Uncovering synthetic steroid biotransformations in gut and environmental bacteria

Synthetic steroids are widely used in both pharmaceuticals and veterinary medicine. Designed for increased potency and resistance to metabolism, these compounds tend to persist in the environment, raising ecological concerns such as disruption of aquatic ecosystems and impacts on plant growth. While several environmental bacteria have shown the ability to degrade natural steroids, such as testosterone and estrone, the degradation of synthetic analogs remains poorly understood. In this study, we screened four environmental species and eight gut bacteria to investigate the biotransformation of 20 steroids and steroid prodrugs, including synthetic estrogens, progestogens, and corticosteroids. Through the development of a tandem mass spectrometry-based pipeline, we identified key steroid biotransformation intermediates and products, revealing distinct steroid metabolizing pathways for the different bacteria tested. These findings illustrate the specific roles in steroid metabolism that environmental and gut bacteria play and suggest that prior steroid exposure to gut microbiota may affect the fate of synthetic steroids in the environment. To uncover the enzymes and enzyme pathways involved, we employed genetic screens and homology searches. Our developed approach provides a robust platform to identify steroid metabolites and select enzymes of specific biocatalytic interest for further characterization, as well as allows for mapping the network of genes, proteins, and species capable of steroid biotransformations within specific ecosystems, such as the human gut and environmental habitats.