Production of an antimalarial drug in engineered S. cerevisiae Ana Virginia Frota Guimaraes PBIO 4500 Dec/04/14

Malaria Malaria is global health problem: 207 million cases of malaria occurred globally in 2012 and 627,000 deaths. (WHO, 2013) It is caused by the parasite Plasmodium spp. that commonly infects a certain type of mosquito which feeds on humans. (cdc.gov) Anopheles mosquito.  cdc.gov Plasmodium falciparum Wikipedia.com

Artemisinin Sesquiterpene lactone endoperoxide extracted from Artemisia annua (Ro, 2006); Highly effective against multi-drug-resistant Plasmodium spp. (Ro, 2006); Its total synthesis is difficult and costly (Schmid, 1983). Artemisia annua gobotany.newenglandwild.org Artemisinin ehow.com anamed.net

Artemisinic acid Immediate precursor for Artemisinin; Its production can be cost-effective, high-quality, environmentally friendly and reliable; Simple and inexpensive purification process. (Ro, 2006) rsc.org

Artemisinic-acid-producing yeast Saccharomyces cerevisiae Three steps production: 1- engineering farnesyl pyrophosphate (FPP) biosynthetic pathway; 2- introducing amorphadiene synthase gene (ADS); 3- cloning and expressing a novel cytochrome P450 monooxygenase (CYP71AV1) and its redox partner NADPH:cytochrome P450 oxidoreductase (CPR) FPP Artemisinic acid Amorpha-4,11-diene ADS P450 three-step oxidation

Modifications Introduction of ADS gene. Expressed under the control of GAL1 promoter in the pRS425 plasmid Selection of transformants done in SD-LEU plates. snapgene.com

153mg/l of amorphadiene produced. Modifications Engineered mevalonate pathway producing more FPP, thus more amorphadiene. Upregulate and downregulate gene expression (plasmids pδ-HMGR, pδ- UPC2, pRS-ERG9 and pδ-ERG20). Selection done in SD-LEU-URA plates, SD-LEU-HIS-MET plates and SD-LEU-URA-HIS-MET plates. Schematic representation of the engineered artemisinic acid biosynthetic pathway in S. cerevisiae strain EPY224 expressing CYP71AV1 and CPR. (Ro, 2006)

Description of plasmids used Name Gene expressed Plasmid type Marker pRS425ADS ADS 2-micron replicon LEU2 pδ-HMGR tHMGR Integrating URA3 pδ-UPC2 upc2-1 pRS-ERG9 PMET3-ERG9 HIS3 pδ-ERG20 ERG20 EPY224 BY4742 pRS425ADS PGAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20 (ura-) (Supplementary material. Ro, 2006)

Modifications Transformed strains with pESC-URA plasmids containing CPR and CYP71AV1. Transformants selected in defined medium lacking histidine, leucine and uracil, and supplemented with galactose and dextrose. genomics.agilent.com

Results Production was analyzed by gas chromatography mass spectrophotometry (GC-MS). Culture medium and cell pellet were analyzed More than 96% of synthesized artemisinic acid was removed from the cell pellet Purification done with a single silica gel column chromatographic separation (more than 95% pure artemisinic acid) GC-MS analysis of artemisinic acid produced from A. annua and transgenic yeast. (Ro, 2006)

Results The transgenic yeast produced artemisinic acid at a biomass fraction of 4.5% dry weight and A. annua produced 1.9% dry weight artemisinic acid and 0.16% artemisinin The production in S. cerevisiae occurred over a much shorter time (4–5 days for yeast versus several months for A. annua). news.softpedia.com commons.wikimedia.org

Perspectives Industrial scale-up and optimization; Reduce artemisinic combination therapies to significantly below their current prices. twitter.com nyas.org 2.uol.com.br

References CDC and Malaria (April, 2014). Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria. Retrieved from: cdc.gov/malaria. Paddon, C. & Keasling, J. Semi-synthetic artemisinin: a model for use of synthetic biology in pharmaceutical development. Nature Reviews. 12, 355-367 (2014). Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943 (2006). Schmid, G. & Hofheinz, W. Total synthesis of Qinghaosu. J. Am. Chem. Soc. 105, 624–-625 (1983). World Health Organization. World Malaria Report 2013. (WHO, 2013)

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