CLONING AND HETEROLOGOUS EXPRESSION OF THE MEVALONATE PATHWAY FOR ARTEMISININ PRECURSOR PRODUCTION IN ESCHERICHIA COLI

• Main Author: Bayu Satriawan, Ahmad
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71133
• Institution: Institut Teknologi Bandung

The World Health Organization (WHO) recommends the artemisinin as the main treatment for malaria infection by Plasmodium falciparum. This compound is used together with one or more drugs in the artemisinin combination therapy (ACTs). Semi-synthetic production is a potential alternative to produce artemisinin. In a previous study, an attempt was made to increase amorphadiene production through heterologous expression of the mevalonate pathway in B. subtilis. However, the upper mevalonate pathway still does not work optimally. Thus, optimization is needed to increase the expression of the upper pathway as a supply of mevalonate intermediate products. In this study three key genes composing the mevalonate pathway (atoB, mvaS, mvaA) were cloned into the pDR111 plasmid using the circular polymerase extension cloning (CPEC) method. Based on the results of restriction analysis and sequencing, cloning was successfully carried out. The results of the SDS-PAGE expression analysis showed that there was protein expression at the size of the target proteins atoB (40.35 kDa), mvaS (43.22 kDa) and mvaA (46.18 kDa). Metabolite analysis by LC-MS/MS showed the presence of mevalonate metabolites formed in cultures containing recombinant plasmids.