Gut bacteria from the Bacteroidota phylum encode conserved methyltransferases that convert diverse carboxyl-containing drugs into prodrugs through methylation, which enhances intestinal permeability and systemic drug exposure in vivo, establishing a novel microbial mechanism that influences individual pharmacokinetics and drug disposition.

The human gut microbiome has been increasingly recognized as a metabolic compartment that influences health and therapeutic responses. While recent studies have cataloged diverse sets of drug biotransformation reactions as a result of microbial metabolism, the pharmacological relevance of the resulting drug metabolites remains mostly uncharacterized. We investigated whether certain microbial drug metabolites can act as functional prodrugs. By systematically mining 871 putative drug metabolites produced by gut bacteria, we identified microbial drug metabolites with key physicochemical properties of prodrugs. Using bacterial culturing, a genetic gain-of-function screen coupled to mass spectrometry and comparative genomics, we uncover that Bacteroidota bacteria encode conserved methyltransferases that can generate prodrugs through carboxyl methylation. As a case in point, we demonstrate that bezafibrate gets converted to a prodrug by a distinct bacterial enzyme, increasing epithelial drug permeability in vitro and systemic drug exposure in vivo in a gnotobiotic mouse model. Additionally, we tested 170 structurally and clinically diverse drugs, and demonstrate that microbial drug–prodrug conversion is a common result of gut bacterial drug biotransformation. Altogether, our findings suggest a novel mechanism of how the gut microbiota influences an individual’s pharmacokinetics, and may generally cause interpersonal differences in microbiota–host metabolic interactions.