Engineering a yeast cell factory for biosynthesis of therapeutic bile acid ursodeoxycholic acid (UDCA)

Abstract

Microbial bile acids (MBAs) are gut microbiota-derived metabolites with diverse structures and promising therapeutic potential. However, their extremely low natural abundance has hindered systematic investigation and drug development. To address this limitation, we aimed to establish a sustainable biosynthetic platform in \textit{Saccharomyces cerevisiae} for the production of ursodeoxycholic acid (UDCA), a representative therapeutic MBA, and its derivatives. This work reconstructed three modular sub-pathways in yeast: (1) de novo biosynthesis of cholesterol, (2) multi-step oxidative transformation from cholesterol to chenodeoxycholic acid (CDCA), and (3) microbial conversion of CDCA to UDCA and its taurine-conjugated form TUDCA. Proof-of-concept was achieved for de novo cholesterol production by rewiring the yeast sterol pathway. Eleven heterologous genes were successfully integrated for the CDCA sub-pathway, and intermediate bottlenecks were identified using a series of partial gene cluster strains and untargeted metabolomics. In parallel, the final conversion module demonstrated efficient CDCA-to-UDCA epimerization (>1 g/L) and enabled TUDCA production by co-expressing taurine biosynthesis and conjugation modules. The platform also allowed successful production of GUDCA, a glycine-conjugated UDCA derivative. Our results highlight key challenges in reconstructing complex eukaryotic pathways, including cofactor imbalance, subcellular localization, and regulatory constraints. Nevertheless, we establish a modular and extensible platform for MBA biosynthesis in yeast, paving the way for MBA drug discovery and scalable therapeutic compound production.