No abstract

Terpenoid indole alkaloid (TIA) biosynthetic pathway of Catharanthus roseus possesses the major attention in current metabolic engineering efforts being the sole source of highly expensive antineoplastic molecules vinblastine and vincristine. The entire TIA pathway is fairly known at biochemical and genetic levels except the pathway steps leading to biosynthesis of catharanthine and tabersonine. To increase the in-planta yield of these antineoplastic metabolites for the pharmaceutical and drug industry, extensive plant tissue culture-based studies were performed to provide alternative production systems. However, the strict spatiotemporal developmental regulation of TIA biosynthesis has restricted the utility of these cultures for large-scale production. Therefore, the present study was performed to enhance the metabolic flux of TIA pathway towards the biosynthesis of vinblastine by overexpressing two upstream TIA pathway genes, tryptophan decarboxylase (CrTDC) and strictosidine synthase (CrSTR), at whole plant levels in C. roseus. Whole plant transgenic of C. roseus was developed using Agrobacterium tumefaciens LBA1119 strain having CrTDC and CrSTR gene cassette. Developed transgenic lines demonstrated up to twofold enhanced total alkaloid production with maximum ninefold increase in vindoline and catharanthine, and fivefold increased vinblastine production. These lines recorded a maximum of 38-fold and 65-fold enhanced transcript levels of CrTDC and CrSTR genes, respectively.

Catharanthus roseus today occupies the central position in ongoing metabolic engineering efforts in medicinal plants. The entire multi-step biogenetic pathway of its very expensive anticancerous alkaloids vinblastine and vincristine is fairly very well dissected at biochemical and gene levels except the pathway steps leading to biosynthesis of monomeric alkaloid catharanthine and tabersonine. In order to enhance the plant-based productivity of these pharma molecules for the drug industry, cell and tissue cultures of C. roseus are being increasingly tested to provide their alternate production platforms. However, a rigid developmental regulation and involvement of different cell, tissues, and organelles in the synthesis of these alkaloids have restricted the utility of these cultures. Therefore, the present study was carried out with pushing the terpenoid indole alkaloid pathway metabolic flux towards dimeric alkaloids vinblastine and vincristine production by over-expressing the two upstream pathway genes tryptophan decarboxylase and strictosidine synthase at two different levels of cellular organization viz. callus and leaf tissues. The transformation experiments were carried out using Agrobacterium tumefaciens LBA1119 strain having tryptophan decarboxylase and strictosidine synthase gene cassette. The callus transformation reported a maximum of 0.027% dry wt vindoline and 0.053% dry wt catharanthine production, whereas, the transiently transformed leaves reported a maximum of 0.30% dry wt vindoline, 0.10% catharanthine, and 0.0027% dry wt vinblastine content.