HETEROLOGOUS EXPRESSION OF CYP124 IN ESCHERICHIA COLI

• Main Author: Marta Gunawan, Catherine
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/74209
• Institution: Institut Teknologi Bandung

Artemisinin is a first-line therapy for malaria produced naturally by Artemisia annua L. The low and unstable production of artemisinin leads to unmet high demand for artemisinin, so it is necessary to increase production by metabolic engineering in microorganisms. CYP124 from Mycobacterium tuberculosis can hydroxylate farnesyl pyrophosphate (FPP) which further become the precursor for amorphadiene synthase (ADS) producing dihydroartemisinic acid. This alternative pathway would reduce the biosynthetic steps on artemisinin production. In this study, optimization of the expression of CYP124 protein cloned on pET16b plasmid transformed into Escherichia coli DH5? and subcloning cyp124 by circular polymerase extension cloning (CPEC) on pCWORI plasmid transformed into E. coli DH5?. Subcloning of cyp124 in pCWORI was carried out because pCWORI is commonly used for CYP450 expression in E. coli. Optimization of CYP124 protein expression was carried out by comparing Luria-Bertani (LB) and Terrific Broth (TB) media, incubation temperatures (18oC, 25oC, and 37oC), and with or without IPTG induction. CYP124 protein production could be expressed better on TB medium with incubation temperature of 25oC and IPTG induction compared to LB medium. Further confirmation of CYP124 protein expression on pET16b plasmid and continue cloning the cyp124 gene in the pCWORI plasmid can be done.