Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus

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.

Confirmation of “pre-plasmolysis mediated ex-osmosis hypothesis” to obtain shoot bud morphogenesis in Catharanthus roseus

“…Control leaf explants treated only with CPW solution (without mannitol) showed poor effect over regeneration response (12–14%). The obtained results of leaves pre-plasmolysis treatment with high mannitol concentration very badly affected the viability and organogenetic responses of leaves [ 11 , 13 ].…”

“…Verma and Mathur [ 13 ] also proposed that plasmolysis of leaves with hypertonic mannitol solution before culturing over the shoot induction media to get the support of ex-osmosis to reduce toxic metabolites inside the cells. Later the same approach was used to generate transgenic C. roseus from pre-plasmolyzed leaves undergoing dedifferentiation and redifferentiation process for de novo shoot bud organogenesis [ 11 , 14 ]. This approach can have wider implications in tissue culturing of all those medicinal plants that are known to accumulate cytotoxic molecules in their leaves or other explants.…”

“…Catharanthus roseus is an important medicinal plant due to its property to synthesize two anticancer phytomolecules monoterpenoid indole alkaloids (MIAs) vinblastine and vincristine [ 10 , 11 ]. In the Indian traditional medicinal system, C. roseus plant parts like seeds, leaves, flowers, and roots are often used to cure diabetes, hypertension, menorrhagia, and tumor growth, whereas in the modern pharmacopeia, the plant is appreciated for two leaf-derived antineoplastic dimeric MIAs vincristine and vinblastine and root-derived antihypertensive monomeric MIAs ajmalicine and serpentine [ 3 , 12 ].…”

“…suspension and hairy root cultures, elicitation, and especially genetic engineering studies were carried out on C. roseus to enhance the production of these pharmaceutically important molecules. However, suspension and hairy root culture systems failed to enhance antineoplastic MIAs due to their limited cellular-organ specific complexity, whereas C. roseus due to its recalcitrant nature was not found a very acquiescent system for whole plant genetic engineering through plant regeneration-based transgenic research [ 11 ]. Secondary metabolites are generally synthesized for plant defense response and hence may have cytotoxic nature, therefore, stored in the specified cellular sites/compartments.…”

Cell Type Matters: Competence For Alkaloid Metabolism Differs In Two Seed-Derived Cell Strains of Catharanthus Roseus.

“…Genetic transformation of the plants as well as the synthetic and/or semi-synthetic routes of production of these metabolites, have been employed with variable and modest success. Only recently, overexpression of CrTDC and CrSTR in C. roseus plants have allowed to enhance vinblastine production vefold (Sharma et al 2018). Irrespective of the political controversy about the agricultural use of transgenic plants, extending this transgenic production platform su ciently to meet the growing demands could be challenging.…”