Cloning of Gene Encoding Amorphadiene Synthase in Saccharomyces cerevisiae BY4741 Aiming for Production of Artemisinin Precursor

• Main Author: Auliya Aksa, Annisa
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38019
• Institution: Institut Teknologi Bandung

Antimalarial drug resistance is one of the greatest threats in controlling the disease. Resistance to all classes of antimalarial drugs has emerged, with the exception of the artemisinin. Semi-synthetic artemisinin has become an alternative source of artemisinin used in malaria treatment. Semi-synthetic artemisinin is produced by engineering microorganisms to produce the precursor of artemisinin. Saccharomyces cerevisiae has had half of the overall enzyme required in the biosynthesis of artemisnin in Artemisia annua. To complete the synthesis of artemisinin in Saccharomyces cerevisiae, several genes encoding enzymes needed in artemisinin biosynthesis need to be cloned. Amorphadiene synthase, ADS, (EC 4.2.3.24) catalyzes the synthesis of amorpha-4,11-diene (amorphadiene), the first cyclic intermediete in biosynthesis of antimalarial artemisinin from its natural source, Artemisia annua. In this project, cloning of yeast-optimized ADS encoding gene ( ~ 1,6 kb) to Saccharomyces cerevisiae BY4741 was performed to obtain a yeast strain producing amorphadiene which, in further processes, can be converted to artemisinin. pBEVY-GU plasmid (~6 kb) was used as the expression vector in S. cerevisiae BY4741. Restriction analyses using EcoRI, NheI, and PstI restriction enzymes was performed to characterize the constructed recombinant plasmid. DNA fragments resulted from restriction with the three enzymes have the same sizes with fragments from pBEVY-GU_ads (~7,6 kb) as predicted. This shows that the construction of pBEVY-GU_ads recombinant plasmid have been successfully performed. pBEVY-GU_ads then used in transformation of S. cerevisiae BY4741 by electroporation and confirmed wtih colony PCR. Colony PCR results as showed by gel electrophoregram confirmed the amplification of 1,6 kb fragment, consistent with the ads gene size. Thus, ADS-encoding gene has been successfully cloned in S. cerevisiae BY4741. However, SDS-PAGE analysis of whole protein extract from transformed yeast still shows no expression of the gene.