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Expression of two key enzymes of artemisinin biosynthesis FPS and ADS genes in saccharomyces cerevisiae
Elfahmia, Hapsari R.A.a, Chrysanthy T.a, Synthiarini V.a, Masduki F.F.a, Setiawan A.b, Muranaka T.b
a School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia
b Departement of Biotechnology, Graduate School of Biotechnology, Osaka University, Japan
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]© 2021 The Author(s).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.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Amorpha-4,11-diene synthase,Artemisinin,Farnesyl phosphate synthase,Malaria,Saccharomyces cerevisiae[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text]The author would like to thanks the Directorate of Research and Community services, Ministry of Research, Technology and Higher Education, Republic of Indonesia and Bandung Institute of Technology for financial support of the research. We also would like to thank Dr. Dessy Natalia’s Lab for their kindness in sharing and supporting the blot transfers equipment and chemical for western blot.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.34172/apb.2021.019[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]