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

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.Dietary fats are metabolized not only by humans but also by microbes in our gastrointestinal tracts. Microorganisms in the gastrointestinal tract interact with their host in many ways and contribute significantly to the maintenance of host health (1). Lipid metabolism by gastrointestinal microbes generates multiple fatty acid species, such as conjugated fatty acids and trans-fatty acids, that can affect host lipid metabolism (2). However, lipid metabolism by gastrointestinal microbes has not been explored in detail. Saturation metabolism of polyunsaturated fatty acids, a representative mode of lipid metabolism by gastrointestinal microbes, is a detoxifying metabolism of anaerobic bacteria, such as lactic acid bacteria, that reside in colon and intestine. This process transforms growth-inhibiting free polyunsaturated fatty acids into less toxic free saturated fatty acids (3). This saturation metabolism generates characteristic fatty acids (e.g., conjugated fatty acids and trans-fatty acids, which are well known to present in ruminant-derived foods and exert various physiological activities).“Conjugated fatty acid” is a collective term for positional and geometric isomers of fatty acids with conjugated double bonds. In particular, conjugated linoleic acids (CLAs), such as cis-9,trans-11-CLA and trans-10,cis-12-CLA, reduce carcinogenesis (4), atherosclerosis (5), and body fat (6). With regard to lipid metabolism, CLA is a potent peroxisome proliferator-activated receptor (PPAR)α agonist (7), and treatment with CLA increases the catabolism of lipids in the liver of rodents (8). Based on these findings, CLA is now commercialized as a functional food for control of body weight, especially in the United States and European countries.On the other hand, consumption of trans-fatty acids increases the risk of coronary heart disease by increasing LDL and reducing HDL cholesterol levels (9). Consequently, trans-fatty acids are considered to be harmful for health, and nutritional authorities have recommended that consumption of trans-fatty acids be reduced to trace amounts (10). Therefore, it is important to control fatty acid saturation processes that generate these fatty acids (11); however, the precise metabolic pathway and enzymes involved have not been clearly identified.Our analyses on conjugated fatty acid synthesis in representative gut bacteria, the lactic acid bacteria (12–15), demonstrated that Lactobacillus plantarum AKU 1009a (AKU Culture Collection, Faculty of Agriculture, Kyoto University) can transform the cis-9,cis-12 diene structure of C18 fatty acids such as linoleic acid, α-linolenic acid, and γ-linolenic acid into the conjugated diene structures cis-9,trans-11 and trans-9,trans-11 (16–21). In addition, this strain can saturate these conjugated dienes into the trans-10 monoene. Our subsequent metabolic analysis indicated that 10-hydroxy-12-octadecenoic acid is an intermediate of CLA synthesis, and further investigations of hydroxy fatty acid metabolism by lactic acid bacteria revealed that CLA is produced from hydroxy fatty acids such as ricinoleic acid in castor oil (22–25). In cell-free extracts from this strain, we identified the enzymes involved in CLA synthesis (26). Three enzymes, CLA-HY, CLA-DH, and CLA-DC, are necessary for synthesis of conjugated fatty acids such as CLA. Only the combined action of these three enzymes can generate CLA from linoleic acid, with 10-hydroxy-cis-12-octadecenoic acid arising as an intermediate (Fig. 1B, 3). The reactions catalyzed by each enzyme, however, were not revealed in those studies. Through genomic analysis in L. plantarum WCFS1, we found that cla-dh (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004567","term_id":"380031102","term_text":"NC_004567"}}NC_004567; region: 59613-60473) and cla-dc (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004567","term_id":"380031102","term_text":"NC_004567"}}NC_004567; region: 60505-61350) are located in a cluster with another gene, cla-er (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004567","term_id":"380031102","term_text":"NC_004567"}}NC_004567; region: 61378-62031) (Fig. 1A). In light of this, we tried to identify the function of the gene product (CLA-ER) together with those of CLA-HY, CLA-DH, and CLA-DC.Open in a separate windowFig. 1.Gene clusters for polyunsaturated fatty acid metabolism and GC chromatograms. (A) Gene clusters for fatty acid metabolic enzymes in L. plantarum. (B) GC chromatograms of substrate (1); reaction with CLA-HY (2); reaction with CLA-HY, CLA-DH, and CLA-DC together with FAD and NADH (3); and reaction with CLA-HY, CLA-DH, CLA-DC, and CLA-ER together with FAD and NADH (4). I.S., internal standard.