Metabolic Engineering in Catharanthus Roseus using RNAI as a Tool

Abstract

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