HETEROLOGOUS EXPRESSION OF SIX KEY ENZYMES INVOLVED IN THE PRODUCTION OF ARTEMISININ PRECURSORS IN SACCHAROMYCES CEREVISIAE BY4741

• Main Author: Purnomo, Indra
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/52080
• Institution: Institut Teknologi Bandung

Malaria, one of the infectious diseases that still contributes to a very large global mortality rate per year, which is around 435,000 people with the number of cases reaching 219 million in 2017 spread across 87 countries. In Indonesia, the total number of malaria cases recorded in 2019 there were 250,644 cases. Artemisinin combination therapy (ACT) is the latest method suggested by WHO for malaria treatment. Regrettably, the amount of artemisinin produced naturally is very low therefore scientists are challenged to find a new way to overcome this problem. In this research, we performed heterologous expression of six key enzymes that involved in the artemisinin precursor biosynthesis namely farnesyl pyrophosphate synthase (FPS), amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 reductase (CPR), artemisinic aldehyde ?11 (13) reductase (DBR2), and artemisinic aldehyde dehydrogenase (ALDH1) in Saccharomyces cerevisiae BY4741. The expression of these key enzymes is expected to change the metabolic pathways in yeast to produce a precursor metabolite of semi-synthetic artemisinin, namely artemisinic acid. This research was conducted using four recombinant plasmids as gene carriers, which are pBEVY-GL_fps, pBEVY-GU_ads_cyp71av1, pESC-HIS_cpr, and pBEVY- L_dbr2_aldh1. The transformation process was carried out by the electroporation method on S. cerevisiae BY4741 wildtype. Confirmation was carried out using auxotrophs selection media and PCR colony to prove that all plasmids had successfully transformed. Furthermore, the transformant were expressed then tested by being given a treatment that is induced by using glucose and galactose as carbon sources to compare the metabolite results. The final results prove that S. cerevisiae BY4741 transformant can produce metabolites presumed one of them to be artemisinic acid which is the final precursor before the formation of semisynthetic artemisinin with presences of galactose as an inducer compared to glucose.