Proteomics-Guided Mining and Characterization of Epoxidase Involved in Camptothecin Biosynthesis from Camptotheca acuminata

The detailed metabolic map for camptothecin (CPT) biosynthesis in Camptotheca acuminatahas been proposed according to our combined omics results. However, the CYP450-mediated epoxidation step in CPT biosynthesis remains unexplored. A proteomics-guided approach was used to identify and annotate the proteins enriched during the vigorous CPT metabolism period in mature C. acuminataand seedlings. Comparative analyses revealed that the CPT and flavonoid biosyntheses were vigorous in stems and all of the samples except the leaves, respectively. The CYP71BE genes were screened based on their enrichment patterns at the transcriptomic–proteomic level and biochemically characterized in Saccharomyces cerevisiaeWAT11. Four CYP71BE proteins exhibited in vitro isoliquiritigenin epoxidase activity. Additionally, CYP71BE206 showed epoxidase activity toward strictosamide, the critical precursor for CPT biosynthesis, both in vitro and in Nicotiana benthamiana. In planta functional verification suggested that CYP71BE206 is involved in CPT biosynthesis. Their catalytic conditions were optimized, and the enzymatic parameters were determined. This study provides valuable insight into the CYP71BE-mediated epoxidation step for CPT biosynthesis and offers evidence to verify that the newly characterized epoxidase (CYP71BE206) is simultaneously responsible for the biosynthesis of CPT and the flavonoid in this plant. An evolution event probably happened on ancestral CYP71BE, resulting in the neofunctionalization of CYP71BE206.