EKSPRESI AMORFA-4,11-DIEN SINTASE (ADS) DAN SITOKROM P450 MONOOKSIGENASE (CYP71AV1), ENZIM KUNCI DALAM BIOSINTESIS ARTEMISININ PADA Saccharomyces cerevisiae

• Main Author: Setianingrum, Sulis
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44090
• Institution: Institut Teknologi Bandung

Malaria is an endemic disease caused by Plasmodium parasite through a bite of an infected female Anopheles mosquito. In 2002, World Health Organization (WHO) recommended an effective treatment for malaria patient, namely artemisinin-based combination therapy (ACT). ACTs is suggested due to highly Plasmodium resistance toward previous antimalarial drug, such as chloroquine and sulfadoxine-pyrimethamine. Artemisinin is a sesquiterpene lactone obtained from Artemisia annua. The production of artemisinin in A. annua is quite low so the production of artemisinin could be increased by a combinatorial biosynthesis of artemisinin in Saccharomyces cerevisiae. S. cerevisiae provides the precursors from the mevalonic pathway, farnesyl pyrophosphate, converts to amorphadiene and followed with further oxidation to artemisinic acid by ADS and CYP71AV1 enzymes, respectively. The codon-optimized synthetic genes ads and cyp71av1 for S. cerevisiae and Escherichia coli respectively were used for cloning. Cloning was carried out directly in S. cerevisiae using pBEVY-GU, a bi-directional expression vector under regulatable galactose promotor. Expression of ADS-his6 (63.9 kDa) and CYP71AV1 (53.8 kDa) was cultivated with variations in galactose concentration (0.1%, 1%, and 2%), incubation time (24, 42, and 48 h), and carbon sources (2% raffinose or galactose) but the expressions were not significantly different. Thus, yeast harboring the pBEVY- GU_cyp71av1eo_ads plasmid was grown on SD (Synthetic Defined) selection media without uracil supplemented with 2% galactose and induced for 24 hours. A Full-length protein of ADS-his6 is successfully expressed confirmed by Western blot using anti-his antibody.