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Engineering Microbial Metabolism for
Production of Anti-Malarial Drugs
Jay D. Keasling
Joint BioEnergy Institute
Lawrence Berkeley National Laboratory
University of California, Berkeley
Malaria
• Caused by
Plasmodium, a single-
cell protozoan
–Transmitted by
Anopheles mosquito
–Destroys red blood cells
Malaria
• Economists have proposed that malaria
decreases the GDP of affected countries by
as much as 50%.
• 1-3 million
people die of
malaria every year
–90% are children
• 300-500 million
people infectedSource: Roll Back Malaria
World Malaria Report 2005
• Most widely-
used drugs to
treat malaria
Quinine-based drugs
• Plasmodium in many parts of the world is largely resistant to chloroquine
Source: Roll Back Malaria
World Malaria Report 2005
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)
1972 Active ingredient (artemisinin) isolated
Artemisinin is produced in oil sacs
on Artemisia annua leaves
O
O
O
O
O
H
HH
Artemisinin... ...is produced by trichomes... ...found on
Artemisia annua leaves...
Current process
Artemisinin
O
O
O
O
O
H
HH
Plant synthesis
Purification
Artesunate
O
O
H
H HH
OO
OH
O
OH
OO
O
H
H HH
OO
OH
O
OH
ArtelinateArteetherArtemether
O
O
OMe
O
O
H
HHO
O
OEt
O
O
H
HH
Chemical
Conversions
Artemisinin demand analysisDemand predicted to grow significantly
Source: Boston Consulting Group
Predicted Demand(various scenarios)
Millions of treatments
50
100
150
200
250
300
350
03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20
Artemisinin
Treatments
delivered
Artemisinin price swingsFood is more profitable than artemisinin
Artemisinin prices
($/kg)Food prices
(indexed to Jan 2002)
Artemisininprices
Commodityfood prices
2002 2003 2004 2005 2006 2007 2008 2009
Source: Boston Consulting Group
Artemisinin supplyHuge shortfalls predicted 2010 and beyond
50
100
150
200
250
300
350
03 04 05 06 07 08 10 11 12 13 14 15 16 17 18 19 20
Tonnes of
Artemisinin
09
Source: Boston Consulting Group
Produced
Stockpiled Demand
Minimum
supply
Goal
Reduce the cost of artemisinin-based anti-
malarial drugs by an order of magnitude.
Approach
Engineer a microorganism
to produce artemisinin from
an inexpensive, renewable
resource.
Microbial process
Artemisinin
O
O
O
O
O
H
HH
Microbial synthesis
Purification
Artesunate
O
O
H
H HH
OO
OH
O
OH
OO
O
H
H HH
OO
OH
O
OH
ArtelinateArteetherArtemether
O
O
OMe
O
O
H
HHO
O
OEt
O
O
H
HH
Chemical
Conversions
Process design
ispHispG idi ispAdxrdxs ispD
DXP Pathway
OPP
FPPPyruvate
+ G3P
Microbial synthesis
Artemisinic
Acid
H
H
O
HO
Purification
Artemisinin
O
O
O
O
O
H
HHHO
O
H
H
HOO
HO
O
H
H
H
Dihydroartemisinic
Acid
Dihydroartemisinic
Acid Hydroperoxide
ReductionPeroxidationOxidation and
Ring-Closure
O
O
OH
O
O
H
HH
Dihydroartemisinin Artesunate
(Artesunic Acid)
O
O
H
H HH
OO
OH
O
OH
OO
O
H
H HH
OO
OH
O
OHArtelinate
(Artelinic Acid)ArteetherArtemether
O
O
OMe
O
O
H
HHO
O
OEt
O
O
H
HH
Chemical
Conversions
Reduction
Amorphadiene Artemisinic
Acid
ADS
H
H
p450
Synthase Hydroxylase Unit
CPR
H
H
O
HO
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Constructing an artemisinic acid-
producing microbe
ispHispG idi ispAdxrdxs ispD
DXP Pathway
OPP
FPPPyruvate
+ G3P
Microbial synthesis
Amorphadiene Artemisinic
Acid
ADS
H
H
p450
Synthase Hydroxylase Unit
CPR
H
H
O
HO
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
E. coli as our chassis
Microbial synthesis
Quinones
ispHispG idi ispAdxrdxs ispD
DXP Pathway
OPP
FPPPyruvate
+ G3PispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Many of the parts are available only in
the native producer
ispHispG idi ispAdxrdxs ispD
DXP Pathway
OPP
FPPPyruvate
+ G3P
Microbial synthesis
Amorphadiene Artemisinic
Acid
ADS
H
H
p450
Synthase Hydroxylase Unit
CPR
H
H
O
HO
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Artemisia annua
Amorphadiene synthase from A. annua
ispHispG idi ispAdxrdxs ispD AmorphadieneADS
DXP Pathway
H
HOPP
Synthase
FPPPyruvate
+ G3P
Microbial synthesis
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Artemisia annua
5-epi-aristolochene synthase
as a model
ispHispG idi ispAdxrdxs ispD 5-epi-aristolocheneEAS
DXP Pathway
OPP
Synthase
FPPPyruvate
+ G3P
Microbial synthesis
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Tobacco
EAS
PPO
5-epi-aristolocheneFPP
Production of a model isoprenoid
in E. coli
Very low production of isoprenoid
resulted when using the native gene
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Iso
pre
no
id (
mg
/L)
ispH ispG idi ispA dxr dxs ispD 5-epi-aristolochene EAS
DXP Pathway
OPP
Synthase
FPP Pyruvate
+ G3P ispF ispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Amorphadiene synthase by design
ispHispG idi ispAdxrdxs ispD AmorphadieneADS
DXP Pathway
H
HOPP
Synthase
FPPPyruvate
+ G3P
Microbial synthesis
ispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
ADS
H
H
PPO
AmorphadieneFPP
Martin. 2003. Nat. Biotech. 21:796-802.
Gene synthesis improves amorphadiene
production
142-fold improved
production!
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Iso
pre
no
id (
mg
/L)
ispH ispG idi ispA dxr dxs ispD Amorphadiene ADS
DXP Pathway
H
HOPP
Synthase
FPP Pyruvate
+ G3P
Microbial synthesis
ispF ispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
Microbially-derived artemisinin
ispHispG idi ispAdxrdxs ispD AmorphadieneADS
DXP Pathway
H
HOPP
Synthase
FPPPyruvate
+ G3PispFispE
OPP
OPP
Central metabolism
OHC
OH
O P
O
O-
O-
H3C CO2H
O
The DXP pathway produces
quinones in E. coliG6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
DXP pathway
IPP DMAPP
FPP AmorphadieneQuinones
Pathways for isoprenoid precursor
biosynthesis
MEV
Mevalonate pathway
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
DXP pathway
IPP DMAPP
FPP AmorphadieneQuinones
Acetate
Acetyl-CoAP
HMGS tHMGRatoBMevT
Mevalonate
FPP
MBISP
PMK MPDMK idi
Mevalonate
ispA
Construction of synthetic mevalonate
pathway operons
Martin. 2003. Nat. Biotech. 21:796-802.
Constructing the mevalonate
pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
MEV
Exogenous
MEV
Quinones
X
The yeast mevalonate pathway improves
yields ~90-fold
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Iso
pre
no
id (
mg
/L)
PMK MPD MK idi ispA HMGS atoB tHMGR Amorphadiene
Mevalonate pathway (TOP)
H
H
Mevalonate pathway
(BOTTOM)
OPP
Synthase
FPP Mevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
Martin. 2003. Nat. Biotech. 21:796-802.
Optimizing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Sesquiterpene
MEV
Exogenous
MEV
Optimizing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
MEV
Exogenous
MEV
Sesquiterpene
Increasing concentrations of mevalonate
inhibit growth
5 mM
10 mM
20 mM
40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Ce
ll G
row
th (
OD
60
0)
Martin. 2003. Nat. Biotech. 21:796-802.
Optimizing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Sesquiterpene
MEV
Exogenous
MEV
Co-expression of sesquiterpene
cyclase alleviates growth inhibition
FPP
AmorphadieneAmorphadiene
Cyclase (ADS)
OPP
No ADS With ADS
5 mM
10 mM
20 mM
40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Ce
ll G
row
th (
OD
60
0)
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Ce
ll G
row
th (
OD
60
0)
Optimizing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
MEV
Exogenous
MEV
IPP DMAPP
FPP Sesquiterpene
Martin. 2003. Nat. Biotech. 21:796-802.
Optimizing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
MEV
Exogenous
MEV
IPP DMAPP
FPP Sesquiterpene
Martin. 2003. Nat. Biotech. 21:796-802.
High expression of amorphadiene synthase
relieves inhibitionG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Sesquiterpene
MEV
Exogenous
MEV
Balancing expression is difficult with
inducible promoters
• Typically, we use inducible promoters to control
expression of genes in a metabolic pathway
• Inducers are expensive
• Tuning expression can be challenging
PMKMK PMD Idi IspA FPP ADSMev Terpene
IPTG Arabinose
Sensing toxic intermediates to balance
metabolic pathway fluxes
• Can we use the toxicity of FPP to regulate the
metabolic pathways that produce and consume it?
• Can we reduce accumulation of toxic
intermediates?
• How do we regulate the pathway dynamically … in
the face of changing environmental conditions?
PMKMK PMD Idi IspA FPP ADSMev Terpene
Identification of FPP-responsive
promoters
• No terpene synthase to consume
FPP
• Examine differences in gene
expression in the presence and
absence of mevalonate
PMKMK PMD Idi IspAMev
FPPPMKMK PMD Idi IspA
FPP Gene X
Mev
Two FPP-responsive promoters
HMGSatoB tHMGR PMKMK PMD Idi IspA FPP ADS
IPTG
PlacPlacPlac
HMGSatoB tHMGR PMKMK PMD Idi IspA FPP ADS
PrstAPgadEPgadE
Inducible system
Dynamically-controlled system
FPP-responsive promoters
improve terpene production
0300600900
120015001800
pA
DS
prs
tA-A
DS
pA
DS
prs
tA-A
DS
lacUV5-MevT-MBIS pgadE-MevT-MBIS
Am
orp
had
ien
e
Pro
du
cti
on
(m
g/L
)
-IPTG
+IPTG
FPP ADS
PrstA
FPP ADS
PrstA
ADS
IPTG
Plac
ADS
IPTG
Plac
HMGS atoB tHMGR PMK MK PMD Idi IspA
IPTG
Plac Plac
HMGS atoB tHMGR PMK MK PMD Idi IspA FPP
PgadE PgadE
High product titers
with inducer-free control
Constructing the mevalonate pathwayG3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Sesquiterpene
MEV
Growth inhibition by MevT pathway
MevalonatetHMGRAtoB
Acetyl-CoAAcetoacetyl
-CoAHMG-CoA
HMGS
pBad33
pHMGS
pHMGSR
pBad33MevT
pHMGS(C159A)
pMevT(C159A)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 2 4 6 8 10 12
Time (hours)
Ce
ll G
row
th (
OD
60
0) pBad33
pHMGS
pHMGSR
pBad33MevT
pHMGS(C159A)
pMevT(C159A)
pBad33
pHMGS
pHMGSR
pBad33MevT
pHMGS(C159A)
pMevT(C159A)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 2 4 6 8 10 12
Time (hours)
Ce
ll G
row
th (
OD
60
0)
atoB HMGS tHMGR
atoB HMGS tHMGR
atoB HMGS tHMGR
atoB HMGS tHMGR
Accumulation of the toxic intermediate
HMG-CoA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS
H
HOPP
FPPMevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
HMG-CoA inhibits fatty acid production
PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS
Mevalonate pathway
(TOP)
H
H
Mevalonate pathway
(BOTTOM)
OPP
Synthase
FPPMevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
HMG-CoA
Fatty acid
biosynthetic
pathway
HMG-CoA inhibits fatty acid production
PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS
Mevalonate pathway
(TOP)
H
H
Mevalonate pathway
(BOTTOM)
OPP
Synthase
FPPMevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
HMG-CoA
Fatty acid
biosynthetic
pathway
Addition of fatty acids to the growth
medium restores growth
PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS
Mevalonate pathway
(TOP)
H
H
Mevalonate pathway
(BOTTOM)
OPP
Synthase
FPPMevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
HMG-CoA
HO
OFatty acid addition to the
growth medium
Accumulation of the toxic intermediate
HMG-CoA
mRNA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
P
HMGS tHMGRatoB
Balancing the mevalonate pathway by tuning
mRNA stability and translation efficiency
mRNA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
P1
HMGS tHMGRatoB
RNase E
site
RNase E
site
RBS RBS RBS
Combinatorial design of intergenic
regionsD
CA
B
EE E E
atoB hmgS hmgR
E E
P1
HMGS tHMGRatoB
Pfleger. 2006. Nat. Biotech. 24:1027
How do we choose?
atoB hmgS hmgRE E
atoB hmgS hmgRE E
atoB hmgS hmgREE
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR
A fluorescent screen for improved
mevalonate production
MEV
MEV
Ac-CoA
MEV
Biomass
E. coli
GFP-engineered
MEV auxotroph
E. coli
MEV Producer
Mutant
pathway
Pfleger. 2006. Nat. Biotech. 24:1027
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
Down-regulation of the last
two genes relieves toxicity,
increases Mev production,
and increases Ac-CoA
concentrationRBS
RBSatoB hmgS hmgR
Pfleger. 2006. Nat. Biotech. 24:1027
Synthetic scaffolds:
another way to solve the problem
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
tHmgRHmgSAtoB
Connecting metabolic pipes with
synthetic scaffolds
Connecting the enzymes in some way might
reduce loss of intermediate to the bulk
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
tHmgRHmgSAtoB
Connecting metabolic pipes with
synthetic scaffolds
Adding additional copies of rate-limiting
enzymes might increase pathway flux
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
tHmgRHmgSAtoB
There are no standards
for connecting enzymes together
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
tHmgRHmgSAtoB
Synthetic scaffolds co-localize pathway
enzymes and reduce intermediate runoff
tHmgRHmgSAtoB
Ato
B
Hm
gR
Hm
gS
Dueber. 2009. Nat. Biotech. 27:753.
Synthetic scaffolds co-localize pathway
enzymes and reduce intermediate runoff
AtoB HmgS HmgR Scaffold
Ptet PBAD
Ato
B
Hm
gR
Hm
gS
Dueber. 2009. Nat. Biotech. 27:753.
Synthetic scaffolds can control relative
enzyme ratios and optimize flux
Ato
B
Hm
gS
Hm
gR
Hm
gR
Hm
gR
tHmgRHmgSAtoB
Dueber. 2009. Nat. Biotech. 27:753.
Variation in the number of
HmgS and HmgR on the scaffold
Ato
B
Hm
gS
Hm
gR
Hm
gR
Hm
gR
Hm
gR
Ato
B
Hm
gS
Hm
gR
Hm
gR
Ato
B
Hm
gS
Hm
gR
Ato
B
Hm
gS
Hm
gR
Hm
gS
Ato
B
Hm
gS
Hm
gR
Hm
gR
Hm
gS
Ato
B
Hm
gS
Hm
gS
Hm
gRH
mg
S
Hm
gS
Hm
gR
Hm
gR
Hm
gR
Ato
B
Hm
gS
Hm
gS
Hm
gR
Hm
gR
Hm
gR
Hm
gR
Ato
B
Hm
gS
Hm
gS
Hm
gRH
mg
S
Hm
gS
Hm
gR
Ato
B
Hm
gS
Hm
gS
Hm
gS
Hm
gS
Hm
gR
Hmg
R
HmgS
Dueber. 2009. Nat. Biotech. 27:753.
Synthetic scaffolds have a dramatic
effect on the mevalonate pathway
HmgS
HmgR
AtoB
n = 1
Dueber. 2009. Nat. Biotech. 27:753.
Increases in Yield are Scaffold Dependent
HmgR
x=1
y
Z
AtoB
HmgS
AtoB HmgS HmgR Scaffold
Ptet PBAD
A: Optimized scaffold
Ptet PBAD
AtoB HmgS HmgR Scaffold GFP
B: Scaffold competitor
AtoB HmgS HmgR Scaffold
Ptet PBAD
GFP
PBAD
C: More scaffold competitor
AtoB HmgS HmgR Scaffold
Ptet PBAD
D: Enzymes without peptide ligands
80
60
40
20
0
Fo
ld in
cre
ase
ov
er
no
scaff
old
A B C D
Dueber. 2009. Nat. Biotech. 27:753.
Synthetic scaffolds can control relative
enzyme ratios and optimize flux
tHmgR
Ato
B
Hm
gS
Hm
gR
Hm
gR
Hm
gS
tHmgRHmgS
HmgSAtoB
Dueber. 2009. Nat. Biotech. 27:753.
Component optimization and debugging
yields another 50 fold
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Iso
pre
no
id (
mg
/L)
PMK MPD MK idi ispA HMGS atoB tHMGR Amorphadiene
Mevalonate pathway (TOP)
H
H
Mevalonate pathway
(BOTTOM)
OPP
Synthase
FPP Mevalonate
OH
OHCH3
HO2CH3C SCoA
O
Acetyl-
CoA
Fermentation optimization pushes yields
beyond 25 g/L!
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Iso
pre
no
id (
mg
/L)
Fermentation and final microbe
optimization done by Amyris.
H
H
O
HO
Identify final enzyme in pathway (P450/AMO)
and transplant into E. coli
PMK MPDMK idi ispAHMGSatoB tHMGR Amorphadiene Artemisinic
Acid
ADS
Mevalonate pathway
(TOP)
H
H
Mevalonate pathway
(BOTTOM)
p450
OPP
Synthase Hydroxyase
Unit
FPPMevalonate CPR
OH
OHCH3
HO2C
H3C SCoA
O
Acetyl-
CoA
A. annua
H
H
H
H
O
HO
p450CPR
Amorphadiene Artemisinic Acid
Proposed artemisinin
biosynthetic pathwayCytochrome P450
monooxygenase
Alcohol
dehydrogenase
Aldehyde
dehydrogenase
CH2OH
OHC
HOOC
CH2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
Artemisinin
An experimental path
for identifying the P450
Amorph-OH
Gene 1
Gene 2
Gene 3
Gene 4
Amorph
Amorph
Amorph
Amorph
Yeast
Amorph-OH
E. coli
Amorph
The mevalonate pathway in yeast
produces ergosterol
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
ErgosterolParadise. 2008. Biotech. Bioeng. 100:371
Production of amorphadiene is very low
when the gene is introduced into yeast
G6P
FDP
G3P
PEP
PYR
AcCoA
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
Ergosterol
Amorphadiene 0
20
40
60
80
Construct
Am
orp
ha
die
ne
(m
g/L
)
A
L
Paradise. 2008. Biotech. Bioeng. 100:371
Overexpression of the HMG-CoA reductase
gene improves production
G6P
FDP
G3P
PEP
PYR
AcCoA
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
Ergosterol
0
20
40
60
80
Construct
Am
orp
ha
die
ne
(m
g/L
)
Amorphadiene
A
L
Paradise. 2008. Biotech. Bioeng. 100:371
Expression of a transcription factor
enhances expression of several
mevalonate pathway genes
G6P
FDP
G3P
PEP
PYR
AcCoA
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
Ergosterol
0
20
40
60
80
Construct
Am
orp
ha
die
ne
(m
g/L
)
Amorphadiene
A
L
Paradise. 2008. Biotech. Bioeng. 100:371
Turning down ergosterol production
enhances amorphadiene production
G6P
FDP
G3P
PEP
PYR
AcCoA
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
Ergosterol
0
20
40
60
80
Construct
Am
orp
ha
die
ne
(m
g/L
)
XAmorphadiene
A
L
Paradise. 2008. Biotech. Bioeng. 100:371
Expression of FPP synthase further
enhances amorphadiene production
G6P
FDP
G3P
PEP
PYR
AcCoA
A
L
CIT
IPP DMAPPMEV
Mevalonate pathway
FPP
Ergosterol
0
20
40
60
80
Construct
Am
orp
ha
die
ne
(m
g/L
)
XAmorphadiene
Paradise. 2008. Biotech. Bioeng. 100:371
Lettuce, chicory, and sunflower produce
isoprenoids like artemisininGermacrene A
(Chicory, sunflower and lettuce)Amorphadiene
(Artemisia annua)
H
H
O
O
O
OH
HO
OH
OH
O
H
H
O
O
O
H
H
OO
O
OO
H
lettucenin Aniveusin A Artemisinin
OHO
O
P450’s involved
CH2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
P450 candidate produces
artemisinic acid
121
24893
18879105 216136
16217314555 67
201 233
121
93 248
79 188105 136 216
16217314555 67 201 233
Re
lati
ve
Io
n A
bu
nd
an
ce
Peak 1
Yeast product
Peak 2
Artemisinic acid
m/z
Ro. 2006. Nature 440:940-943
HO
O
H
H
H
HO
O
H
H
H
Amorphadiene
Artemisinic
Acid
H
H
H
H
O
HO
HO
O
H
H
H
HO
O
H
H
H
H
H
O
HO
HO
O
H
H
H
H
H
O
HO
Artemisinic acid is
transported out of the cell
Ro. 2006. Nature 440:940-943
N-terminal parts for functionally
expressing P450s in E. coli
Chang. 2007. Nat. Chem. Biol. 3:274-277
FPP
CadS CadH,O2
CPR, NADPHH H
OH
cadinene 8-hydroxycadinene
Public-Private Partnerships
• Royalty-free, exclusive licenses for
production of artemisinin for the
Developed World
• Must be produced at cost
Patents
Patents
Licenses Licenses
Licenses
Acknowledgements
Funding
Department of Energy
National Science Foundation
Office of Naval Research
University of California Discovery Grant
Bill & Melinda Gates Foundation (OneWorld Health)
Keasling lab
Jennifer Anthony
Michelle Chang
Howard Chou
Jeffrey Dietrich
John Dueber
Connie Kang
Lance Kizer
Jim Kirby
Taek Soon Lee
Vincent Martin
Karyn Newman
Farnaz Nowroozi
Mario Ouellet
Eric Paradise
Chris Petzold
Brian Pfleger
Doug Pitera
Dae-Kyun Ro
Christina Smolke
Sydnor Withers
Gabriel Wu
Yasuo Yoshikuni
Amyris#
Jack Newman
Chris Paddon
Kinkead Reiling
Rika Regentin
Neil Renninger
#Jay Keasling has a financial
interest in Amyris & LS9.
Joint Genome Institute
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