Unambiguous de-novo clarification of microbial metabolic pathways

The gut microbiota produces numerous small-molecule metabolites that influence host health, but the biosynthetic pathways of many remain unclear. To address this, we developed a Multiomics-driven Reverse Metabolic Pathway Extension approach for de novo reconstruction of microbial metabolic pathways. Using indole-3-acetate (IAA) as a model, we mapped the complete oxidative pathway from tryptophan, uncovering new intermediates (IAA-CoA and P-IAA) and confirming pathway function through engineered E. coli and mouse studies. We further applied MRMPE to decipher hydrogenobutyrate and indolepropionate pathways in Weissella and Clostridium, demonstrating its broad applicability. This strategy offers a powerful tool for uncovering and harnessing gut microbial metabolites for therapeutic purposes.