Produção de Artemisinina em Leveduras

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Isoprenoids 
isoprene unit 
 (C5H8)n 
monoterpenoids 
n=2 
geraniol 
limonene 
menthol 
n=1 
hemiterpenoids 
sesquiterpenoids 
abscisic acid 
artemisinin 
humulene 
n=4 
diterpenoids 
cafestol taxadiene 
n=5 
sesterterpenoids 
ophiobolin A 
n=3 
n=6 
triterpenoids 
cholesterol 
n=8 
tetraterpenoids 
β-carotene 
n>8 
polyterpenoids 
rubber 
http://upload.wikimedia.org/wikipedia/commons/b/bb/Cafestol.svg
http://upload.wikimedia.org/wikipedia/commons/8/80/Ophiobolin_A.svg
Isoprenoids 
Pathway transfer from plant to microorganism 
vs. 
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2 metabolic pathways 
mevalonate pathway 
(MVA pathway) 
 
eukaryotes 
plants (cytosol) 
archaea, few eubacteria 
 
precursor: 
acetyl-CoA 
 
1.5 glucose per IPP 
 
 
 
non-mevalonate pathway 
(DXP pathway, MEP pathway) 
 
eubacteria 
plants (plastides) 
apicomplexa 
 
precursors: 
pyruvate + glyceraldehyde-3-phosphate 
 
1 glucose per IPP 
 
Isoprenoids 
O P
O
O
O P
O
O
O
IPP 
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Partow et al. (2012) PLoS ONE 7:e52498 
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Isoprenoids 
vs. 
Advantage E. coli: more efficient pathway -> higher theoretical yields 
 
Advantage S. cerevisiae: better expression host for auxiliary enzymes 
 (P450 monooxygenases) 
 
=> choice may depend on product 
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Artemisinin 
Antimalarial drug precursor 
 
Derivatives used in antimalarial combination therapies 
 
Derived from sweet wormwood plant (Artemisia annua) 
 
Fluctuating price and availability 
 
 
 
⇒ Semi-synthetic artemisinin project: 
 
Started in 2004 
 
Funded by Bill and Melinda Gates Foundation 
 
Collaboration between UC Berkeley, Amyris, Institute for OneWorld Health 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
Plant pathway: 
 
Conversion of FPP tp amorphadiene 
via amorphadiene synthase (ADS) 
 
2 oxidation products: artemisinic acid, 
dihydroartemisinic acid 
 
spontaneous conversion of 
dehydroartemisinic acid to artemisinin 
 
 
Project aim: 
 
Production of artemisinic acid in E. coli 
 
Chemical conversion to artemisinin 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
1. Amorphadiene production in E. coli 
 
• Use of plant terpene synthases with limited success 
• Overexpression of rate-limiting DXP pathway enzymes with limited success 
 (probably due to internal regulatory mechanisms) 
 
 
amorphadiene 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
1. Amorphadiene production in E. coli 
 
• Use of plant terpene synthases with limited success 
• Overexpression of rate-limiting DXP pathway enzymes with limited success 
 
⇒ Expression of codon-optimised ADS 
⇒ Expression of yeast MVA pathway -> 0.5 g/l amorphadiene 
 
amorphadiene 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
1. Amorphadiene production in E. coli 
 
Problem: Imbalanced enzyme expression leading to HMG-CoA accumulation and growth 
inhibition 
 
Solution: Balanced expression of HMGS and HMGR, replacement of yeast enzymes with 
bacterial enzymes 
Fermentation optimisation -> 25 g/l amorphadiene 
amorphadiene 
HMGS HMGR 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
2. Switch to yeast 
 
 
a) Identification of enzyme converting 
amorphadiene to artemisinic acid: 
 
CYP71AV (P450 monooxygenase) 
 
-> 100 mg/l artemisinic acid in primarily engineered 
strain 
 
 
 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
2. Switch to yeast 
 
 
b) Pathway optimisation 
 
• Overexpression of all MVA pathway genes 
 
• Downregulation of first step of ergosterol formation (gene under regulation of 
methionine- or copper-regulatable promoter) 
 
• Fermentation optimisation -> 40 g/l amorphadiene 
 
but: amorphadiene production 10x higher than artemisinic acid production 
 
 
 
 
amorphadiene artemisinic acid 
ergosterol 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
2. Switch to yeast 
 
 
c) Optimisation of oxidation step 
 
Engineered strains with low viability due to oxidative stress 
 (poor coupling between reductase CPR1 and CYP71AV1) 
 
=> Reduction of CPR1 expression, additional expression of 
cytochrome b5 
 
Accumulation of oxidation intermediates 
 
⇒ Additional expression of A. annua aldehyde dehydrogenase 
ALDH1 and alcohol dehydrogenase ADH1 
 
 
-> 25 g/l artemisinic acid 
Paddon et al. (2013) Nature 496:528-532 
Paddon & Keasling (2014) Nature Rev 12:355-367 
Artemisinin 
Conclusion (Part I) 
Yeast as a suitable host for production of high-value compounds 
 
Engineering strategies often product specific and time consuming 
 
 
⇒ Need for better predictability, universal strategies, platform strains 
Systems biology for the 
improvement of microbial 
fermentations 
 
Verena Siewers 
Systems and Synthetic Biology 
Chalmers University of Technology 
 
Campinas, 12 October 2014 
Design Strain Construction 
Fermentation 
Phenotypic characterization 
Reference 
Plasmid 
Chromosome 
integrated 
modification 
Time 
Metabolic Eng. (2004) 6:204-211 
The metabolic engineering cycle 
	Bioenergy beyond biofuels:�High-value, low-volume compounds
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	Systems biology for the improvement of microbial fermentations 
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	Evaluation of point mutations
	Thermotolerant strains showed trade-off at 30C
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