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Engineering microbes for production of low-cost, effective, anti-malarial drugs
Jay D. Keasling
MCB 113
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 infected
Source: Roll Back MalariaWorld Malaria Report 2005
• Most widely-used drugs to treat malaria
NCl
NHH3C
H3C
H3C
Chloroquine-based drugs
• Plasmodium in many parts of the world is largely resistant to chloroquine
Source: Roll Back MalariaWorld Malaria Report 2005
Disease burden on the rise as resistance grows
Trends in Africa Particularly Troubling
Source: Roll Back Malaria
Ann
ual d
eath
s (in
mill
ions
)
Treatment:Quinine only
Heavy use ofDDT spraying
Resistance increasing
0.0
1.0
2.0
3.0
4.0
1900 1930 1950 1970 1990 2000
Africa
Americas
China & NE Asia
S. Asia & Middle East
ArtemisininArtemisia annua
O
O
O
OO
H
HH
Artemisinin is produced by trichomes in Artemisia annua leaves
O
O
O
OO
H
HH
Artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...
A brief history of artemisinin
Active ingredient (artemisinin) isolated 1972
Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments)
Fevers (malaria)
340 A.D.
Recipes For 52 Kinds Of Diseases found in the Mawangdui Han Dynasty tomb
Hemorrhoids
168 B.C.
Isoprenoids
Essential oils
OHMenthol
C-10 Monoterpene
Carotenoids
LycopeneC-40 Tetraterpene
Chemotherapeutics
TaxolC-20 Diterpene
EleutherobinC-20 Diterpene
> 50,000 known molecules
Isoprenoid metabolic pathwaysOPO(OH)OP(OH)2O OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2OFarnesyl pyrophosphate(FPP)
Geranyl pyrophosphate(GPP)
Dimethylallyl pyrophosphate(DMAPP)
Isopentenyl pyrophosphate(IPP)
OPO(OH)OP(OH)2OGeranylgeranyl pyrophosphate
(GGPP)
Monoterpenes
Sesquiterpenes
DiterpenesCarotenoids
Artemisinin metabolic pathwaysOPO(OH)OP(OH)2O OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
Amorphadiene
Farnesyl pyrophosphate(FPP)
Geranyl pyrophosphate(GPP)
Dimethylallyl pyrophosphate(DMAPP)
Isopentenyl pyrophosphate(IPP)
ArtemisininO
O
O
OO
H
HH
Pathways for isoprenoid precursor biosynthesis
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
FPP
MEV
Mevalonate pathway
DXP
DXP pathway
GGPP
Monoterpenes
Sesquiterpenes
Diterpenes
TCACycle
Gly
coly
sis
Artemisinin-based drugs• The current cost for an artemisinin-
based drug is approximately $2.50.–Artemisinin generally adds $1.00-
1.50 to the cost for drugs–Most developing countries spend
less than $4/person/year on health care
• As many as 10-12 treatments are needed for each person annually
• World Health Organization estimates that 700 tons will be needed annually
O
O
O
OO
H
HH
A chemical synthesis route to
artemisinin
H HOH
H OH
H
O
HI
H
OH
H
O
HI
H
OH
H
O
Si((SiMe)3)3
O
OH
H
O
H
OH
H
O
H
O
OH
H
O
H
SS
+
+ +
OH
H
O
H
SS
OMeH
H
O
HO
SS H
OMeH
H
O
O
SS H
OMeH
H
O
O
SS H
H3C
+
OMeH
H
O
O
O H
H3C O
O
O
OO
H3C
CH3
H
CH3
H
H
Artemisinin
OH
H
O
HI
OH
H
O
H
O
GoalReduce the cost of artemisinin-based anti-malarial drugs by an order of magnitude.
ApproachEngineer a microorganism to produce artemisinin from an inexpensive, renewable resource.
Designing a microbe to synthesize artemisinin Identify the chemistry
CH 2OHCH 2OH
OHCOHC
HOOCHOOC
CH 2OHCH 2OH
OHCOHC
HOOCHOOC
FPP5
6 7 811
12 13
4
Find and clone (or synthesize) the genes
Designing a microbe to synthesize artemisinin
Supply of intracellular precursors
Designing a microbe to synthesize artemisinin
Well characterized geneticcontrol system (chassis, parts, devices)
Designing a microbe to synthesize artemisinin
Microbial production of artemisinin
• Production not affected by weather conditions
• Consistent supply• Pure product can be made (free of other
contaminating terpenes)• Access to artemisinin can be controlled to
minimize production of mono-therapies.
Advantages
Microbial production of artemisinin
• Need the genes for all of the enzymes in the pathway
• Not always simple to express in microbes the genes from very different organisms
• Need to balance metabolic pathways to optimize production
• Need a platform host and a host of metabolic engineering tools
Challenges
Designing a microbe to synthesize artemisinin Identify the chemistry
CH 2OHCH 2OH
OHCOHC
HOOCHOOC
CH 2OHCH 2OH
OHCOHC
HOOCHOOC
FPP5
6 7 811
12 13
4
CH 2OH
OHC
HOOC
CH 2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
Artemisinin
Bertea (2005) Planta Med. 71:40-47
Proposed artemisininbiosynthetic pathway
Find and clone (or synthesize) the genes
Synthesis of artemisinin inmicrobes
CH 2OH
OHC
HOOC
CH 2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
Artemisinin
Bertea (2005) Planta Med. 71:40-47
Proposed artemisinin biosynthetic pathway
Production of a model isoprenoid in E. coli
Tobacco as an early model
5-epi-aristolochene
IPP DMAPP
GPP
DXP pathway
FPP
Production of a model isoprenoid in E. coli
Very low production of isoprenoid resulted when using the native gene
5-epi-aristolochene
IPP DMAPP
GPP
DXP pathway
FPP
0.0001
0.001
0.01
0.1
1
10
100
1000
ConstructIs
opre
noid
(mg/
L)
Production of the artemisinin precursor (amorphadiene) in E. coli
amorphadiene
IPP DMAPP
GPP
DXP pathway
FPP
Gene synthesis
atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagaagtctttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaagtcggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatcgcttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagataacaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttctttttgtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttcccggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgtcgg
atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagactttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaaggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagacaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttcttgtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgt
• Remove rare codons• Insert restriction sites
Gene resynthesis improves amorphadiene production
142-fold improved production!
amorphadiene
IPP DMAPP
GPP
DXP pathway
FPP
0.0001
0.001
0.01
0.1
1
10
100
1000
Construct
Isop
reno
id (m
g/L)
DXP PathwayG6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
DXP pathway
IPP DMAPP
GPP
FPP AmorphadieneQuinones
Eliminating bottlenecks in the DXP pathway
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
FPP
DXP
Amorphadiene
DXS
IdI
IspA
Engineering E. coli’s native pathway improves production
Three-fold improvement in production
P
P
R
A
CIT
IPP DMAPP
GPP
FPP
DXP
Amorphadiene
DXS
IdI
IspA
0.0001
0.001
0.01
0.1
1
10
100
1000
ConstructIs
opre
noid
(mg/
L)
Enhancing all of the steps in the DXP pathway
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
FPP
DXP
Amorphadiene
IdI
IspA
pyridoxinethiamine
Pathways for isoprenoid precursor biosynthesis
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
FPP
MEV
Mevalonate pathway
DXP
DXP pathway
AmorphadieneTCA
Cycle
Gly
coly
sis
Recruiting the mevalonate pathway from yeast
Yeast
G3P
PEP
PYR
cCoA
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
DXP
DXP pathway
FPPCAycle
Amorphadiene
Acetyl-CoAP
HMGS tHMGRatoBMevTMevalonate
FPP
MBISP
PMK MPDMK idi
Mevalonate
ispA
Construction of synthetic mevalonate pathway operons
Constructing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Amorph
MEV
Exogenous MEV
The yeast mevalonate pathway improves yields ~90-fold
G3P
PEP
PYR
cCoA
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
DXP
DXP pathway
FPPCAycle
Amorphadiene0.0001
0.001
0.01
0.1
1
10
100
1000
ConstructIs
opre
noid
(mg/
L)
Acetyl-CoAP
HMGS tHMGRatoBMevTMevalonate
FPP
MBISP
PMK MPDMK idi
Mevalonate
ispA
Optimizing the bottom part of the mevalonate pathway
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Amorph
MEV
Exogenous MEV
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
MEV
Exogenous MEV
Amorphadiene
Increasing concentrations of mevalonate inhibit growth
5 mM10 mM20 mM40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
Martin et al. 2003. Nat. Biotechnol. 21:796-802.
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Amorph
MEV
Exogenous MEV
Co-expression of sesquiterpene cyclase alleviates growth inhibition
FPPAmorphadiene
AmorphadieneCyclase (ADS)
OP P
No ADS With ADS
5 mM
10 mM20 mM40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
MEV
Exogenous MEV
IPP DMAPP
FPP Amorphadiene
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
MEV
Exogenous MEV
IPP DMAPP
FPP Amorphadiene
High expression of amorphadiene synthase relieves inhibition
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
FPP
DXP
Amorph
MEV
Exogenous MEV
0
100
200
300
400
500
600
700
0 20 40 60 80 100
Am
orph
adie
ne (m
g/L)
Time (hours)
Mevalonate kinase limits amorphadiene production
MBISP
PMK MPDMK idi ispA
ispA
idi
MPD
PMK
MK
Optimizing the mevalonate pathway
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
DXP
MEV
Exogenous MEV
IPP DMAPP
FPP Amorph
Pathway mutation to prevent accumulation of toxic intermediates?
Mini-summary: Two lessons
• Even native metabolic intermediates can be toxic at high concentrations.
• “Pulling” on a pathway is just as important as “pushing”.
Acetyl-CoAP
HMGS tHMGRatoBMevTMevalonate
FPP
MBISP
PMK MPDMK idi
Mevalonate
ispA
Optimizing the top part of the mevalonate pathway
The top part of the mevalonate pathway
MevalonatetHMGR
Diffusesout of cell
AtoBAcetyl-CoA Acetoacetyl
-CoA HMG-CoAHMGS
HMGS tHMGRatoBMevT
Growth inhibition by MevT pathway
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)
Cel
l Gro
wth
(OD
600)
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)
Cel
l Gro
wth
(OD
600)
HMGS tHMGRatoB
HMGS tHMGR
tHMGR
HMGS
MevalonatetHMGRAtoB
Acetyl-CoA Acetoacetyl-CoA HMG-CoA
HMGS
HMGSatoB
Accumulation of the toxic intermediate Hmg-CoA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS tHmgR
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)
Cel
l Gro
wth
(OD
600)
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)
Cel
l Gro
wth
(OD
600)
0
10
20
30
40
50
60
0 2 4 6 8Time (hours)
HM
G-C
oA (n
M/O
D 600
) D
0
10
20
30
40
50
60
0 2 4 6 8Time (hours)
HM
G-C
oA (n
M/O
D 600
) D
C159A mutation in HMGS knocks out activity
HMGS tHMGRatoB
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB tHmgR
E E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D V E G I D T T N A C Y G G T A AQ D S G N T D I E G I D T T N A C Y G G T A A
Human fibroblastHuman adrenalChinese hamster ovaryRat liverChicken liverRat liver – mitochondrial
S. cerevisiae L F G E N T D V E G I D T L N A C Y G G T N A
E E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D V E G I D T T N A C Y G G T A AQ D S G N T D I E G I D T T N A C Y G G T A A
Human fibroblastHuman adrenalChinese hamster ovaryRat liverChicken liverRat liver – mitochondrial
S. cerevisiae L F G E N T D V E G I D T L N A C Y G G T N A
Comparison of active pathway, inactive pathway, and vector only
HMGS tHMGRatoB
HMGS tHMGRatoB
HMG-CoA stress only
Protein expression stress only
Protein andHMG-CoA stress
Inactive pathway
Active pathway
Vector only
Functional genomics pipeline
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
Metabolomics
Proteomics
T0
T2T1
Gro
wth
(OD
)
Time
Transcriptomics
AnalysisBiomass production
Functional genomics pipeline
Denature and reduce cysteine bridges
Cation Exchange Cleanup
Analyze by LC-MS/MS
Digest
114 115 116 117
Digest Digest Digest
Pool
Proteins
Transcripts
Metabolites
15 20 25 30 35 40 Time [min]15 20 25 30 35 40 Time [min]
Methionine Sulfone (ISD)
Valine Betaine
Glutamine
Lysine
Arginine
Analysis
Biomass production
MevT up-regulates genes involved in fatty acid biosynthesis
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACP
FabD
FabHAccD, AccB
Acetoacetyl-ACP
ACPSH, CO2
FabBAccC, AccA FabB
Proteomics
Transcriptomics
Saturated fatty acids
Unsaturated fatty acids
FabBFabA
UPDOWN
Stress regulons up-regulated in MevT strain
HMG-CoA
Fe-S ClusterRepair
Sideophores
Hydroperoxidases
NADPH Generation
Oxidative Stress
ChaperonesProteases
Inclusion BodyProteins
Protein Misfolding
Protectants
PSPPorins Porins
Membrane/Osmotic Stress
Proteomics
Transcriptomics
HMG-CoA and malonyl-CoAaccumulation
Active pathway
Inactive pathway
HMG-CoA Malonyl-CoAActive
pathway
Inactive pathway
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
Metabolomics
Saturatedfatty acids
Unsaturatedfatty acids
Presence of MevT enriches unsaturated fatty acids
Inactive
Inactive
Active
Active
Pathway
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
Metabolomics
MevT up-regulates fatty acid biosynthesis
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACP
FabD
FabHAccD, AccB
Acetoacetyl-ACP
ACPSH, CO2
FabBAccC, AccA FabB
Saturated fatty acids
Unsaturated fatty acids
FabBFabA
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
0 5 10 15 20 25 Time [min]
AspPheGlu
Pro
Ile
Leu
Lys
Arg Val
His Met
Metabolomics
Proteomics
Transcriptomics
UPDOWN
Three additional pieces of information
• Malonyl-CoA is very similar in structure to HMG-CoA
• HMG-CoA reductase (tHMGR) activity increases with induction, then decreases sharply.
• HMG-CoA accumulation in organisms that produce it natively is known to cause oxidative stress.
HMG-CoAMalonyl-CoA
AccD, AccB
AccC, AccA
A model for MevT effects on fatty acid biosynthesis
UPDOWN
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACP
FabD
FabH
Acetoacetyl-ACP
ACPSH, CO2
FabB FabB
Saturated fatty acids
Unsfat
FabBFabA
AccD, AccB
AccC, AccA
A model for MevT effects on fatty acid biosynthesis
UPDOWN
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACP
FabD
FabH
Acetoacetyl-ACP
ACPSH, CO2
FabB FabB
Saturated fatty acids
Unsfat
FabBFabA
HMG-CoA
Mev
AtoB
HmgS
tHmgR
Acetoacetyl-CoA
AccD, AccB
AccC, AccA
A model for MevT effects on fatty acid biosynthesis
UPDOWN
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACP
FabD
FabH
Acetoacetyl-ACP
ACPSH, CO2
FabB FabB
Saturated fatty acids
Unsfat
FabBFabA
HMG-CoA
Mev
AtoB
HmgS
tHmgR
Acetoacetyl-CoA
FabD
A model for MevT effects on fatty acid biosynthesis
HMG-CoA
Mev
AtoB
HmgS
tHmgR
Acetoacetyl-CoA
UPDOWN
Acetyl-CoA
Malonyl-CoA Malonyl-ACP
Acetyl-ACPFabH
AccD, AccB
Acetoacetyl-ACP
ACPSH, CO2
FabBAccC, AccA FabB
Saturated fatty acids
Unsfat
FabBFabA
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 MevAtoB HmgS tHmgR
P1
HMGS tHMGRatoB
RNase Esite
RNase Esite
RBS RBS RBS
Combinatorial design of intergenicregions D
CA
B
EE E E
atoB hmgS hmgRE E
P1
HMGS tHMGRatoB
How do we choose?atoB hmgS hmgR
E E
atoB hmgS hmgRE E
atoB hmgS hmgREE
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR
A fluorescent screen for improved mevalonate production
MEVMEV
Ac-CoA
MEV
Biomass
E. coliGFP-engineeredMEV auxotroph
E. coliMEV Producer
Mutant pathway
B.)B.)
Best producers make about 7X more mevalonate
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
Down-regulation of last two genes relieves toxicity, increases Mev production, and increases Ac-CoA concentration
RBS
RBSatoB hmgS hmgR
Mini-summary: Two more lessons
• Functional genomics is a useful tool for diagnosing problems in engineered cells.
• Pathway balancing is much harder than pathway construction!
Large-scale production of amorphadiene O2
G3P
PEP
PYR
cCoA
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
DXP
DXP pathway
FPPCAycle
Amorphadiene
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14
Time (Hours)
Am
orph
adie
ne
conc
entra
tion
(mg/
L)Amorphadiene is volatile
and is lost from bioreactors O2
Amorphadiene is lost from bioreactors O2
Condenser
Amorphadiene
Amorphadiene production in a two-phase fermentation O2
Oil
Amorphadiene production in a two-phase fermentation O2
0
100
200
300
400
500
0 12 24 36 48 60 72
Time (hr)
Am
orph
adie
ne
conc
entr
atio
n (m
g/L)
A two-phase fermentation collects all of the amorphadiene
1 million fold improved production of amorphadiene
G3P
PEP
PYR
cCoA
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
DXP
DXP pathway
FPP
Amorphadiene0.0001
0.001
0.01
0.1
1
10
100
1000
ConstructIs
opre
noid
(mg/
L)
Completing the pathway
Amorphadiene
ArtemisinicAcid
H
H
H
H
O
HO
Artesunate(Artesunic Acid)
O
O
H
H HH
OO
OH
O
OH
O
HO
O
H
H
H
ArtemisinicAcid
O
O
H
H HH
OO
OMeH
O
O
H
H HH
OO
OEtH
O
O
H
H HH
OO
OH
O
OH
Artelinate(Artelinic Acid)ArteetherArtemether
Proposed artemisinin
biosynthetic pathway
Cytochrome P450 monooxygenase
Alcohol dehydrogenase
Aldehyde dehydrogenase
CH 2OH
OHC
HOOC
CH 2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
Artemisinin
Cytochrome P450’s• Heme-containing oxygenases• Important for metabolizing
toxic compounds in the human body
• Often required as one of the initial steps in natural products synthesis
• Plant- and animal-derived versions require additional proteins for function– Cytochrome P450 reductase
partners• Often bound to the membrane
http://www.p450.kvl.dk/gallery/CYP3A4.jpg
Cloning the remaining genes in the Artemisinin biosynthetic pathway• Cytochrome P450’s are traditionally difficult to
express in E. coli
• A good host for cytochrome P450 expression and screening would aid in elucidating the remaining steps in the biosynthetic pathway–A Saccharomyces cerevisiae (yeast) strain
capable of producing amorphadiene would be very beneficial
An experimental path for identifying the P450
Amorph-OH
Gene 1
Gene 2
Gene 3
Gene 4
Amorph
Amorph
Amorph
Amorph
YeastAmorph-OH
E. coli
Amorph
Construction of a yeast that produces amorphadiene
Amorph
Amorph
Amorph
Amorph
The mevalonate pathway in yeast produces ergosterol
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
Ergosterol
Production of amorphadiene is very low when the gene is introduced
into yeast
G6P
FDP
G3P
PEP
PYR
AcCoA
AA
L
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
ErgosterolAmorphadiene 0
20
40
60
80
Construct
Am
orph
adie
ne (m
g/L)
Overexpression of the HMG-CoA reductase gene improves production
G6P
FDP
G3P
PEP
PYR
AcCoA
AA
L
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
Ergosterol0
20
40
60
80
Construct
Am
orph
adie
ne (m
g/L)
Amorphadiene
Expression of a transcription factor enhances expression of many of the
genes in the mevalonate pathway
G6P
FDP
G3P
PEP
PYR
AcCoA
AA
L
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
Ergosterol0
20
40
60
80
Construct
Am
orph
adie
ne (m
g/L)
Amorphadiene
Turning down ergosterol production enhances amorphadiene production
G6P
FDP
G3P
PEP
PYR
AcCoA
AA
L
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
Ergosterol0
20
40
60
80
Construct
Am
orph
adie
ne (m
g/L)
XAmorphadiene
Expression of FPP synthase further enhances amorphadiene production
G6P
FDP
G3P
PEP
PYR
AcCoA
AA
L
CIT
IPP DMAPP
GPP
MEVMevalonate pathway
FPP
Ergosterol0
20
40
60
80
Construct
Am
orph
adie
ne (m
g/L)
X
Amorphadiene production by yeast is 1/10th of that in E. coli … but good enough to screen for P450’s.
Amorphadiene
The genes encoding the P450 and redox partners must be present in
the same cell
Amorph-OH
Gene 3Amorph
Cytochrome P450 redoxpartners from A. annua
Finding the remaining genes in the biosynthetic pathwayGene 1
Gene 2
Gene 3
Gene 4
Lettuce, chicory, and sunflower produce isoprenoids like artemisinin
Germacrene A(Chicory, sunflower and lettuce)
Amorphadiene(Artemisia annua)
H
H
OO
OOH
HO
OH
OH
O
H
H
O
O
O
H
H
OO
O
OO
H
lettucenin Aniveusin A Artemisinin
OHOO
P450’s involved
Alignment of known terpene hydroxylases
71C171C271C3
71C471D12
71D1371D18
71D1671D20
71AV173A1
98A379F1
701A393C1
75A175B2
76B6706B1
84A188A3
90A190B1
50 changes
100100
90
74
79
91
86
100
90
66
92
100
90
9785
88
60
86100
100
71C171C271C3
71C471D12
71D1371D18
71D1671D20
71AV173A1
98A379F1
701A393C1
75A175B2
76B6706B1
84A188A3
90A190B1
50 changes
100100
90
74
79
91
86
100
90
66
92
100
90
9785
88
60
86100
100
P450 candidate is functional in yeast!
YeastCPR/CYP71AV1
YeastCPR
A. Annua
28.0
628
.06
IS
20 35Time (min)
1
2
*
*
Tota
l Ion
Col
lect
ion
P450 candidate produces artemisinic acid!
121
2489318879
105 21613616217314555 67 201 233
121
93 24879 188
105 136 21616217314555 67 201 233
Rel
ativ
e Io
n A
bund
ance
Peak 1Yeast product
Peak 2Artemisinic acid
m/z
YeastCPR/CYP71AV1
YeastCPR
A. Annua
28.0
628
.06
IS
20 35Time (min)
1
2
*
*
Tota
l Ion
Col
lect
ion
CH 2OH
OHC
HOOC
FPP
Amorphadiene
Artemisinic acid
Proposed artemisininbiosynthetic pathway
121
2489318879
105 21613616217314555 67 201 233
121
93 24879 188
105 136 21616217314555 67 201 233
Rel
ativ
e Io
n A
bund
ance
Peak 1Yeast product
Peak 2Artemisinic acid
m/z
HO
O
H
H
H
HO
O
H
H
H
Amorphadiene
ArtemisinicAcid
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
Expression of amorphadiene oxidase in yeast
• Amorphadiene oxidase catalyzes all three steps–amorphadiene alcohol aldehyde
artemisinic acid• The amorphadiene oxidase is functional in
vivo• Artemisinic acid is secreted from yeast and
adsorbs to the outside of the cells• Artemisinic acid can readily released from the
cells by a pH change
Production of artemisinic acid in E. coli
Amorphadiene
ArtemisinicAcid
H
H
H
H
O
HO
Artesunate(Artesunic Acid)
O
O
H
H HH
OO
OH
O
OH
O
HO
O
H
H
H
ArtemisinicAcid
O
O
H
H HH
OO
OMeH
O
O
H
H HH
OO
OEtH
O
O
H
H HH
OO
OH
O
OH
Artelinate(Artelinic Acid)ArteetherArtemether
Production of hydroxylated terpenes in E. coli
FPP
CadS CadH,O2
CPR, NADPHH H
OH
cadinene 8-hydroxycadinene
Completing the biosynthetic pathway in E. coli
p450
Amorphadiene Artemisinic Acid
1 2 3
>25 g/LCurrent titer
in E. coli(lab scale)
> 1 g/L
P450/AMO Catalyzes 3 Separate Oxidations
Research, Development & Delivery
Keasling Laboratory
Amyris Biotechnologies
Institute forOneWorldHealth
Artemisinin costs
$1.00/gCurrent cost of API
$.10/gCost with new process
Conclusions• E. coli has been engineered to produce
amorphadiene at yields in excess of 0.5 g/L.• S. cerevisiae has been engineered to produce
amorphadiene at yields of approximately 0.1 g/L.• A cytochrome P450 and its redox partners can
oxidize amorphadiene to artemisinic acid.• S. cerevisiae expressing amorphadiene oxidase
produces artemisinic acid, which is secreted from the cells.
• E. coli can functionally express cytochrome P450’s that oxidize terpenes.
Acknowledgements
FundingNational Science FoundationOffice of Naval ResearchMaxygenDiversaUniversity of California Discovery GrantBill & Melinda Gates Foundations (OneWorld Health)
Keasling labJennifer AnthonyMichelle ChangConnie KangLance KizerJim KirbyVincent MartinKaryn NewmanFarnaz NowrooziMario Oulet
Eric ParadiseChris PetzoldBrian PflegerDoug PiteraDae-Kyun RoChristina SmolkeSydnor WithersYasuo Yoshikuni
Amyris Biotechnologies#
Jack NewmanChris PaddonKinkead ReilingRika RegentinNeil Renninger
#Jay Keasling has a financial interest in Amyris Biotechnologies.
Joint Genome Institute