Abstract Detail

Phytochemical Section

De Luca, Vincenzo [1].

Completion of the 7 step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.

Anti-tumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amount in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors, catharanthine and vindoline. While catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical level. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3- hydroxy-16-methoxy-2,3-dihydrotabersonine that are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, since the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the reaction product of the T3R reaction is transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells in order to complete the biosynthesis of these MIAs. With these two genes the complete 7-gene pathway was engineered in yeast to produce vindoline from tabersonine.

1 - Brock University, Biological Sciences, 500 Glenridge Avenue, St. Catharines, ON, Canada

Keywords:
Monoterpenoid Indole Alkaloids
Virus-induced gene silencing
Pathway discovery
metabolic engineering.

Presentation Type: Oral Paper:Papers for Sections
Session: 30
Location: Salon 13/The Shaw Conference Centre
Date: Wednesday, July 29th, 2015
Time: 8:45 AM
Number: 30004
Abstract ID:869
Candidate for Awards:None