Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots

Camptothecin (CPT) is a natural compound with remarkable antitumor activities due to its special mechanism of inhibiting DNA topoisomerase I. Ophiorrhiza pumila was found to produce camptothecin. By comparative transcriptome profile analysis of O.pumila plantlet roots and hairy roots with suspension cell culture (CSC), two key CPT biosynthetic genes including OpG10H and OpSLS with significantly higher expression level were selected for metabolic engineering. Camptothecin content in hairy root lines mediated by CRISPR-Cas9 system (OpG10H or OpSLS knock-out lines) was obviously reduced, suggesting that both OpG10H and OpSLS play pivotal roles in CPT biosynthesis. Meanwhile individual overexpression of OpG10H (G line) or OpSLS (S line) in O.pumila hairy roots significantly elevated the production of CPT, the highest content was 2.40 mg/g in G line and 3.28 mg/g in S line. Subsequently co-introduction of OpG10H and OpSLS (SG line) resulted in higher concentration of CPT, reaching 3.5 mg/g in SG-17 line, showing cooperative effect than single gene. Besides, camptothecin crude extract from transgenic hairy roots exhibited excellent anti-tumor activity. In summary, the present study shows genetic manipulation of OpG10H and OpSLS revealed by comparative transcriptome analysis is an effective strategy for production of CPT in O.pumila, which sheds new insights for enhancement of other valuable bioactive products.