title: 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-α(S)-strictosidine as its central MIA intermediate and instead uses an alternative seco-iridoid 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Radin
authors.identifier: 0000-0002-8412-2579
authors.initials: R
authors.lastname: Sadre
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Maria
authors.initials: M
authors.lastname: Magallanes-Lundback
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Sujana
authors.initials: S
authors.lastname: Pradhan
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Vonny
authors.initials: V
authors.lastname: Salim
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Alex
authors.initials: A
authors.lastname: Mesberg
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: A Daniel
authors.initials: AD
authors.lastname: Jones
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 dellapen@msu.edu.
authors.forename: Dean
authors.initials: D
authors.lastname: DellaPenna
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis*; D029769:Camptotheca / Q000378:metabolism*; D002166:Camptothecin / Q000378:metabolism*; D002243:Carbolines / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010940:Plant Proteins / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D002243:Carbolines; D006027:Glycosides; D010940:Plant Proteins; C551725:strictosidinic acid; D002166:Camptothecin