KONSTRUKSI DAN EKSPRESI CYP124 DALAM ESCHERICHIA COLI SEBAGAI BAGIAN DARI PRODUKSI PREKURSOR ARTEMISININ

• Main Author: Mayrene Kurniawan, Monica
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/69271
• Institution: Institut Teknologi Bandung

Semisynthetic production of artemisinin requires recombinant microorganisms. Amorpha-diene synthase (ADS) as a key enzyme in artemisinin biosynthesis can accept hydroxylated farnesyl diphosphate substrate. This allows noncanonical biosynthesis of artemisinin precursors without involving CYP71AV1 from Artemisia annua. However, an enzyme that has the ability to hydroxylate farnesyl diphosphate (FDP) is needed. It is known that CYP124 of Mycobacterium tuberculosis has the ability to hydroxylate methyl branched fatty acids on C? and isoprenoids, including FDP. This research was conducted in order to construct and express CYP124 as an initial step in increasing the production of artemisinin precursors. In this study, cyp124 was cloned on pET16b plasmid which was transformed into Escherichia coli DH5? competent cells. There are 2 methods of cloning, namely, ligation dependent and ligation independent (circular polymerase extension cloning). Through the CPEC process, several colonies were confirmed to carry the cyp124 gene in the pET16 plasmid. Confirmation was done by colony PCR reaction, restriction analysis, and DNA sequence analysis. CYP124 protein expression was carried out under 2 incubation conditions, 25°C and 37°C, with IPTG induction and without IPTG induction. CYP124 protein is thought to be expressed, but further testing is needed to confirm the expression.