Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila

Abstract

Background The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.

Overexpression of SmSCR1 Promotes Tanshinone Accumulation and Hairy Root Growth in Salvia miltiorrhiza

“…After further centrifugation at 6,500 × g for 10 min, the samples were filtered separately through 0.22 μM organic membranes (Jinten, China) using a 1 ml sterile syringe ( Huang et al, 2019 ). All metabolites were quantified by high-performance liquid chromatography (HPLC), as described previously (Agilent Technologies, Santa Clara, CA, United States) ( Zhou et al, 2016 ; Sun et al, 2019 ; Yang et al, 2021 ).…”

Integrative Analysis of Elicitor-Induced Camptothecin Biosynthesis in Camptotheca acuminata Plantlets Through a Combined Omics Approach

“…Over the last 55 years, 10 genes have been characterized based on the proposed biosynthetic pathways for CPT. These genes include CaIPI1 and CaIPI2 ( Pan et al, 2008 ), CaDXR ( Yao et al, 2008 ), CaCPR ( Qu et al, 2015 ), CaGES ( Chen et al, 2016 ), CaIS ( Sadre et al, 2016 ), CaGPPS ( Yang et al, 2017 ), CaDL7H/CaSLAS1 and CaDL7H/CaSLAS2 ( Yang et al, 2019 ), Ca10HGO ( Awadasseid et al, 2020 ) (involved in the iridoid pathway), CaTDC ( López-Meyer and Nessler, 1997 ) (involved in generating tryptamine), Ca10OMT ( Salim et al, 2018 ) (involved in the methyl modification of 10-hydroxycamptothecin), and 3 CaSTR ( Yang et al, 2021 ) (responsible for the formation of strictosidine). However, the genes responsible for the downstream oxidation steps for CPT biosynthesis remain unknown.…”

“…Thus, comparative omics analyses have served as an effective way to prioritize functional genes from numerous candidate genes. Strictosidinic acid and strictosamide have been identified as critical bio-precursors for CPT in C. acuminata ( Sadre et al, 2016 ; Pu et al, 2020 ; Yang et al, 2021 ). The formation of strictosamide is followed by a sequence of enzymatically catalyzed reactions, including 2,7-epoxidation, epoxidation hydrolysis, dehydrogenation oxidation, intramolecular re-cyclization, 2-dehydration, 7-carbonyl reduction and dehydration, 16-hydroxylation and double-bond shift, glucosyl hydrolysis, 22-dehydrogenation oxidation, and 20-hydroxylation ( Figure 1 ).…”

Genome-Wide Identification and Comparative Analysis of the Teosinte Branched 1/Cycloidea/Proliferating Cell Factors 1/2 Transcription Factors Related to Anti-cancer Drug Camptothecin Biosynthesis in Ophiorrhiza pumila

“…Additionally, a number of other CPT derivatives have shown promising results in preclinical and clinical trials (Xie et al, 2016). Despite CPT with pharmacological relevance, its biosynthesis pathway is complex and partially deciphered (Sadre et al, 2016;Yang et al, 2021). CPT is produced by the iridoid and shikimate pathways, which supply the important precursors, such as secologanin and tryptamine.…”

“…At present, genomes and transcriptome data of many monoterpenoid indole alkaloidproducing plants, such as O. pumila, Catharanthus roseus, Camptotheca acuminata, and Nothapodytes nimmoniana, provided the foundation for understanding the biosynthesis and regulatory components of plant-specialized metabolites, followed by molecular characterization and functional validation of candidate genes (Kellner et al, 2015;Rather et al, 2018;Kang et al, 2021;Rai et al, 2021). At present, a few genes-encoding key enzymes, such as OpTDC, OpTDC2, OpSTR, OpCPR, OpG10H, OpSLS, OpLAMT, in the CPT biosynthetic pathway and several transcription factors (TFs), such as OpMYB1, OpWRKY1/2/3, and OpERF1/2/3, have been cloned and analyzed in O. pumila (Kai et al, 2015;Rohani et al, 2016;Udomsom et al, 2016;Shi et al, 2020;Hao et al, 2021;Yang et al, 2021;You et al, 2021). For example, OpWRKY2 acted as a positive regulator of CPT biosynthesis by directly binding and activating the gene OpTDC (Hao et al, 2021).…”