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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 semisynthetic 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-ß-D-1-thiogalactopyranoside (IPTG) induction and characterized by sodium dodecyl sulfatepolyacrylamide 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. [ABSTRACT FROM AUTHOR]

Purpose: Artemisinin, a secondary metabolite in Artemisia annua is one of primary choice for the treatment of malaria, it is naturally produced in low concentration from this plant. This study was aimed to clone key enzymes of artemisinin production in order to enhance its production through the semi-synthetically production in Saccharomyces cerevisiae. Methods: Two key enzymes in artemisinin biosynthetic pathway which are farnesyl phosphate synthase (fps) and amorpha-4,11-diene synthase (ads) genes were transformed into S. cerevisiae using pBEVY vector. Successful transformation was checked by polymerase chain reaction (PCR) method and sequencing analysis Results: Recombinant plasmids which are pBEVY-GU_ads and pBEVY_GL_fps were successfully constructed. The optimized ads gene was amplified using PCR with a couple of primers that are designed in order to provide the homolog recombination between ads gene with the expression plasmid of pBEVY-GU respectively. While the A. annua optimized fps gene was cloned using classical method. Transformants were grown in selective media Synthetic Defined (SD) without leucine for transformants contain plasmid pBEVY-GL_fps and media without uracil for transformants contain plasmid pBEVY-GU_ads. Confirmation of colonies was done by PCR with primers to amplify fps and ads. DNA from yeast was isolated from positive colonies then transformed to E. coli. Plasmid from E. coli was isolated for restriction analysis and sequencing. Protein expression was induced by cultivating the yeast in the media with 2% galactose. Conclusion: Based on PCR, restriction and sequencing analysis, it could be concluded that fps and ads genes were successfully constructed, transformed and expressed in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Purpose: The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens , and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads - p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads - p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads - p19 up to 2.57 folds compared to the untransformed leaves, while for p19 , cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua. [ABSTRACT FROM AUTHOR]