Metabolite Diversity in Alkaloid Biosynthesis: A Multilane (Diastereomer) Highway for Camptothecin Synthesis in Camptotheca acuminata

Abstract

Camptothecin is a monoterpene indole alkaloid (MIA) used to produce semisynthetic antitumor drugs. We investigated camptothecin synthesis in Camptotheca acuminata by combining transcriptome and expression data with reverse genetics, biochemistry, and metabolite profiling. RNAi silencing of enzymes required for the indole and seco-iridoid (monoterpene) components identified transcriptional crosstalk coordinating their synthesis in roots. Metabolite profiling and labeling studies of wild-type and RNAi lines identified plausible intermediates for missing pathway steps and demonstrated nearly all camptothecin pathway intermediates are present as multiple isomers. Unlike previously characterized MIA-producing plants, C. acuminata does not synthesize 3-a(S)-strictosidine as its central MIA intermediate and instead uses an alternative secoiridoid pathway that produces multiple isomers of strictosidinic acid. NMR analysis demonstrated that the two major strictosidinic acid isomers are (R) and (S) diastereomers at their glucosylated C21 positions. The presence of multiple diastereomers throughout the pathway is consistent with their use in synthesis before finally being resolved to a single camptothecin isomer after deglucosylation, much as a multilane highway allows parallel tracks to converge at a common destination. A model "diastereomer" pathway for camptothecin biosynthesis in C. acuminata is proposed that fundamentally differs from previously studied MIA pathways.

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population

“…As plant metabolites provide indispensable resources for human nutrition, energy and medicine (Butelli et al ., ; Chen et al ., ), dissecting the mechanism of metabolite biosynthesis in plants draws extreme interest (Saito and Matsuda, ; Cardoso et al ., ; Quadrana et al ., ; Zhao et al ., ; Fernie and Tohge, ; Perchat et al ., ; Tian et al ., ). In recent years, the rapid development of analysis approaches for metabolomes and multiomics techniques have greatly improved our knowledge of the naturally occurring metabolic variation in plants and its underlying genetic determinants in several species (Keurentjes et al ., ; Shang et al ., ; Sadre et al ., ; Tohge et al ., ; Wen et al ., ; Fernie and Tohge, ; Rai et al ., ; Westhues et al ., ; Xiao et al ., ; Zhu et al ., ). As one of the most essential crop species, rice ( Oryza sativa L.) not only feeds approximately half of the human population worldwide but also serves as a nutrition source.…”

Metabolic GWAS‐based dissection of genetic bases underlying the diversity of plant metabolism

“…Deciphering the genetic control of plant metabolic diversity has attracted a lot of interest, because this topic is essential for enhancing our understanding of the plant metabolome, as well as for human health. Much effort has been made to dissect metabolic pathways, including screening of mutant libraries (Li et al ., ; Yonekura‐Sakakibara et al ., ), analysis of gene families (Yamamura et al ., ; Sadre et al ., ), and comparative genomics studies (Denoeud et al ., ; Huang et al ., ). To obtain deeper insights into genetic and biochemical bases of diversity in the metabolome and to artificially produce natural products with synthetic biology, linkage mapping, and association mapping based on next‐generation sequencing have been applied to metabolomics studies (Luo, ; Alseekh and Fernie, ).…”

Integrated omics analysis of specialized metabolism in medicinal plants

“…The authors were able to capture different metabolic processes and their distribution/biosynthesis across selected plant species, and provided an important metabolic resource for the study of BIA biosynthesis in planta. Metabolite profiling of camptothecin-producing medicinal plants provided an overview of the diverse monoterpenoid indole alkaloids produced (Yamazaki et al, 2003Sadre et al, 2016). Similarly, metabolic profiling of various medicinal plants across different plant families has shown a large diversity of bioactive metabolite classes including flavonoids, saponins and alkaloids.…”

“…Using comprehensive metabolite analysis the authors were able to show that camptothecin is derived by the combination of the 2-C-methyl-D-erythritol-4-phosphate (MEP) and shikimate pathways, and not from the mevalonate pathway. A recent study using a metabolite labeling approach across wild-type and RNAi lines of C. accuminata proposed an alternate seco-iridoid pathway for the biosynthesis of monoterpenoid indole alkaloid (Sadre et al, 2016). The authors showed that unlike previously characterized MIAproducing plants such as O. pumila, C. accuminata does not synthesize 3-a-(S)-strictosidine but instead synthesizes strictosidinic acid composed of a mixture of glucoside distereomers as the central MIA intermediate.…”