Characterization of two key cytochrome P450 enzymes in Isodon amethystoides and de novo production of 3α,15β-dihydroxy-ent-kaurene in yeast

Glaucocalyxin A (GLA), a well-known ent- kaurene diterpenoid, has emerged as a high-value bioactive natural product in pharmaceutical research owing to its potent and broad-spectrum activities. To date, its total synthesis has proven extremely challenging due to poor yield and intricate routes, and the biosynthetic pathway of GLA remains poorly understood. Herein, we identified two enzymes, IamCYP71D761 and IamCYP706V18, in Isodon amethystoides , which specifically mediate the C3α- and C15β-hydroxylation of ent- kaurene with high stereoselectivity and regioselectivity, respectively, generating 3α,15β-dihydroxy- ent- kaurene ( 3 ), a key precursor in the biosynthesis of GLA. In addition, we established an efficient de novo biosynthesis platform for 3 in Saccharomyces cerevisiae . Briefly, high-efficiency isozyme screening, protein engineering, increased acetyl-CoA synthesis, and copy number enhancement were applied to the ent- kaurene biosynthesis module. Subsequently, promoter optimization, competitive pathway knockout, and electron transfer optimization were introduced, resulting in production of 3 to 24.0 ± 0.2 mg/L in shake flask. In summary, this work highlights the pivotal roles of IamCYP71D761 and IamCYP706V18 in the heterologous biosynthesis of 3 , offering valuable insights for the further pathway reconstruction and de novo production of GLA, GLB, and other ent- kaurene diterpenoids.