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

Antitumor 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 amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. 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, which 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, as 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 subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine. Significance Bioinformatics and virus-induced gene silencing (VIGS)-guided gene discovery combined with biochemical enzyme assays show that tabersonine 3-oxygenase (T3O) and tabersonine 3-reductase (T3R) are required to form 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, an intermediate in the formation of anticancer drug precursor vindoline from tabersonine. In the absence of T3R, tabersonine is converted by T3O to a series of byproducts that can no longer be used by T3R, suggesting a concerted reaction mechanism. Engineering the seven-gene pathway in yeast demonstrated a prototype platform of high potential for industrial production of the anticancer drug precursor vindoline.