Abstract
Purpose: Artemisinin, a secondary metabolite in Artemisia annua is one of primary choice for the treatment of malaria, it is naturally produced in low concentration from this plant. This study was aimed to clone key enzymes of artemisinin production in order to enhance its production through the semi-synthetically production in Saccharomyces cerevisiae.
Methods: Two key enzymes in artemisinin biosynthetic pathway which are farnesyl phosphate synthase (fps) and amorpha-4,11-diene synthase (ads) genes were transformed into S. cerevisiae using pBEVY vector. Successful transformation was checked by polymerase chain reaction (PCR) method and sequencing analysis
Results: Recombinant plasmids which are pBEVY-GU_ads and pBEVY_GL_fps were successfully constructed. The optimized ads gene was amplified using PCR with a couple of primers that are designed in order to provide the homolog recombination between ads gene with the expression plasmid of pBEVY-GU respectively. While the A. annua optimized fps gene was cloned using classical method. Transformants were grown in selective media Synthetic Defined (SD) without leucine for transformants contain plasmid pBEVY-GL_fps and media without uracil for transformants contain plasmid pBEVY-GU_ads. Confirmation of colonies was done by PCR with primers to amplify fps and ads. DNA from yeast was isolated from positive colonies then transformed to E. coli. Plasmid from E. coli was isolated for restriction analysis and sequencing. Protein expression was induced by cultivating the yeast in the media with 2% galactose.
Conclusion: Based on PCR, restriction and sequencing analysis, it could be concluded that fps and ads genes were successfully constructed, transformed and expressed in S. cerevisiae.