Reconstruction of de novo verazine biosynthesis in Saccharomyces cerevisiae and computational analysis of cholesterol-binding geometry for 22R hydroxylation by VcCYP90B27 to dissect the cyclopamine biosynthetic pathway.

Cyclopamine, a typical isosteroidal alkaloid identified as a hedgehog pathway inhibitor, serves as a crucial molecular scaffold for the semi-synthesis of drugs treating nevoid basal cell carcinoma. Verazine is a key intermediate in the biosynthetic pathway of cyclopamine. In this study, we reconstructed the biosynthetic pathway of verazine in engineered Saccharomyces cerevisiae BY-SQ1, obtaining a strain that produced verazine with a yield of 265.38 μg/L. Furthermore, during a 1,000-ns classical MD trajectory, cholesterol retained a broadly consistent heme-facing pose within VcCYP90B27. The modeled pro-R C22 hydrogen remained closer to the heme Fe center on average than the pro-S hydrogen (4.01 vs 4.65 Å), revealing a persistent orientational bias in the bound ensemble. MM-GBSA and contact analyses further indicated that hydrophobic packing around the steroid scaffold and side chain may help maintain this asymmetric presentation. This research not only lays a foundation for the biosynthesis of cyclopamine and other steroidal alkaloids, but also provides a structural rationale for how cholesterol recognition by VcCYP90B27 may contribute to the observed 22R hydroxylation and identifies candidate residues for experimental evaluation.