乳酸菌の不飽和脂肪酸代謝に関する生化学的研究とその応用

京都大学0048新制・課程博士博士(農学)甲第19046号農博第2124号新制||農||1032(附属図書館)学位論文||H27||N4928(農学部図書室)31997京都大学大学院農学研究科応用生命科学専攻(主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

αC including a novel three- component non-heme diiron monooxygenase system

Please click Additional Files below to see the full abstrac

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). Please click Additional Files below to see the full abstract

Indigo-Mediated Semi-Microbial Biofuel Cell Using an Indigo-Dye Fermenting Suspension

Aizome (Japanese indigo dyeing) is a unique dyeing method using microbial activity under anaerobic alkaline conditions. In indigo-dye fermenting suspensions; microorganisms reduce indigo into leuco-indigo with acetaldehyde as a reductant. In this study; we constructed a semi-microbial biofuel cell using an indigo-dye fermenting suspension. Carbon fiber and Pt mesh were used as the anode and cathode materials, respectively. The open-circuit voltage (OCV) was 0.6 V, and the maximum output power was 32 µW cm−2 (320 mW m−2). In addition, the continuous stability was evaluated under given conditions starting with the highest power density; the power density rapidly decreased in 0.5 h due to the degradation of the anode. Conversely, at the OCV, the anode potential exhibited high stability for two days. However, the OCV decreased by approximately 80 mV after 2 d compared with the initial value, which was attributed to the performance degradation of the gas-diffusion-cathode system caused by the evaporation of the dispersion solution. This is the first study to construct a semi-microbial biofuel cell using an indigo-dye fermenting suspension

Mechanistic Insights into Indigo Reduction in Indigo Fermentation : A Voltammetric Study

Indigo is one of the oldest natural blue dyes. Microorganisms and their enzymatic activities are deeply involved in the traditional indigo staining procedure. To elucidate the mechanism of the microbial indigo reduction, we directly performed cyclic voltammetry on alkaline fermenting dye suspensions. A pair of characteristic redox peaks of leuco-indigo was observed in a supernatant fluid of the fermenting dye suspension. On the other hand, it was found that the indigo/leuco-indigo redox couple mediated two-way microbially catalyzed oxidation and reduction in a sediment-rich suspension of the fermenting suspension. Acetaldehyde was supposed to be the electron donor and acceptor of the catalytic reactions. In order to verify the bioelectrocatalytic reaction, we isolated indigo-reducing bacterium K2-3′ from the fermenting suspension, and the two-way bioelectrocatalysis was successfully restaged in a model system containing K2-3′ and methyl viologen (as a soluble mediator instead of indigo) as well as acetaldehyde at pH 10

Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of parasitism and endosymbiosis

Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of Escherichia coli where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction

A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes

地球外有機化合物に対する微生物代謝の解明から全く新規な酵素系を発見 --生命分子進化の理解や産業応用に期待--. 京都大学プレスリリース. 2021-01-20.The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C–H bond in methyl groups need to be activated

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)