A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.

Bryonolic acid, a characteristic triterpenoid in Trichosanthes cucumerina L., is well-known due to its pharmacological activities. The biosynthetic pathway of this compound is similar to other triterpenoids, which are known to be initiated by oxidosqualene cyclase for squalene cyclization and cytochrome P450s for oxidation. However, the final step in this pathway has not been identified. This study presents a discovery of a novel enzyme, CYP712D39, with the remarkable ability to catalyze the crucial C-29 oxidation step in bryonolic acid production. We utilized previous transcriptome analysis with eight promising P450 candidates, exhibiting greater expression levels in callus tissue, which shows a high bryonolic acid production. Functional characterization experiment confirmed its capacity to convert isomultiflorenol into bryonolic acid in the WAT11 yeast system. Furthermore, we also predicted key amino acids, including F113, crucial for binding with C3-OH of isomultiflorenol for C29-oxidation. This discovery fills a critical knowledge gap and offers significant biotechnological potential for pharmaceutical and agricultural applications.