Functional identification of two cytochrome P450 enzymes involved in diterpenoid oxidation in Ginkgo biloba

Abstract

Ginkgo biloba is a globally renowned medicinal plant. 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. The contrasting product profiles of these two enzymes demonstrate the distinct catalytic activities of the CYP7005C and CYP720B families in G. biloba toward different diterpene scaffolds and extend our knowledge of P450-mediated early diterpenoid oxidation in gymnosperms. Our study expanded the substrate profile of CYP720Bs by showing that the ginkgo CYP720B acts on bicyclic diterpenes. This is different from previously characterized conifer CYP720Bs, which act on tricyclic diterpenes. These results provide new insights into the evolutionary diversification of this enzyme family. Collectively, this study highlights the value of the engineered yeast chassis for accelerating the discovery of plant diterpenoid oxidases and provides new enzymatic tools for the future biosynthetic production of structurally diverse oxidized diterpenoids.