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

• Main Author: Fredrika Sibarani, Sherin
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49867
• Institution: Institut Teknologi Bandung

A sesquiterpene endoperoxide artemisinin isolated from Artemisia annua is recommended by WHO as the effective antimalarial treatment via artemisinin-based combination therapy (ACT). One production strategy to increase artemisinin production is metabolic engineering for the biosynthesis of artemisinin in bacteria. In this study carried out a construction effort in alternative biosynthesis path for the production of artemisinin precursors using Bacillus subtilis. Utilization of the versatility of amorphadiene synthase (Ads) to accept a hydroxylated farnesyl diphosphate substrate is used in the biosynthesis phase of dihydroartemisinic aldehyde as a precursor of artemisinin. Cytochrome CYP124 Mycobacterium tuberculosis selected to be able to supply 12- hydroxyfarnesyl diphosphate. CYP requires ferredoxin/flavodoxin–NADPH reductase and ferredoxin/flavodoxin. Therefore, in this research conducted the construction for coding genes of flavodoxin (ykuN dan ykuP) and flavodoxin reductase (yumC) from B. subtilis along with the cyp124 M. tuberculosis to produce dihydroartemisinic aldehyde. In the literatur study, the author described that flavodoxin of B. subtilis (YkuN dan YkuP) can work as elektron bearer in a sequence of redox reactions with cytochrome P450 BioI. The elektron transfer rate (Kred) of these two flavodoxin is faster than E. coli flavodoxin (FldA dan FldB). Flavin mononucleotide (FMN) that is bound to YkuN dan YkuP proteins affects the stability of flavodoxin structure. One strategy to increase the activity of CYP is through the rerouting of NADPH synthetic pathway. This metabolic engineering succeeded in increasing the production of protopanaxadiol (a terpenoid compound) more than 11 times from the yeast strain without optimization of NADPH. K