Priyanka Verma

Genetic engineering approach using early Vinca alkaloid biosynthesis genes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus

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.

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

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.

In Vitro Manipulations for Value Addition in Potent Herbal Insecticidal Activities of Chrysanthemum cinerariaefolium

The Chrysanthemum genus belongs to the genus Asteraceae which covers up to 15% of all the species of this genus. The natural insecticidal compound pyrethrin is found in C. cinerariaefolium and mostly found in the aerial parts such as achenes of the flowers. Chemically pyrethrin is a set of six structurally close monoterpene esters formed by esterification of two monoterpenic acids (chrysanthemic acid and pyrethric acid) with three ketone alcohols (pyrethrolone, cinerolone and jasmolone). The side effects of the chemical analogue of this molecule and eco-friendly action of pyrethrin such as rapid degradation into the environment and swift action against insects make this molecule the ultimate choice for scale-up industries thus far making this plant system of uttermost importance which needs biotechnological intervention. The in vitro research in this plant system is not completely achieved as the whole pathway-level understanding is not fully understood. Apart from it, the regeneration-level protocols in C. cinerariaefolium have not been established to the par, and Agrobacterium-based genetic transformation studies are limited which could have paved the way for better pathway-level studies, gene transfer studies and new variety development with higher pyrethrin content. The present chapter discusses the present scenario and future prospects of in vitro pyrethrin production.

Biotechnological Interventions to Modulate Terpenoid Indole Alkaloid Pathway in Catharanthus roseus Using In Vitro Tools and Approaches

Catharanthus roseus plant is valued for harboring more than 130 bioactive terpenoid indole alkaloids (TIAs) including the two of its leaf derived bisindole alkaloids—vinblastine and vincristine which are indispensible constituents of anti-neoplastic drugs used in metastatic malignancy associated with acute lymphoblastic leukaemia’s and Hodgkin’s/Non-Hodgkin’s lymphomas. The extremely low in planta occurrence of TIAs in C. roseus plants resulting in high commercial demand and exorbitant price have brought this herb in focus of an intense scientific scrutiny in last 30 years. Research efforts have so far advanced in two major directions: towards understanding the enzymology and genetic regulation of the concerned metabolic pathway(s) leading to TIAs biosynthesis in plant and; secondly, exploring the possibility of developing cell/tissue culture based platforms for in vitro TIAs production to meet the industry’s demand. Designing plants, free from such metabolic constraints, can be a possible approach to enhance the production of plant based medicines. This subject of plant metabolic engineering is gaining lot of attention these days. Pathway manipulation using the modern tools of genetic engineering to over-express a limiting enzyme or to suppress the expression of an enzyme using a shared substrate of a branched pathway are attractive options of metabolic engineering for diverting the metabolic flux towards the synthesis of a desired end product. Knowledge, thus gained, indicates that TIAs biogenetic route is characterized by extensive metabolic cross-talk and shuttling of at least 35 intermediates synthesized via 30 enzymatic reactions occurring in four different types of tissues (epidermis, internal phloem parenchyma, idioblasts and leticifers) and five different sub-cellular compartments (cytosol, vacuole, thyllakoid membrane, nucleus and endoplasmic reticulum). The complexity is further compounded by extremely high level of recalcitrancy of C. roseus plant for regeneration and Agrobacterium-mediated genetic transformation for pathway engineering. As a consequence, all genetic modulation efforts so far made in C. roseus are confined to cell suspension and transformed hairy root cultures that lack the required level of cyto- and tissue-differentiation essential for the expression of entire TIAs pathway genes and enzymes. A perusal of published work in C. roseus clearly suggests that inspite of several pathway manipulation/engineering attempts, the level of TIAs production in cell/tissue/hairy root cultures of this herb could never be enhanced to the level of expectations. The enzymatic, developmental and environmental rigidity/complexities associated with the biosynthetic pathway of these alkaloids have often been cited as possible reasons for these disappointing outcomes Therefore, three major areas of investigation are in focused attention of Catharanthus researchers’ the world over are: (1) how to select or design the starting cells or tissue(s) to realize the full potential of applying metabolic engineering tools for up-regulating the TIAs pathway in them; (2) how to overcome the strong recalcitrancy of Catharanthus plant tissues for de novo organogenesis and in vitro plant regeneration for whole plant-level expression of a transgene coding either for a limiting pathway enzyme or a transcription factor that can control the global expression of several pathway genes and; (3) how to overcome the inability of non-differentiated cell cultures to execute those pathway steps that are expressed only in specialized tissues/cells of C. roseus plants. Various biotechnological approaches and generation of novel tissue types have been discussed in the present chapter for the modulation and increased TIAs flux in C. roseus.

Differential rubisco content and photosynthetic efficiency of rol gene integrated Vinca minor transgenic plant: Correlating factors associated with morpho-anatomical changes, gene expression and alkaloid productivity

Transgenic plants obtained from a hairy root line (PVG) of Vinca minor were characterized in relation to terpenoid indole alkaloids (TIAs) pathway gene expression and vincamine production. The hairy roots formed callus with green nodular protuberances when transferred onto agar-gelled MS medium containing 3.0mg/l zeatin. These meristematic zones developed into shoot buds on medium with 1.0mg/l 2, 4-dichlorophenoxyacetic acid and 40mg/l ascorbic acid. These shoot buds subsequently formed rooted plants when shifted onto a hormone-free MS medium with 6% sucrose. Transgenic nature of the plants was confirmed by the presence of rol genes of the Ri plasmid in them. The transgenic plants (TP) had elongated internodes and a highly proliferating root system. During glass house cultivation TP consistently exhibited slower growth rate, low chlorophyll content (1.02±0.08mg/gm fr. wt.), reduced carbon exchange rate (2.67±0.16μmolm-2s-1), less transpiration rate (2.30±0.20mmolm-2 s-1) and poor stomatal conductance (2.21±0.04mmolm-2 s-1) when compared with non-transgenic population. The activity of rubisco enzyme in the leaves of TP was nearly two folds less in comparison to non-transgenic controls (1.80milliunitsml-1mgprotein-1 against 3.61milliunits ml-1mgprotein-1, respectively). Anatomically, the TP had a distinct tetarch arrangement of vascular bundles in their stem and roots against a typical ployarched pattern in the non-transgenic plants. Significantly, the transgenic plants accumulated 35% higher amount of total TIAs (3.10±0.21% dry wt.) along with a 0.03% dry wt. content of its vasodilatory and nootropic alkaloid vincamine in their leaves. Higher productivity of alkaloids in TP was corroborated with more than four (RQ=4.60±0.30) and five (RQ=5.20±0.70) times over-expression of TIAs pathway genes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) that are responsible for pushing the metabolic flux towards TIAs synthesis in this medicinal herb.