Completing the Biosynthesis of the Clinically Important Diterpenoid Andrographolide in <i>Andrographis Paniculata</i>

Abstract Andrographolide is a prominent labdane diterpenoid extracted from Andrographis paniculata with exceptional anti‐inflammatory properties. Commercial production of andrographolide relies exclusively on extraction from plant resources. Although the scaffold of andrographolide ent ‐copalol has previously been biosynthesized, further oxidative modifications remain elusive. In this study, by taking an integrated analysis of transcriptomes and metabolomes, we were able to identify four cytochrome P450 enzymes constituting the minimal set of andrographolide biosynthetic genes. Specifically, ApCYP71D587 catalyzes the conversion of ent ‐copalol to 19‐hydroxy‐ ent ‐copalol. Subsequently, ApCYP71BE50 mediates the formation of the lactone ring, ultimately yielding andrograpanin. Then ApCYP706U5 accomplishes the third step by mediating the C‐3 hydroxylation reaction, thereby allowing the formation of 14‐deoxyandrographolide. Ultimately, ApCYP72F1 completes the biosynthetic generation of andrographolide with C‐14 hydroxylation of the lactone and rearrangement of the olefin bond. In addition, co‐expression of the minimal gene set in N. benthamiana engineered to produce ent ‐copalol feasibly produces andrographolide, thus establishing an innovative metabolic engineering strategy to produce this medicine of historical importance, circumventing the need for plant extraction.