Indole-3-acetic acid production is rare among gut bacteria and reflects OFOR-driven amino acid oxidation in acetogens

Indole-3-acetic acid (IAA) is a tryptophan-derived gut microbial metabolite with reported anti-inflammatory activities, but the organisms and anaerobic pathways that support robust production remain unclear. Screening 206 human gut bacterial isolates by LC-MS revealed that IAA production is rare: only five strains exceeded the limit of quantitation, and high-capacity production was confined to the acetogens <i>Blautia hydrogenotrophica</i> and <i>Intestinibacter bartlettii</i>. Across growth conditions, IAA was a minor product that rose alongside carbohydrate-sensitive, OFOR-linked catabolism of multiple amino acids, generating abundant branched-chain and aromatic organic acids. In gnotobiotic mice mono-colonized with <i>I. bartlettii</i>, these metabolites were produced <i>in vivo</i> but showed distinct host handling, with branched-chain fatty acids largely extracted between portal and peripheral plasma, whereas aromatic acids and their glycine conjugates appeared in plasma and urine. Genomic analyses and heterologous enzyme assays identified expanded repertoires of 2-oxoacid:ferredoxin oxidoreductases (OFORs) with activities spanning pyruvate/oxaloacetate, branched-chain, and aromatic 2-oxoacids, including indolepyruvate conversion to indoleacetyl-CoA, a putative intermediate <i>en route</i> to IAA. Finally, position-specific ¹³C tracing showed that CO₂ released during amino acid oxidation is reassimilated into acetate via reductive acetogenesis, indicating that gut acetogens can maintain redox balance without fermenting partner strains. Together, these findings show that high IAA output is restricted to select gut acetogens and linked to a broader OFOR-driven anaerobic metabolism that generates additional metabolites that are absorbed by the host.