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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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)

9. 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

10. 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)

11. 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

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

13. 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, (2014).
Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, (2006).
Schmid, G. & Hofheinz, W. Total synthesis of Qinghaosu. J. Am. Chem. Soc. 105, 624–-625 (1983).
World Health Organization. World Malaria Report (WHO, 2013)

14. Thank you!
Any questions?