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

The detailed metabolic map for camptothecin (CPT) biosynthesis
in Camptotheca acuminata has 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. acuminata and 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 cerevisiae WAT11. 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.