Michiki Takeuchi

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.

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.

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.

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.

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.

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.

Hydratase, Dehydrogenase, Isomerase, and Enone Reductase Involved in Fatty Acid Saturation Metabolism

Summary We revealed the complex metabolic pathway of the PUFA saturation metabolism in lactic acid bacteria in detail. The enzyme system was found to consist of four enzymes. The concerned action of these enzymes accomplishes the saturation of a C=C double bond and generated unique fatty acid molecular species such as hydroxy fatty acids, oxo fatty acids, and conjugated fatty acids showing unique activities.

CHAPTER 5:Recent Advances in the Production of CLA and Conjugated Vegetable Oils: Microbial and Enzymatic Production of Conjugated Fatty Acids and Related Fatty Acids in Biohydrogenation Metabolism

Lactic acid bacteria produced CLA from linoleic acid. The produced CLA comprised a mixture of cis-9, trans-11-octadecadienoic acid (18:2) and trans-9, trans-11-18:2. Using washed cells of Lactobacillus plantarum AKU 1009a as a catalyst, CLA production from linoleic acid reached 40 mg/ml under the optimized conditions. Similar reactions were observed with C18 fatty acids with cis-9, cis-12 non-conjugated diene system such as α-linolenic acid, γ-linolenic acid, and stearidonic acid. Lactic acid bacteria transformed ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) to CLA. Castor oil, which is rich in the triacylglycerol form of ricinoleic acid, was also found to act as a substrate with the aid of lipase-catalysed triacylglycerol hydrolysis. Clostridium bifermentans saturated C20 PUFAs of arachidonic acid and EPA into corresponding partially saturated fatty acids with conjugated isomers of arachidonic acid and EPA as intermediates, respectively. The conjugated fatty acid synthesis was found to be a part reaction of biohydrogenation and the complex metabolic pathway was revealed. The enzyme system catalysing the biohydrogenation was found to consist of four enzymes, i.e., hydratase, dehydrogenase, isomerase and enone reductase. These enzymes are useful for the production of unique PUFA species, such as hydroxy, oxo, conjugated and partially saturated fatty acids.