KLONING DAN KONSTRUKSI GEN FLAVODOKSIN DAN FLAVODOKSIN REDUKTASE DARI ESCHERICHIA COLI UNTUK KOEKSPRESI DENGAN CYP124 MYCOBACTERIUM TUBERCULOSIS DALAM RANGKA PRODUKSI ALDEHID DIHIDROARTEMISINAT

• Main Author: Samantha Puteri, Patricia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48381
• Institution: Institut Teknologi Bandung

Malaria is a common endemic disease in the tropical region. WHO has recommended artemisinin combination based therapy to alleviate the Plasmodium falciparum infection in the chloroquine resistance cases.The main problems that arise in the use of artemisinin are the high demand for ACT from various countries and fluctuations in the price and production of artemisinin. In this study, an alternative pathway for artemisinin biosynthesis was proposed by utilizing amorphadiene synthase flexibility to receive 12-hydroxyparnesyl diphosphate as its modified substrate. This alternative pathway might shorten the natural biosynthesis pathway of artemisinin by generating dihydroartemisinic aldehyde from farnesyl diphosphate using two instead of three steps of reactions. The product requires one step oxidation reaction to produce dihydroartemisinic acid as the closest precursor of artemisinin. Theoretically, the hydroxylation of farnesyl diphosphate requires CYP124 from Mycobacterium tuberculosis. The cytochrome activity requires ferredoxin / flavodoxin and ferredoxin / flavodoxin - NADPH reductase which play a role in the delivery of electrons to the cytochrome reaction center. This research is focused on the cloning process of the CYP124, flavodoxin, and flavodoxin reductase gene of Escherichia coli. The construction of CYP124, flavodoxin and flavodoxin reductase gene in the plasmid was carried out using the polymerase chain reaction (PCR) and circular polymerase extention cloning (CPEC) methods. Gene confirmation upon the isolation and cloning process was conducted using gel agarose electrophoresis. Based on the DNA electrophoresis migration, it was confirmed that the isolated genes have comparable size to E. coli flavodoxin and flavodoxin reductase available in the reference. In addition, the effectiveness of cytochrome fusion with its reductase pair is discussed. Comparisons were made between CYP725A4-CPR cytochrome fusion and separate expressions of the two, with the amount of oxidized taxadiene product as parameter. The results showed that the expression of CYP725A4 separately from CPR gave a much higher amount of oxidized taxadiene than the fusion of the two.