Si-Bum Park

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.

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.

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.

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

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. Graphical abstract Oxidative cleavage of oxo unsaturated fatty acid into dicarboxylic acid by laccase.

Estimating catechin concentrations of new shoots in the green tea field using ground-based hyperspectral image

Hyperspectral camera was applied to establish the models of catechin concentration for green tea. The possibility of improvement for the models was checked by the multi-year models and the mutual prediction. ECg, EGCg and the ester catechin (ECg and EGCg) decreased with the growth but EC, EGC and the free catechin (EC and EGC) were changed by the covering. In partial least square regression (PLSR) models for each catechin, R2 (Relative Error for validation) was more than 0.785 (13.4%) for a single year data, 0.723 (13.3%) for two years data, and 0.756 (13.6%) for three years data except several catechins. It was possible to improve the precision and accuracy of models using the combination of catechin (free and ester type) or the combination of multi-year data. When each and each type of catechin model was predicted by the other year data, the accuracy of two years model improved comparing with it of a single year data. It means that the multi-year models might be more accurate than a single year models to predict the unknown data.