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

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 O₂ and H₂O 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.