Bacterial metabolism of synthetic steroids across ecosystems reveals diverse biotransformation products, reactions, and enzymes

Biotransformation of synthetic steroid drugs by gut and environmental bacteria affects their activity within the host, persistence, and environmental fate of these widely used pharmaceuticals. However, the spectrum of bacterial transformation of synthetic steroids, the enzymes involved, and the role of cooperative microbial metabolism in these processes remain poorly understood. We systematically mapped the biotransformation of 22 steroids, comprising 20 synthetic and 2 natural compounds and spanning clinically used estrogens, progestogens, corticosteroids, and prodrugs, across 12 bacterial species (8 intestinal and 4 environmental), yielding 264 steroid-bacteria combinations. We identified 97 unique biotransformation products and found that the tested bacteria catalyze diverse reactions including ester hydrolysis, oxidation-reduction chemistry, and steroid side-chain cleavage. Notably, the environmental bacterium Sphingobium herbicidovorans catalyzed desmolase-like steroid side-chain cleavage under aerobic conditions, a transformation previously reported solely for anaerobic bacteria. Combining sequence homology searches, a genetic gain-of-function screen, and expression proteomics we identified six steroid-transforming enzymes in S. herbicidovorans . We further demonstrate that distinct bacterial species cooperatively metabolize synthetic steroids through cross-feeding, enabling sequential activation and metabolism of corticosteroids across microbial communities. Together, our findings uncover previously unrecognized bacterial enzymes and community-level interactions involved in synthetic steroid metabolism. By directly linking steroid metabolites, enzymes, and microbial community interactions, this study provides molecular-level mechanistic insights into microbial steroid biotransformation. Such insights are essential for ultimately predicting metabolic interactions within and across microbial communities, as well as their interactions with the host and the environment.