Differing functions of two CYP716 genes for saikosaponin biosynthesis in Bupleurum chinense

Saikosaponins are the major pharmacologically active components in Bupleurum genus and exhibit significant application potential in multiple fields such as immune regulation and anti-tumor activity. To elucidate the biosynthetic pathway of saikosaponins, we identified two cytochrome P450 monooxygenases, CYP716A41 and CYP716Y4, in Bupleurum chinense. These enzymes catalyze the C-28 oxidation and C-16 hydroxylation of oleanane-type triterpene skeletons, respectively. The catalytic efficiency of CYP716A41 from a southern B. chinense variety was significantly higher than that from a northern variety. Molecular docking and mutagenesis experiments revealed that amino acid residues at sites 9 and 35 may contribute to this difference in catalytic efficiency. Additionally, under cold stress, the expression levels of both CYP450 genes and the saikosaponin contents in the leaves of southern varieties were significantly higher compared to those in northern varieties. The variation in the catalytic efficiency of CYP716A41 and the differential expression of the two CYP450 genes under cold stress during winter are associated with the differences in saikosaponin biosynthesis in the leaves of southern and northern B. chinense varieties. This is consistent with the distinct medicinal usage practices observed between southern and northern China.