Gut Clostridia and Gammaproteobacteria metabolize dietary linoleic acid to produce a signature of microbial polyunsaturated fatty acids and associated lipids

Unlike the well-studied gut fermentation of fibres and proteins, that of dietary lipids remains underexplored. Here, we investigated gut microbial metabolism of linoleic acid (LA), a predominant dietary fatty acid (FA), by the human gut microbiota, integrating isotopically labeled LA (ILLA), isotope tracking lipidomics, metagenomics, and pathway analyses. The findings were further validated within a dynamic gut-simulating environment using the Simulator of Human Intestinal Microbial Ecosystem (SHIME) and secondary analysis of a human dietary intervention trial. LA exposure led to a broad spectrum of lipids, including LA-derived microbial lipids and others produced as adaptive responses. These included various polyunsaturated FAs (PUFAs) like α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid. ILLA tracing indicated that LA was primarily routed to β-oxidation in several species within the class Clostridia and Gammaproteobacteria, and the intermediates likely facilitated FA synthesis. Temporal patterns revealed that Clostridia (e.g., Enterocloster spp.) and Pseudomonas aeruginosa dominated early-stage LA metabolism, while Gammaproteobacteria (e.g., Klebsiella spp.) played later roles, producing overlapping lipids that primarily relied on de novo FA synthesis. The SHIME confirmed LA bioprocessing over the long term and indicated that a high protein-to-fibre feeding increased PUFA concentration in the colonic condition through its stimulating effects on Gammaproteobacteria. The effect of high protein-to-fibre ratio diet on gut microbial PUFA production was confirmed by the results of the human trial. Overall, this study maps the microbial taxa, pathways, and microbial lipids associated with LA, expanding the knowledge of monoculture experiments and predicting how one of the most abundant FAs is utilized by the human gut microbiota. Our atlas of LA-derived microbial lipids provides a foundation for exploring bioactive microbial lipids that act either locally by exerting their bioactivity on the intestine while concomitantly modulating gut microbes, or systemically, once absorbed, and then released in the bloodstream to target different organs.