Poster Presentation First Malaria World Congress 2018

Cloning and Expression of Artemisia annua Farnesyl Pyrophosphate Synthase (FPS) in Saccharomyces cerevisiae for Production of Semi-Synthetic Artemisinin   (#459)

Fifi Fitriyah Masduki 1 2 , Vaniarta Shintyarani 2 3 , Annisa Aulia Aksa 2 , Rizqiya Astri Hapsari 2 , Dessy Natalia 1 2 , Elfahmi Elfahmi 2 3
  1. Biochemistry Division, Faculty of Mathematics and Natural Science Institut Teknologi Bandung, Bandung, West Java, Indonesia
  2. Bioscience and Biotechnology Research Center , Institut Teknologi Bandung, Bandung, West Java, Indonesia
  3. School of Pharmacy, Institut Teknologi Bandung, Bandung, West Java, Indonesia

Antimalarial drug resistance is one of the greatest threats in controlling the disease. Resistance to all classes of antimalarial drugs has emerged, with the exception of the artemisinin. Semi-synthetic artemisinin has become an alternative source of artemisinin used in malaria treatment. Semi-synthetic artemisinin is produce by engineering microorganisms to produce the precursor of artemisinin. Saccharomyces cerevisiae has had half of the overall enzyme required in the biosynthesis of artemisinin in Artemisia annua. One of the main precursors in artemisinin biosynthesis is farnesyl pyrophosphate (FPP). Derived from the general terpenoid biosynthesis, two molecules of isopentenyl diphosphate (IPP) and one dimethylallyl diphosphate (DMAPP) are condensed by farnesyl pyrophosphatesynthase (FPS; EC:; MW 39.5 kDa) into FPP. S. cerevisiae naturally produce FPS. In order to increase FPP production, we overexpressed FPS by cloning the FPS gene to S. cerevisiae. pBEVY-GL plasmid was used as the expression vector compatible with S. cerevisiae expression system. The recombinant plasmid pBEVY-GL_fps was transformed into S. cerevisiae BY4741. The FPS gene was expressed using galactose 2% as an inducer for 18 hours on 30oC. SDS-PAGE analysis showed an increase in the thickness of protein band around 39.5 kDa in induced culture compared to wild type and non-induced cell of recombinant S. cerevisiae. This result suggest that FPS is successfully overexpressed. The presence of FPP was analyzed by thin layer chromatography. Culture cell was lysed and extracted using ethyl acetate, and its chromatogram from thin layer chromatography using n-hexane:ethyl acetate (6:4) as the mobile phase was analyzed. A spot with Rf 0.35 detected using sprayed annisaldehyde and H2SOwas shown a bit thicker qualitatively in induced culture. The spots were considered as FPP. Based on these results, it could be concluded that FPS gene had been successfully overexpressed and also increased the production of FPS in S. cerevisiae BY4741.




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