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 falciparumWikipedia.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).

Artemisinin ehow.com

anamed.net

gobotany.newenglandwild.org

Artemisia annua

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 yeastSaccharomyces 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 acidAmorpha-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

Schematic representation of the engineered artemisinic acid biosynthetic pathway in S. cerevisiae strain EPY224 expressing CYP71AV1 and CPR. (Ro, 2006)

Modifications 153mg/l of amorphadiene produced.

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.

EPY224 BY4742 pRS425ADS PGAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20 (ura-)

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 Integrating URA3

pRS-ERG9 PMET3-ERG9 Integrating HIS3

pδ-ERG20 ERG20 Integrating URA3

(Supplementary material. Ro, 2006)

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.

Modifications

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).

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Perspectives

Industrial scale-up and optimization; Reduce artemisinic combination therapies to significantly

below their current prices.

nyas.org

2.uol.com.br

twitter.com

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)

Thank you!

Any questions?