Nahoko Kitamura

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

Significance Microorganisms in the gastrointestinal tract interact with their host in many ways. Lipid metabolism by gastrointestinal microbes generates multiple fatty acid species that can affect host health. In the representative gut bacterium Lactobacillus plantarum, we revealed a fatty acid metabolism, saturation metabolism of polyunsaturated fatty acid, that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, fatty acid analysis in mice suggests that the fatty acid metabolism by gastrointestinal microbes modifies fatty acid composition of the host. Therefore, functional investigations of lipid metabolisms of gastrointestinal microbes may provide new methods for improving our health by altering lipid metabolism related to the onset of metabolic syndrome. 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.

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.

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.

An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)-Actinol in a One-Pot Reduction System

(4R,6R)‐Actinol can be stereo‐selectively synthesized from ketoisophorone by a two‐step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)‐levodione reductase. However, (4S)‐phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p‐HBA, and introduced point mutations into the substrate‐recognition loop. The most effective mutant (P295G) showed two‐ and 12‐fold higher catalytic activities toward ketoisophorone and (4S)‐phorenol, respectively, than the wild‐type, and improved the yield of the two‐step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate‐recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

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

UNLABELLED 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,β-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.

Efficient enzymatic production of hydroxy fatty acids by linoleic acid Δ9 hydratase from Lactobacillus plantarum AKU 1009a

This study aims to produce hydroxy fatty acids efficiently.

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 β-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.

Structure and reaction mechanism of a novel enone reductase

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.

Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708.

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 angstrom resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 angstrom. The Matthews coefficient (V(M) = 1.76 angstrom(3) Da(-1)) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit.

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 a‐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‐β 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. FASEB J. 32, 304‐318 (2018). www.fasebj.org