52 research outputs found
Basics and applications of gut bacterial lipid-metabolizing enzymes- A tribute to the late Professor Hideaki Yamada
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Enzymes involved in polyunsaturated fatty acid saturation metabolism in lactic acid bacteria and its application for functional lipid synthesis
Polyunsaturated fatty acids and probiotic lactic acid bacteria are reported to be effective to prevent metabolic syndrome. The mechanism, however, was not clear yet. We revealed the polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a, which converted linoleic acid into conjugated linoleic acid (CLA)1).
The enzyme system for this saturation metabolism was found to consist of four enzymes (hydratase2), dehydrogenase3), isomerase, enone reductase4)) and generate hydroxy fatty acids, oxo fatty acids, and conjugated fatty acids as intermediates. The homologous genes encoding these four enzymes were found in genome sequences of many gut microorganisms. Therefore, acting in concert, gut microbiota may mediate the unsaturated fatty acid saturation metabolism in gastrointestinal tract.
Furthermore, we confirmed the existence of these fatty acids in host tissues depending on the existence of gut microbes using specific pathogen free (SPF) mouse and germ free mouse1). Successive analysis revealed health promoting activity of these hydroxy and oxo fatty acids, i.e., intestinal epithelial barrier protection5), anti-obesity6), and anti-diabetic activity7), etc. Therefore, we developed novel production system for these fatty acid metabolites using the enzymes from probiotic lactic acid bacteria8,9,10).
10-hydroxy-cis-12-octadecenoic acid (HYA), an initial intermediate of linoleic acid saturation, has immunomodulatory activity and ameliorates intestinal epithelial barrier impairment, etc5). HYA was found in foods such as cheese, bacon, milk and vegetable pickles, but at low level. We developed hydroxy fatty acid production process using fatty acid hydratase in probiotic lactic acid bacteria. HYA was produced from safflower oil rich in linoleic acid (approximately 75%) with high conversion rate of approximately 50% with Lactobacillus plantarum. We achieved the industrial scale production using 2,000 L fermenter and 500 L reactor. Other C18 Δ9 unsaturated fatty acids such as oleic acid, α-linolenic acid, and γ-linolenic acid were also converted to corresponding 10-hydroxy fatty acids. The various hydroxy fatty acids provided by this technology using fatty acid hydratase in probiotic lactic acid bacteria are promising as novel functional fatty acids.
These studies could open a new application of the enzymes involved in polyunsaturated fatty acid saturation in lactic acid bacteria to novel functional lipid production.
1) S. Kishino, et al. : Proc. Natl. Acad. Sci. USA, 110, 17808 (2013).
2) M. Takeuchi, et al. : J. Biosci. Bioeng. 119, 636 (2015).
3) M. Takeuchi, et al. : J. Mol. Catal., B Enzym. 117, 7 (2015).
4) H. Feng, et al. : FEBS Journal, 282, 1526-1537 (2015).
5) J. Miyamoto, et al. : J. Biol. Chem., 290, 2902 (2015).
6) T. Nanthirudjanar, et al. : Lipids, 50, 1093-1102 (2015).
7) T. Goto, et al. : Biochem. Biophys. Res. Commun., 459, 597 (2015).
8) H. Sakurama, et al. : J. Lipid Res., 55, 1855 (2014).
9) M. Takeuchi, et al. : J. Appl. Microbiol., 120, 1282-1288 (2016).
10) M. Takeuchi, et al. : Biosci. Biotechnol. Biochem., 80, 2132-2137 (2016)
Eicosapentaenoic acid conversion by cytochrome P450 BM-3 and its mutants to bio- active epoxide derivatives
Oxidized polyunsaturated fatty acids such as resolvin and protectin are promising functional lipids because they have strong anti-inflammatory effect1). Recently, dietary eicosapentaenoic acid (EPA) was found to exert anti-allergic effect through the conversion to 17,18-epoxyeicosatetraenoic acid in the gut2). These findings promoted the studies on enzymatic EPA epoxydation to bio-active derivatives. We screened P450 BM-3 and its mutants with rationally modified substrate binding site for conversion of EPA with existence of NADPH regeneration system and ROS decomposing system, catalase. Through the screening, some mutants were found to produce several products (UK1, 2, 3, and 4). Then, these products were purified and identified with LC-MS, NMR, and GC-MS. Finally, these products were identified: UK1 was 14,15:17,18-diepoxy-eicosatrienoic acid (14,15:17,18-DEpETr), UK2 was 17,18-epoxy-eicosatetraenoic acid (17,18-EpETe), UK3 was 14,15-epoxy-eicosatetraenoic acid (14,15-EpETe), UK4 was 11,12-epoxy-eicosatetraenoic acid (11,12-EpETe). The reaction conditions were optimized with P450 BM-3 mutants, and under the optimized conditions, mutant Al4_Ile converted 0.5 mg/ml EPA to 0.20 mg/ml 11,12-EpETe (conversion rate: 38.0% mol/mol). Mutant F87A converted 0.5 mg/ml EPA to 0.19 mg/ml 14,15-EpETe (conversion rate: 36.1% mol/mol). Wild type P450 BM-3 converted 0.5 mg/ml EPA to 0.38 mg/ml 17,18-EpETe (conversion rate: 72.2% mol/mol). Mutant L7V converted 0.5 mg/ml EPA to 0.075 mg/ml 14,15:17,18-DEpETr (conversion rate: 13.5% mol/mol).
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Development of P450-BM3 using molecular dynamics simulations- A tribute to the late Professor Hideaki Yamada
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Gut microbial metabolites of linoleic acid are metabolized by accelerated peroxisomal β-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 hours. 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 β-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 β-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 β-oxidation activity
Enzyme systems involved in glucosinolate metabolism in Companilactobacillus farciminis KB1089
Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin
Lipid production via simultaneous conversion of glucose and xylose by a novel yeast, Cystobasidium iriomotense
バイオマスから油脂を生産する新種の酵母を発見 --油脂製造プロセスの効率化と低炭素社会の実現に貢献--. 京都大学プレスリリース. 2018-10-01.The yeast strains IPM32-16, ISM28-8sT, and IPM46-17, isolated from plant and soil samples from Iriomote Island, Japan, were explored in terms of lipid production during growth in a mixture of glucose and xylose. Phylogenetically, the strains were most closely related to Cystobasidium slooffiae, based on the sequences of the ITS regions and the D1/D2 domain of the LSU rRNA gene. The strains were oleaginous, accumulating lipids to levels > 20% dry cell weight. Moreover, kinetic analysis of the sugar-to-lipid conversion of a 1:1 glucose/xylose mixture showed that the strains consumed the two sugars simultaneously. IPM46-17 attained the highest lipid content (33%), mostly C16 and C18 fatty acids. Thus, the yeasts efficiently converted lignocellulosic sugars to lipids, aiding in biofuel production (which benefits the environment, promotes rural jobs, and strengthens fuel security). The strains constituted a novel species of Cystobasidium, for which we propose the name Cystobasidium iriomotense (type strain ISM28-8sT = JCM 24594T = CBS 15015T)
Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids
The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain
Production of ricinoleic acid-containing monoestolide triacylglycerides in an oleaginous diatom, Chaetoceros gracilis.
実用珪藻ツノケイソウによるリシノール酸の生産に成功. 京都大学プレスリリース. 2016-11-14.Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG
Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation
Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets
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