to the OF hyperspectral reflectance and the OF model was applied to the CF hyperspectral reflectance for mutual prediction, the prediction accuracy was better with the OF models than CF models. The prediction models using both CF and OF data (hyperspectral reflectances, and concentrations of catechins) had a precision of more than 0.76 except for the non-galloyl-type catechins as a group and EGC alone. These results provide useful data for maintaining and improving the quality of green tea.

Characterization of regioselective glycosyltransferase of Rhizobium pusense JCM 16209T useful for resveratrol 4'-O-α-d-glucoside production.

Enzymatic glycosylation is an industrially useful technique for improving the properties of compounds with hydroxy groups, and the biological activities of the resulting glycosides differ depending on the glycosylation position. Therefore, regioselective glycosyltransferases are required for precise synthesis of glycosides. We found that Rhizobium pusense JCM 16209T could catalyze the regioselective glycosylation of resveratrol. To identify the regioselective glycosyltransferase, two α-glucosidases of R. pusense JCM 16209T (RpG I and RpG II) were cloned and expressed in Escherichia coli. The molecular mass of purified recombinant RpG I and II was estimated to be 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RpG I showed strong glycosylation activity toward resveratrol with 4'-selectivity of 98.3%. The enzyme activity was maximized at pH 8.0 and 50 °C, and enhanced in the presence of Cs+ and Li+ ions. The maximum molar yield of resveratrol 4'-O-α-glucoside from resveratrol reached 41.6% at 30 min, and the concentration of the product was 2.08 mmol L-1. Glycosylation activity was observed toward resveratrol as well as toward caffeic acid, ferulic acid, 6-gingerol, flavonoid, and isoflavonoid compounds with high regioselectivity, indicating that RpG I could glycosylate a wide range of substrates. To the best of our knowledge, there are few reports on microbial glycosyltransferases that are useful for regioselective glycosylation. This research could be the first step toward developing technologies for the precise synthesis of glycosides.

Novel multi-component enzyme machinery in lactic acid bacteria catalyzing C=C double bond migration useful for conjugated fatty acid synthesis.

Linoleic acid isomerase was identified as a multi-component enzyme system that consists of three enzymes that exist in both the membrane and soluble fractions of Lactobacillus plantarum. One enzyme (CLA-HY) is present in the membrane fraction, while two enzymes (CLA-DH and CLA-DC) exist in the soluble fraction. Three Escherichia coli transformants expressing CLA-HY, CLA-DH, and CLA-DC were constructed. Conjugated linoleic acid (CLA) and 10-hydroxy-12-octadecenoic acid were generated from linoleic acid only when all these three E. coli transformants were used as catalysts simultaneously. CLA-HY catalyzed the hydration reaction, a part of linoleic acid isomerization, to produce 10-hydroxy-12-octadecenoic acid. This multi-component enzyme system required oxidoreduction cofactors such as NADH and FAD. This is the first report to reveal enzymes genes and the elaborate machinery that synthesizes CLA, especially an important isomer of cis-9, trans-11-CLA, in lactic acid bacteria.

17(S),18(R)-epoxyeicosatetraenoic acid generated by cytochrome P450 BM-3 from Bacillus megaterium inhibits the development of contact hypersensitivity via G-protein-coupled receptor 40-mediated neutrophil suppression.

Dietary intake of ω3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid is beneficial for health control. We recently identified 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) as a lipid metabolite endogenously generated from eicosapentaenoic acid that exhibits potent anti-allergic and anti-inflammatory properties. However, chemically synthesized 17,18-EpETE is enantiomeric due to its epoxy group-17(S),18(R)-EpETE and 17(R),18(S)-EpETE. In this study, we demonstrated stereoselective differences of 17(S),18(R)-EpETE and 17(R),18(S)-EpETE in amelioration of skin contact hypersensitivity and found that anti-inflammatory activity was detected in 17(S),18(R)-EpETE, but not in 17(R),18(S)-EpETE. In addition, we found that cytochrome P450 BM-3 derived from Bacillus megaterium stereoselectively converts EPA into 17(S),18(R)-EpETE, which effectively inhibited the development of skin contact hypersensitivity by inhibiting neutrophil migration in a G protein-coupled receptor 40-dependent manner. These results suggest the new availability of a bacterial enzyme to produce a beneficial lipid mediator, 17(S),18(R)-EpETE, in a stereoselective manner. Our findings highlight that bacterial enzymatic conversion of fatty acid is a promising strategy for mass production of bioactive lipid metabolites.

Gut microbial metabolites of linoleic acid are metabolized by accelerated peroxisomal ß-oxidation in mammalian cells.

Microorganisms in animal gut produce unusual fatty acids from the ingested diet. Two types of hydroxy fatty acids (HFAs), 10-hydroxy-cis-12-octadecenoic acid (HYA) and 10-hydroxy-octadecanoic acid (HYB), are linoleic acid (LA) metabolites produced by Lactobacillus plantarum. In this study, we investigated the metabolism of these HFAs in mammalian cells. When Chinese hamster ovary (CHO) cells were cultured with HYA, approximately 50% of the supplemented HYA disappeared from the dish within 24 h. On the other hand, the amount of HYA that disappeared from the dish of peroxisome (PEX)-deficient CHO cells was lower than 20%. Significant amounts of C2- and C4-chain-shortened metabolites of HYA were detected in culture medium of HYA-supplemented CHO cells, but not in medium of PEX-deficient cells. These results suggested that peroxisomal ß-oxidation is involved in the disappearance of HYA. The PEX-dependent disappearance was observed in the experiment with HYB, but not with LA. We also found that HYA treatment up-regulates peroxisomal ß-oxidation activity of human gastric MKN74 cells and intestinal Caco-2 cells. These results indicate a possibility that HFAs produced from gut bacteria affect lipid metabolism of host via modulation of peroxisomal ß-oxidation activity.

Synthesized enone fatty acids resembling metabolites from gut microbiota suppress macrophage-mediated inflammation in adipocytes.

SCOPE: Recent reports indicate that gut microbiota and their metabolites may regulate host inflammatory conditions, including the chronic inflammation of obese adipose tissues. In this study, we investigated whether specific synthesized fatty acids, identical to the metabolites generated by gut microbiota, act as anti-inflammatory factors in obesity-induced inflammation. METHODS AND RESULTS: We first used lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages to examine the anti-inflammatory effect of fatty acids synthesized to resemble representative polyunsaturated fatty acid metabolites from gut microbiota. Fatty acids containing an enone structure showed the most potent anti-inflammatory activity. Enone fatty acids also displayed anti-inflammatory effects on macrophages cocultured with hypertrophied 3T3-L1 or immortalized primary adipocytes; and macrophages stimulated with 3T3-L1 adipocyte conditioned medium. Consistently, the beneficial outcome was revealed in the case of LPS- and obesity-induced inflammatory cytokine stimulation in ex vivo adipose tissues. Furthermore, these fatty acids recovered the suppression of ß-adrenergic receptor-stimulated uncoupling protein 1 expression and secretion of adiponectin in C3H10T1/2 and 3T3-L1 adipocytes, respectively, under inflammatory conditions, suggesting that enone fatty acids can ameliorate dysfunctions of adipocytes induced by inflammation. CONCLUSION: These findings indicate that synthesized enone fatty acids show potent anti-inflammatory effects, leading to the improvement of inflammation-induced dysfunctions in adipocytes.

Characterization of the linoleic acid Δ9 hydratase catalyzing the first step of polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a.

Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy-cis-12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis-9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent Km value for linoleic acid was estimated to be 92 µM, with a kcat of 2.6∙10(-2) s(-1) and a Hill factor of 3.3. The apparent Km value for HYA was estimated to be 98 µM, with a kcat of 1.2∙10(-3) s(-1).

Structure and reaction mechanism of a novel enone reductase.

UNLABELLED: Recently, a novel gut-bacterial fatty acid metabolism, saturation of polyunsaturated fatty acid, that modifies fatty acid composition of the host and is expected to improve our health by altering lipid metabolism related to the onset of metabolic syndrome, was discovered in Lactobacillus plantarum AKU 1009a. Enzymes constituting the pathway catalyze sequential reactions of free fatty acids without CoA or acyl carrier protein. Among these enzymes, CLA-ER was identified as an enone reductase that can saturate the C=C bond in the 10-oxo-trans-11-octadecenoic acid (KetoB) to produce 10-oxo-octadecanoic acid (KetoC). This enzyme is the sole member of the NADH oxidase/flavin reductase family that has been identified to exert an enone reduction activity. Here, we report both the structure of holo CLA-ER with cofactor FMN and the KetoC-bound structure, which elucidate the structural basis of enone group recognition of free fatty acids and provide the unique catalytic mechanism as an enone reductase in the NADH oxidase/flavin reductase family. A 'cap' structure of CLA-ER underwent a large conformational change upon KetoC binding. The resulting binding site adopts a sandglass shape and is positively charged at one side, which is suitable to recognize a fatty acid molecule with enone group. Based on the crystal structures and enzymatic activities of several mutants, we identified C51, F126 and Y101 as the critical residues for the reaction and proposed an alternative electron transfer pathway of CLA-ER. These findings expand our understanding of the complexity of fatty acid metabolism. DATABASE: The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID 4QLX, 4QLY).

Gut Microbial Fatty Acid Metabolites Reduce Triacylglycerol Levels in Hepatocytes.

Hydroxy and oxo fatty acids were recently found to be produced as intermediates during gut microbial fatty acid metabolism. Lactobacillus plantarum produces these fatty acids from unsaturated fatty acids such as linoleic acid. In this study, we investigated the effects of these gut microbial fatty acid metabolites on the lipogenesis in liver cells. We screened their effect on sterol regulatory element binding protein-1c (SREBP-1c) expression in HepG2 cells treated with a synthetic liver X receptor α (LXRα) agonist (T0901317). The results showed that 10-hydroxy-12(Z)-octadecenoic acid (18:1) (HYA), 10-hydroxy-6(Z),12(Z)-octadecadienoic acid (18:2) (γHYA), 10-oxo-12(Z)-18:1 (KetoA), and 10-oxo-6(Z),12(Z)-18:2 (γKetoA) significantly decreased SREBP-1c mRNA expression induced by T0901317. These fatty acids also downregulated the mRNA expression of lipogenic genes by suppressing LXRα activity and inhibiting SREBP-1 maturation. Oral administration of KetoA, which effectively reduced triacylglycerol accumulation and acetyl-CoA carboxylase 2 (ACC2) expression in HepG2 cells, for 2 weeks significantly decreased Srebp-1c, Scd-1, and Acc2 expression in the liver of mice fed a high-sucrose diet. Our findings suggest that the hypolipidemic effect of the fatty acid metabolites produced by L. plantarum can be exploited in the treatment of cardiovascular diseases or dyslipidemia.

Occurrence of conjugated polyenoic fatty acids in seaweeds from the Indian Ocean.

Three species of red marine macro algae (Rhodophyta) from the Indian Ocean were analysed for the occurrence of conjugated polyenes. The composition of different lipid classes in these seaweeds along with their fatty acid composition has also been reported. Analysis of lipid classes of these seaweeds revealed that both Acanthophora spicifera (Ceramiales, Rhodophyta) and two species of Gracilaria, viz. G. edulis and G. folifera (Gracilariales, Rhodophyta) were rich in glycolipids followed by neutral- and phospholipids. The fatty acid composition of these seaweeds revealed C16:0 as the predominant fatty acid in all three species. However, A. spicifera had significantly higher amounts of eicosapentaenoic acid (EPA) and arachidonic acid (AA) as compared to negligible amount of these fatty acids in both species of Gracilaria. The red seaweed Acanthophora spicifera contained conjugated eicosapentaenoic acid (CEPA) and conjugated arachidonic acid (CAA) in all lipid classes except glycolipids.

Characterization of a trifunctional fatty acid desaturase from oleaginous filamentous fungus Mortierella alpina 1S-4 using a yeast expression system.

A ω3-fatty acid desaturase gene (maw3) which is involved in biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) was previously isolated from Mortierella alpina 1S-4. In this report, we investigated the products of MAW3 catalyzing reaction with endogenous and exogenous fatty acids in the yeast transformant. Two unusual fatty acids de novo synthesized in the yeast transformant expressing maw3 gene were identified as n-4 hexadecadienoic acid (16:2(9cis,12cis)) and n-1 hexadecatrienoic acid (16:3(9cis,12cis,15)) by GC-MS and (1)H NMR analyses. In addition to the desaturation activity at the ω3-position for 18- and 20-carbon PUFAs, MAW3 in the yeast transformant inserted a double bond at Δ12-position of endogenous palmitoleic acid (16:1(9cis)) and further at Δ15-position of the resulting 16:2(9cis,12cis) to result in the formation of 16:3(9cis,12cis,15) leading to a bifunctional Δ12/Δ15-desaturase for 16-carbon fatty acids. Moreover, we evaluated the activity of MAW3 in the yeast transformant under different temperatures. The MAW3 did not have desaturation activities in M. alpina 1S-4 at 28°C but it had in the yeast transformant for various fatty acids. The MAW3 was demonstrated to be a trifunctional Δ12/Δ15/ω3-desaturase, exhibiting Δ12-desaturation for 16:1(9cis), Δ15-desaturation for 16- and 18-carbon fatty acids that had a preexisting cis-double bond at Δ12 position, and ω3-desaturation for 20-carbon fatty acids having that at Δ14-position. It is the first report that the fatty acid desaturase (MAW3) is shown to have Δ12- and Δ15-desaturation activities for a 16-carbon fatty acid, in addition to its major function, ω3-desaturation activity.