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Carqueijeiro IT , Brown S , Chung K , Dang TT , Walia M , Besseau S , Duge de Bernonville T , Oudin A , Lanoue A , Billet K , Munsch T , Koudounas K , Melin C , Godon C , Razafimandimby B , de Craene JO , Glevarec G , Marc J , Giglioli-Guivarc'h N , Clastre M , St-Pierre B , Papon N , Andrade RB , O'Connor S , Courdavault V
Two tabersonine 6,7-epoxidases start synthesis of lochnericine-type alkaloids in Catharanthus roseus
Plant Physiol. 2018 Aug;177(4) :1473-1486
PMID: 29934299    PMCID: PMC6084683    URL: https://www.ncbi.nlm.nih.gov/pubmed/29934299
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Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from stereoselective C6, C7 epoxidation of tabersonine and can be further metabolized to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2, 3 epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to horhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 mainly expressed in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhorhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.
1532-2548 Carqueijeiro, Ines Teto Brown, Stephanie Chung, Khoa Dang, Thu-Thuy T Walia, Manish Besseau, Sebastien Duge de Bernonville, Thomas Oudin, Audrey Lanoue, Arnaud Billet, Kevin Munsch, Thibaut Koudounas, Konstantinos Melin, Celine Godon, Charlotte Razafimandimby, Bienvenue de Craene, Johan-Owen Glevarec, Gaelle Marc, Jillian Giglioli-Guivarc'h, Nathalie Clastre, Marc St-Pierre, Benoit Papon, Nicolas Andrade, Rodrigo B O'Connor, Sarah Courdavault, Vincent Journal Article United States Plant Physiol. 2018 Jun 22. pii: pp.18.00549. doi: 10.1104/pp.18.00549.