Supplementary file 1_Functional identification of two cytochrome P450 enzymes involved in diterpenoid oxidation in Ginkgo biloba.pdf

Ginkgo produces structurally unique diterpene metabolites, of which ginkgolides are the most representative bioactive compounds. Although the ginkgo genome contains a large and diverse family of cytochrome P450s (CYPs), only a few CYPs involved in the early steps of ginkgolide biosynthesis have been identified so far.

As a result, the contribution of CYP-mediated oxidation to the diversity of ginkgo’s diterpene metabolism remains poorly understood. In this study, we used a constructed diterpenoid-producing yeast chassis for rapid functional screening and identified two cytochrome P450 oxidases from G. biloba: Gb_04669 (GbCYP7005C1) and Gb_33836 (CYP720B2). Gb_04669 catalyzed the oxidation of abietane-type diterpenoids, yielding 2α-hydroxyabietadiene and ginkgosinoic acid A. On the other hand, Gb_33836 catalyzed the C-19 oxidation of labdane-related alcohol substrates, producing isocupressic acid at a titer of 211.24 ± 9.98 μg/L in the engineered yeast strain.