Synthetic Production
of Artemisinin
Patrick J. Westfall, Ph.D.
Senior Scientist, Amyris Inc.
CONFIDENTIAL
[1]
Introduction to Amyris, Inc. – World wide locations
CONFIDENTIAL
[2]
Introduction to Amyris, Inc. - Founders
•
•
•
•
Jack Newman – CSO
Kinkead Reiling
Jay Keasling – UC Berkeley
Neil Renninger -CTO
CONFIDENTIAL
[3]
Introduction to Amyris, Inc. – The Power of Synthetic Biology
Isolate Gene
Scale Up
Engineer Host
Identify Single Gene
Product
CONFIDENTIAL
[4]
Introduction to Amyris, Inc. – The Power of Synthetic Biology
Optimize Pathway
Ergosterol
Identify Entire
Metabolic Pathway
CONFIDENTIAL
[5]
Malaria
Female Anopheles Mosquito
•
•
•
•
Plasmodium falciparum
Infected Red Blood Cells
Affects 109 Countries Worldwide
216 Million cases in 2010
~665,000 deaths in 2010
1 child dies every minute of
Malaria in Africa
•
Source: 2010 WHO malaria
fact sheet
CONFIDENTIAL
[6]
A Brief History of Artemisinin
168 B.C Recipes For 52 Kinds Of Diseases found in
the Mawangdui Han Dynasty tomb
Hemorrhoids
340
A.D.
Zhou Hou Bei Ji Fang (Handbook of
Prescriptions for Emergency Treatments)
Fevers (malaria)
1967
China sets up top-secret “Project 523” to
find malaria medicines to help Vietnam
during the war with the U.S.
1972
Active ingredient (artemisinin) isolated.
2004
World Health Organization (WHO)
recommends artemisinin-based
combination therapy (ACT) for
uncomplicated malaria in areas
experiencing chloroquine resistance
CONFIDENTIAL
[7]
Treating Malaria : The Challenges
Treating malaria would require:
189 to 327 million ACT treatments per year
Artemisinin needed:
150 to 200 tons of artemisinin per year
• 2-3X increase in production
• Decrease/stabilization in price
• Elimination of stock-outs
CONFIDENTIAL
[8]
The Artemisinin Project: Significant lives saved due to scalable supply
Artemisinin
extraction
A. annua cultivation
(10-12 month growing cycle)
Fermentation production
(2 week cycle)
Artemisinic
Acid
3-Step Chemical
Synthesis Steps
CONFIDENTIAL
[9]
Primer to Important Compounds
Oxidation/
Reduction
Oxidation/
Reduction
Oxidation/
Reduction
H
H
H
H
H
H
H
H
H
HO
Amorphadiene
Artemisinic Alcohol
HO
O
O
Artemisinic
Aldehyde
Artemisinic Acid
H
Chemistry
O
O
H
Artemisinin
H
O
H
O
CONFIDENTIAL
[ 10 ]
The Mevalonate Metabolic Pathway in S. cerevisiae
O
H 3C
SCoA
ERG 10
E RG13
HM G1
HO C
2
Acetyl-CoA
OH
OH
Mevalonate
OPP E RG20
FPP
HO
H C
3
H
IDI1
E RG19
E RG8
E RG12
H
Ergosterol
Ergosterol
Identify Metabolic
Pathway
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[ 11 ]
Engineering the Mevalonate Pathway in Yeast
tHMGR
O
H3C
ERG10
S CoA
Acetyl-CoA
ERG13
ERG13
thiolase
HMG1
synthase
reductase
tHMGR
H3 C
OH
H O2C
OH
Mevalonate
FPP
OPP
UPC2-1
ERG20
ERG20
FPP
synthase
IDI1
ERG19
isomerase
decarboxylase
ERG8
P-mev
kinase
ERG12
ERG12
mev
kinase
H
ADS
ERG9
squalene
synthase
H
H
HO
Amorphadiene
H
Ergosterol
CONFIDENTIAL
[ 12 ]
160
140
120
100
80
60
40
20
0
S
FP
P
R
M
G
2
x
tH
PC
U
P
(M
E
21
G
9
R
T3
)-E
tH
M
1x
A
G
R
Amorphadiene
concentration
D
S
Amorphadiene (mg/l)
Amorphadiene Production in S. cerevisiae
Genetic modification
From Ro et al Nature Biotech. 2006
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[ 13 ]
Engineering the Mevalonate Pathway in Yeast
tHMGR
O
H3C
ERG10
S CoA
Acetyl-CoA
thiolase
H3 C
ERG13
OH
H O2C
synthase
reductase
tHMGR
OH
Mevalonate
FPP
OPP
ERG20
FPP
synthase
IDI1
ERG19
isomerase
decarboxylase
ERG8
P-mev
kinase
ERG12
mev
kinase
UPC2-1
H
H
CYP71AV1
CYP71AV1
CYP71AV1
AaCPR1
AaCPR1
AaCPR1
ADS
H
H
Amorphadiene
HO
H
H
H
H
HO
O
Art. Acid
H
O
CONFIDENTIAL
[ 14 ]
Production of Artemisinic Acid in S. cerevisiae
30
Target
Product [g/L]
25
20
15
S. cerevisiae Art. Acid
S. cerevisiae Amorph.
10
5
0
0
50
100
150
200
250
Time (hrs)
CONFIDENTIAL
[ 15 ]
Heterologous Expression of the Entire S. cerevisiae Mev Pathway
tHMGR
O
H3C
ERG10
S CoA
Acetyl-CoA
thiolase
H3 C
ERG13
OH
H O2C
synthase
reductase
tHMGR
OH
Mevalonate
FPP
OPP
ERG20
FPP
synthase
IDI1
ERG19
ERG8
ERG12
isomerase
decarboxylase
P-mev
kinase
mev
kinase
UPC2-1
H
H
CYP71AV1
CYP71AV1
CYP71AV1
AaCPR1
AaCPR1
AaCPR1
ADS
H
H
Amorphadiene
HO
H
H
H
H
HO
O
Art. Acid
H
O
CONFIDENTIAL
[ 16 ]
Heterologous Expression of the Entire S. cerevisiae Mev Pathway
Yeast 1.0
CONFIDENTIAL
[ 17 ]
Heterologous Expression of the Entire S. cerevisiae Mev Pathway
tHMGR
O
H3 C
H3C
ERG10
S CoA
Acetyl-CoA
thiolase
tHMGR
ERG13
OH
H O2C
synthase
OH
Mevalonate
tHMGR
FPP
OPP
ERG20
FPP
synthase
IDI1
ERG8
ERG19
isomerase
P-mev
kinase
decarboxylase
ERG12
mev
kinase
H
H
CYP71AV1
CYP71AV1
CYP71AV1
AaCPR1
AaCPR1
AaCPR1
ADS
H
H
H
H
H
H
HO
O
Amorphadiene
HO
Art. Acid
H
O
CONFIDENTIAL
[ 18 ]
Yeast 2.0 Amorphadiene Production
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[ 19 ]
Large “Gap” in Production Between Amorphadiene and Art. Acid
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[ 20 ]
Chemical Conversion of Amorphadiene to Artemisinin (Plan B)
H
H
H
c,d
a,b
76%
H
H
76.4%
H
O
HO
H
H
H
e,f
O
O
H
O
77.7%
H
H
O
H
H
O
Westfall et al. PNAS USA 2012 Jan 17;109(3):E111-8
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[ 21 ]
Possible Sources of Toxicity and Poor P450 Activity
2. Product (AA) toxicity
1. Protein expression toxicity
H
H
H
HO
ER
O
ER
H
?
NADPH
CPR
NADP
5. Poor coupling leads to
excessive NADPH oxidation
P450
?-OH
b5
H2O2
3. Improper oxidation
toxicity
4. Oxidative stress
6. Poor coupling due to lack of CYT b5
22
CONFIDENTIAL & PROPRIETARY - Do Not Distribute Without Authorization
CONFIDENTIAL
[ 22 ]
Artemisinic Acid Production in Shakeflasks
CONFIDENTIAL
[ 23 ]
Heterologous Expression of the Entire S. cerevisiae Mev Pathway
tHMGR
H3 C
O
H3C
ERG10
S CoA
thiolase
Acetyl-CoA
tHMGR
ERG13
OH
H O2C
OH
Mevalonate
synthase
tHMGR
FPP
OPP
ERG20
IDI1
FPP
synthase
isomerase
ERG8
ERG19
P-mev
kinase
decarboxylase
ERG12
mev
kinase
H
CYP71AV1
ADS
H
CYP71AV1
H
CYP71AV1
AaCYB5
AaCYB5
AaCYB5
AaCPR1
AaCPR1
AaCPR1
H
HO
H
H
O
Amorphadiene
H
HO
H
H
Art. Acid
O
CONFIDENTIAL
[ 24 ]
Product Formation in Shakeflasks
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[ 25 ]
Product Formation in Shakeflasks
CONFIDENTIAL
[ 26 ]
Conversion of Amorphadiene to Artemisinic Acid
P450
P450
P450
H
H
H
H
H
H
H
H
HO
Amorphadiene
Artemisinic Alcohol
H
HO
O
O
Artemisinic
Aldehyde
Artemisinic Acid
CONFIDENTIAL
[ 27 ]
Intermediate Build Up
CONFIDENTIAL
[ 28 ]
Collaboration with Dr. Pat Covello, Canadian Natural Resource Council
H
H
HO
AaADH1
H
H
H
Pat Covello CNRC-PBI
AaALDH1
H
HO
H
O
O
CONFIDENTIAL
[ 29 ]
Conversion of Amorphadiene to Artemisinic Acid
P450
AaALDH1
P450
AaADH1
P450
H
H
H
H
H
H
H
H
HO
Amorphadiene
Artemisinic Alcohol
H
HO
O
O
Artemisinic
Aldehyde
Artemisinic Acid
CONFIDENTIAL
[ 30 ]
Product Formation in Shakeflasks
CONFIDENTIAL
[ 31 ]
Heterologous Expression of the Entire S. cerevisiae Mev Pathway
tHMGR
H3 C
O
H3C
ERG10
S CoA
thiolase
Acetyl-CoA
tHMGR
ERG13
OH
H O2C
OH
Mevalonate
synthase
tHMGR
FPP
OPP
ERG20
IDI1
FPP
synthase
isomerase
ERG8
ERG19
P-mev
kinase
decarboxylase
ERG12
mev
kinase
H
ADS
H
CYP71AV1
CYP71AV1
CYP71AV1
AaCYB5
AaCYB5
AaADH1
AaCYB5
AaALDH1
AaCPR1
AaCPR1
AaCPR1
H
H
Amorphadiene
H
HO
H
H
HO
H
H
O
Art. Acid
O
CONFIDENTIAL
[ 32 ]
Product Formation in Bioreactors
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[ 33 ]
Summary

Heterologous expression of the entire mevalonate pathway in yeast
allowed us to exceed the target goal for Amorphadiene production..

High level expression of the P450 and the AaCPR1 causes oxidative
stress in cells resulting in decreased product formation and lower cell
viability.

Although the P450/CPR were capable of performing all three oxidation
steps to convert Amorphadiene to Artemisinic Acid, discovery of
additional pathway genes led reduced buildup of potentially toxic
pathway intermediates.
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[ 34 ]
The Opportunity for Impact
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[ 35 ]
Predicted artemisinin supply by The Boston Consulting Group
(November 2009)
2009
2010
2011
2012
2013
2014
2015
2010
2011
2012-2015
Supply of Artemisinin
likely to be sufficient to
meet ACT demand
Artemisinin shortage
probable unless the
supply situation improves
Significant increase in
supply needed to meet
steady state demand
Some sources have predicted
shortage or tight supply will be tight
• We feel a shortage is unlikely
given the most probable set of
supply data and demand
assumptions
• Wild leafs would be able to make
any potential small gap
Three critical factors:
(1) Amount of safety stock held in
the supply chain today
(2) Amount of Artemisia currently
cultivated
(3) AMFm uptake Phase I countries
Companies will deplete majority of
safety stock to meet 2010 demand
Even with delayed AMFm demand will
outstrip growing projected growing
capacity available in 2011
Significantly increase in area under
cultivation needed in 2010 to meet
expected demand
Cultivated Artemisia needs to increase
to 23,000 hectares (vs 7,000 today)
• Assuming that AMFm ramps to
cover 50% of the private antimalarial market in 38 highest
burden countries
Historically, prices needed to reach
>$1,000 / kg to attract this many
farmers/ extractors to the market
• Unclear what price point would be
required to stimulate this level of
growing in future
New technologies may begin to affect
artemisinin supply in this timeframe
CONFIDENTIAL
[ 36 ]
July 7, 2010 press release
“intent to commercialize ACTs containing the semisynthetic artemisinin in 2012”
CONFIDENTIAL
[ 37 ]
Acknowledgements
UC Berkeley
Eric Paradise
Dae-Kyun Ro
Mario Oulett
Jim Kirby
Michelle Chang
Syd Withers
Jay Keasling
Institute for OneWorld Health
Tue Nguyen
Sanofi-Aventis
Corinne Masson-Brocard
Denis Thibaut
Ronan Guevel
Marc Ferron
Bruno Dumas
Paul Baduel
Henri Farret
Amyris Biotechnologies
Chris Paddon
Hanxiao Jiang
Kirsten Benjamin
Tom Treynor
Diana Eng
Anna Tai
Rika Regentin
Andrew Main
Hiroko Tsuruta
Dan Dengrove
Doug Pitera
Michele Fleck
Jake Lenihan
Gordon Dang
Jack Newman
Stephanie Secrest
Neil Renninger
Tizita Horning
Kinkead Reiling
Karl Fischer
Zygaya
Kay Monroe
Derek McPhee
Consultants
Jasper Rine (UCB)
Hans van Dijken (Bird Engineering)
Paul Ortiz de Montellano (UCSF)
Pat Covello (CNRC)
CONFIDENTIAL
[ 38 ]
Synthetic Biology in Northern California
Institute for OneWorld Health
Amyris
JBEI (Jay Keasling) & SynBERC
Amyris
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[ 39 ]