Unveiling the Complete Biosynthetic Pathway of Azacitidine: A Rare 1,3,5‐Triazine Nucleoside Derived From GTP

ABSTRACT Azacitidine is a rare triazine‐containing nucleoside and a clinically used DNA methyltransferase inhibitor for the treatment of myelodysplastic syndromes. Despite its medical importance, its biosynthetic pathway has remained elusive. Herein, we identified the azacitidine biosynthetic gene cluster ( aza BGC) from Streptomyces mobaraensis IPIO2 using a resistance gene‐guided comparative genomics strategy and elucidated its biosynthetic pathway via in vivo and in vitro studies. Unexpectedly, the bifunctional GTP cyclohydrolase I AzaE generates the triaminopyrimidine intermediate as the major product and H2NTP as a shunt product from GTP, diverging from canonical GCHI enzymes. The cofactor‐independent cupin dioxygenase AzaA then assembles the 1,3,5‐triazine scaffold using both O 2 and H 2 O as oxygen donors, a mechanism unprecedented in nucleoside antibiotic biosynthesis. Subsequent decarboxylation by AzaB/AzaC, phosphoribosyl transfer by AzaD, and dephosphorylation by AzaG together with other endogenous hydrolases yield azacitidine. This work uncovers a distinct biosynthetic logic for triazine‐containing nucleosides and provides a molecular basis for the synthetic biology‐driven production of azacitidine.