Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2022 , Vol 26 , Issue 6
Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli
Tresna LESTARI1,Catur RIANI2,Elfahmi ELFAHMI1,Asep Gana SUGANDA1
1Department of Pharmaceutical Biology, School of Pharmacy, Institut Teknologi Bandung, Bandung, Indonesia
2Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung, Bandung, Indonesia
3Biosciences and Biotechnology Research Center, Institut Teknologi Bandung, Indonesia
4Faculty of Pharmacy, Universitas Bakti Tunas Husada, Tasikmalaya, Indonesia
DOI : 10.29228/jrp.256 Artemisinin combination therapies (ACTs) have become the mainstay of treatment for malaria worldwide. This has led to a high demand for artemisinin precursors as starting materials for artemisinin production and their semi-synthetic derivatives. In this study, heterologous expression of enzymes involved in artemisinin biosynthesis was performed in Escherichia coli to produce artemisinin precursors, i.e., amorphadiene, artemisinic acid, and dihydroartemisinic acid. These enzymes are farnesyl pyrophosphate synthase (FPS), amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1/CYP), artemisinic aldehyde delta-11(13) reductase (DBR2), and aldehyde dehydrogenase 1 (ALDH1). Overexpression of the heterologous 1-deoxy-D-xylulose 5-phosphate (DXP) synthase gene (dxs) from Bacillus subtilis and the native isopentenyl diphosphate delta isomerase (IDI) gene (idi) from E. coli was also performed to enhance isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the methylerithritol phosphate (MEP) pathway in E. coli. All genes were cloned into three plasmids. Gene expression was performed under isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The number of IPTG, incubation temperature, length of incubation time, and culture medium were optimized to find the best conditions for protein expression. It is found that all proteins could be expressed under 0.5 mM IPTG induction at an incubation temperature of 20 °C for 24 hours in Luria-Bertani medium. The cloning of seven key enzymes involved in artemisinin biosynthesis in E. coli has never been described in other studies. By further optimizing the fermentation process, this research offers a promising future for the production of artemisinin precursors in E. coli. Keywords : heterologous expression; MEP pathway; artemisinin; Escherichia coli
Marmara University