Profiling the aromatic amino acid metabolome in the human gut microbiota reveals Clostridioides difficile -specific N -acyl amino acids

Owing to its compositional and chemical complexity, much of the gut microbiota metabolome remains poorly characterized. Aromatic amino acids (AAAs) have a history of being privileged substrates for the biosynthesis of diverse bioactive metabolites and thus represent a potentially rich source of bioactive molecules within the microbiota metabolome. In this study, we leveraged ¹³ C- and ² H-labeled aromatic amino acids and untargeted liquid chromatography-mass spectrometry (LC-MS) to profile AAA-derived metabolites produced by 80 phylogenetically diverse human gut bacterial isolates. Collectively, we found 93 unique LC-MS features, majority of which, predominantly produced by Clostridioides difficile , were identified as N -acyl amino acids. C. difficile produced the highest levels of the AAA-derived phenylacetic acid and phenylpropionic acid, exceeding all Bacteroidetes and Proteobacteria strains in our panel. C. difficile ’ s uniquely diverse N- acyl amino acids have the potential to serve as biomarkers for C. difficile colonization and mediators of C. difficile -specific host interaction.