Metabolic Engineering in CATHARANTHUS ROSEUS using RNAi as a Tool

Abstract

Catharanthus roseus (G. Don), (Apocynaceae family), is a well known medicinal plant, being source of important pharmaceutically active anticancer compounds namely vinblastine, vincristine as well as antihypertensive compounds namely ajmalicine and serpentine all having high commercial value. All these compounds are part of the secondary metabolism of the plant, and arise from monoterpenoid indole alkaloid (MIA) biosynthetic pathway. The importance of these phytochemicals has motivated researchers to carry out extensive studies to elucidate MIA biosynthetic pathway and regulation and use it for metabolic engineering to enhance MIA pathway biosynthesis. In this regards, a potential candidate for metabolic engineering in C. roseus i.e Phenylalanine ammonia lyase (PAL) enzyme remained unexplored for the purposes. It is first and key rate-limiting enzyme of phenylpropanoid pathway catalyzing conversion of phenylalanine to cinnamic acid which is universal precursor for other phenolics in C. roseus including flavonoids, anthocyanins and lignins. Both phenylpropanoid pathway and MIA pathway share chorishmate as precursor pool indicating that there could be competition for carbon pool between both the pathways. With this propspectus in mind, the present project aimed to carry out metabolic engineering in C. roseus using RNA interference (RNAi) approach to downregulate expression of PAL enzyme. We utilized inhp RNAi expression vector to transform C. roseus with RNAi triggering double stranded PAL inverted repeats. PAL gene expression was successfully downregulated to 1.2 fold using RNAi phenomenon in C. roseus cells. Decrease in expression of PAL gene led to 10 fold decrease in synthesis of cinnamic acid in transformed C. roseus cells indicating decrease in precursor pool inflow in the pathway. In parallel, there was no significant increase in ajmalicine content indicating precursor pool did not increase towards MIA pathway in transformed C. roseus cells. It can be suggested that complex tight regulation of MIA pathway counteracted change in its metabolic perturbation as a result of PAL enzyme downregulation.