Multiomics-guided mining and characterization of epoxide hydrolase involved in camptothecin biosynthesis from Camptotheca acuminata.

The 2,7-epoxy hydrolysis step is critical and inevitable for the biosynthesis of camptothecin (CPT). CPT-type drugs have excellent cytotoxic and antitumor activities. However, the genes responsible for this hydrolysis step remain unclear in Camptotheca acuminata Decne. In this study, multiomics resources of C. acuminata Decne have been utilized to mine and screen the genes involved in the epoxide hydrolase step. Three genes (CaEH1-CaEH3) have been identified, and their recombinant CaEH proteins have been prepared in a soluble form. All CaEHs display (S)-styrene oxide, (R)-styrene oxide, and trans-stilbene oxide oxirane ring-opening activities. Notably, CaEH1 displays excellent catalytic performance for (S)- and (R)-styrene oxides but poor enantioselectivity. On the other hand, CaEH2 and CaEH3 display a higher S isomer preference for styrene oxide. Furthermore, CaEH1-CaEH3 display strictosamide epoxide 2,7-epoxy ring opening activity. They exhibit inferior catalytic performance toward strictosamide epoxide compared to "slim" substrates but better catalytic performance for the larger substrates than characterized plant EHs. Functional verification in planta suggests that the newly identified CaEH1-CaEH3 are jointly responsible for CPT biosynthesis. These CaEH genes are expressed in all plantlet tissues and are enriched in the leaves. Evolutionary analysis indicates that CaEH1-CaEH3 originate from different ancestral EH genes. The convergent evolution of these CaEH genes likely results in the homofunctionalization of CaEH1-CaEH3. Overall, this study reveals one of the previously unexplored biosynthetic steps of camptothecin in C. acuminata.