Abstract

Khellin is a prototypical furochromone and was approved in Germany as an anti-anginal drug in 1990. However, its biosynthetic pathway remains elusive. In this study, we identify a polyketide synthase, a prenyltransferase, three O-methyltransferases, and three cytochrome P450 enzymes from Ammi visnaga and thus elucidate the biosynthetic pathway of khellin. Interestingly, the flowers and roots of A. visnaga have different pathways, involving two 5-O-methyltransferases (AvOMT1 and AvOMT2), which catalyze noreugenin and visamminol, respectively. Site-directed mutagenesis reveals that N272 and G327 are the determinant residues for the substrate specificity. Moreover, in A. visnaga, the presence of N272 in AvOMT1 gives rise to a chromone metabolic pathway that is distinctive among Apiaceae plants. This work provides a foundation for the biocatalytic production of khellin and related furochromones.