Engineering Bacillus subtilis as a sustainable platform for the production of functional bile acids

Bile acids and their derivatives are key regulators of host metabolism, immunity, and microbiome interactions, with growing therapeutic potential. Conventional production methods of bile acids relying on animal extraction or chemical synthesis are inefficient, costly, and environmentally unfriendly. Microbial synthesis offers a sustainable alternative but is limited by the absence of genetic tools for native anaerobic producers. Here, we reconstructed the bile acid 7α-dehydroxylation pathway in Bacillus subtilis, a safe and tractable host. We characterized the key Bai Enzymes and performed comprehensive analyses of the bile acids generated through in vitro assays and whole-cell catalysis. Multiple intermediates and end products, including cholyl-CoA, 3-oxo-cholic acid, 3-oxo-4,5-6,7-didehydro-deoxycholic acid, 3-oxo-4,5-dehydro-deoxycholic acid, 3-oxo-deoxycholic acid, and deoxycholic acid, were identified. This work establishes a functional heterologous platform for bile acid biosynthesis, enabling sustainable production and future applications in therapeutics and microbiome engineering.