PENERAPAN BIOSINTESIS KOMBINATORIAL PADA BEBERAPA SENYAWA BAHAN ALAM SERTA DETEKSI ADS DAN CYP71AV1 ARTEMISIA ANNUA L. PADA SACCHAROMYCES CEREVISIAE BY4741

• Main Author: Christabella Sutopo, Natasha
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48375
• Institution: Institut Teknologi Bandung

Based on World Malaria Report 2019 published by World Health Organization, it was estimated that there were around 228 million cases related to malaria globally. Malaria is a disease caused by protozoa parasite, Plasmodium sp., which infected human red blood cells. One of the treatments that has been proven effective treat malaria is Artemisinin-based combination therapies, which is a treatment combining antibiotics and artemisinin-derivatives drugs, which are two active substances with different mechanism of action. Artemisinin is sesquiterpene lactone compound, contained as secondary metabolites in Artemisia annua . Naturally, artemisinin production in A. annua is very limited. Various methods have been done to enhance the production of artemisinin. One of the methods is by using combinatorial biosynthesis method using microbial host, namely Saccharomyces cerevisiae. Artemisinin is produced through mevalonate biosynthesis pathway. By using genetic engineering, key enzymes that play a role in the biosynthesis process can be generated. Amorpha4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) are the key enzymes that play a role in the biosynthesis process of artemisinin. In particular, they take part in transforming farnesyl pyrophosphate (FPP) into amorphadiene and in oxidating amorphadiene into artemisinic acid. In this research, expression level of ADS and CYP71AV1 will be detected using qPCR method. Sample that will be detected are S. cerevisiae wildtype, empty vector pBEVY-GU induced and uninduced by galactose, also recombinant plasmid pBEVY-GU_cyp71av1eo_ads induced and uninduced by galactose using sampling point of 1 hour, 24 hours, and 48 hours after induction. RNA of sampled S. cerevisiae cells are isolated and syntheses to cDNA. cDNA products then will become the sample that will be detected the expression level using qPCR method. Application of combinatorial biosynthesis on natural compound will also be discussed in this paper. From terpenoids group, artemisinin and paclitaxel will be discussed. From alkaloids groups, morphine and vinca alkaloids will be discussed. From phenolic group, flavonoids compound will be discussed. The compounds mentioned are natural compounds that are widely use in the health industries but naturally produces in a very small amount, hence combinatorial biosynthesis is the key to resolve this problem.