Transcript New Tools for Metabolic Engineering of Bacteria

Genetic tools for metabolic enzyme
production in Escherichia coli
Jay D. Keasling
Department of Chemical Engineering
University of California
Berkeley, CA 94720
Terpenoids
Chemotherapeutics
> 50,000 known molecules
Essential oils
OH
Menthol
C-10 Monoterpene
Eleutherobin
C-20 Diterpene
Carotenoids
Lycopene
C-40 Tetraterpene
Taxol
C-20 diterpene
OPO(OH)OP(OH)2O
Isopentenyl pyrophosphate
(IPP)
Terpenoid metabolic
pathways
Dimethylallyl pyrophosphate
OPO(OH)OP(OH)2O
(DMAPP)
OPO(OH)OP(OH)2O
Geranyl pyrophosphate (GPP)
Monoterpenes
OPO(OH)OP(OH)2O
Farnesyl pyrophosphate
(FPP)
Sesquiterpenes
OPO(OH)OP(OH)2O
Geranylgeranyl pyrophosphate
(GGPP)
Diterpenes
Carotenoids
The DXP pathway
pyridoxine
thiamine
Pyruvate
4-diphospho-2Cmethyl-D-erythritol
O
Dxs
CO2H
H3C
OH
P
O
D-glyceraldehyde3-phosphate
(G3P)
HO
O
-
OH
O
IspD
O
O-
P
OH
-
O-
Dxr
O
O
O
O
OHC
OH
O
P
O-
O-
OH
N
HO
2C-methyl-Derythritol-4phosphate (MEP)
1-deoxy-Dxylulose-5phosphate (DXP)
NH2
O
O
OH
O
P
O
O-
OH
P
O
O
N
O
O-
OH OH
IspE
O
O
Isopentenyl
Pyrophosphate
(IPP)
P
O
O-
P
IspF
O
OO
IspH
O
OH
O
O
P
Dimethylallyl
Pyrophosphate
(DMAPP)
O
O
-
O
O-
P
-
O
NH2
O
O
P
O
-
O
IspG
O
O
O OO
P
-
O
1-hydroxy-2-methyl2-(E)-butenyl 4diphosphate
O
O
P
N
O
O
O-
O
OH
P
O-
P
-
OH
P
O-
O
O
P
O
O
N
O
O-
O
OOH OH
OH
OH
2C-methyl-Derythritol
2,4-cyclodiphosphate
4-diphosphocytidyl-2C-methylD-erythritol-2-phosphate
The mevalonate pathway
O
H3C
O
Acetyl-CoA HSCoA
SCoA
acetyl-CoA
Acetyl-CoA
thiolase
O
Acetyl-CoA HSCoA
SCoA
H3C
acetoacetyl-CoA
HMG-CoA
synthase
CH3 OH O
HO2C
S-CoA
3-hydroxy-3-methylglutaryl-CoA
2 NADPH
HMG-CoA
reductase
2 NADP
HSCoA
CH3 OH
HO2C
AT P
AD P
AT P
OP(OH) 2O
Mevalonate
mevalonate 5-phosphate kinase
AD P
CH3 OH
HO2C
OH
mevalonate
Phosphomevalonate
kinase
CH3 OH
HO2C
AT P
OPO(OH)OP(OH)2O
AD P
Pi CO2
OPO(OH)OP(OH)2O
Mevalonate
mevalonate 5-diphosphate
pyrophosphate decarboxylaseIsopentenyl diphosphate (IPP)
Artemisia annua
Artemisinin
O O
O
O
O
Artemisinin-based drugs
O O
O
O
O
• The current cost for an artemisininbased drug is approximately $2.25.
–Artemisinin generally adds $1.001.50 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
Microbial production of artemisinin
• Advantages
– Microbial fermentations are relatively simple to scale up
– Inexpensive starting materials can be used
• Challenges
– 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 good “platform organism” with appropriate
gene expression tools
Synthesis of
artemisinin in E. coli
Identify the enzymes
Synthesis of
artemisinin in E. coli
Clone the genes
Synthesis of
artemisinin in E. coli
Well characterized
parts to control gene
expression
Synthesis of
artemisinin in E. coli
Supply of
intracellular
precursors
Gene expression tools for metabolic
engineering
Plasmid
Plasmid copy number can influence
gene expression levels
High-copy plasmid
A
B
C
Enzyme 1
X
Enzyme 3
Y1
Enzyme 4
Z
Y2
Enzyme 2
Low-copy plasmid
A
Enzyme 1
B
C
Enzyme 2
X
Enzyme 3
Y2
Y1
Enzyme 4
Z
dxs expressed from a
high-copy plasmid
DMAPP
DXS
Pyr + G3P
IPP
Ptac
dxs
High-copy
plasmid
FPP
CrtE
CrtI
CrtY
Pconst crtE
crtI
crtY
Carotenoids
7
6
5
4
3
2
1
0
0
0.3
0.6
0.9
IPTG concentration (mM)
7
6
5
4
3
2
1
0
Cell Growth (OD570)
Carotenoid (mg/ml)
Carotenoid production in cells
expressing dxs from a high-copy
plasmid
Bacterial Artificial
Chromosome (BAC)
0
F 75
plasmid
25
50
ccd
oriV oriS
Tn1000
E
E
rep FIB
5
34
H
BP
flm
par
E
rep FIA
43
45
Native F plasmid of Escherichia coli
48
64 kb
Fraction plasmidbearing cells
BACs are stable indefinitely in the
absence of selection pressure
1
Gene expression
induced
Not induced
0.8
0.6
0.4
0.2
0
0
40
80
120
Culture time (generations)
160
Fraction plasmidbearing cells
Commonly-used high-copy
plasmids are segregatively unstable
1
Not induced
0.8
0.6
0.4
0.2
0
0
Gene expression
induced
40
80
120
Culture time (generations)
160
The auxiliary chromosomes have
improved control of gene expression
BAC
15 units
4,000 units
0.69 hr-1
High-Copy
Plasmid
200 units
12,500 units
0.53 hr-1
dxs expressed from a
bacterial artificial chromosome
DMAPP
DXS
Pyr + G3P
IPP
araC
PBAD
dxs
Bacterial
artificial
chromosome
FPP
CrtE
CrtI
CrtY
Pconst crtE
crtI
crtY
Carotenoids
Cell growth (OD600nm)
Lycopene (mg/ml)
Carotenoid production in cells
expressing dxs from a BAC
10.0
8.0
6.0
4.0
2.0
0.0
0
0.013
0.133
1.33
13.3
Arabinose concentration (mM)
Carotenoid production in cells
expressing dxs
Gene expression tools for metabolic
engineering
Reproducible
promoter
control
The arabinose-inducible PBAD
promoter
Chromosome
Plasmid
araC
inside
outside
The arabinose-inducible PBAD
promoter
Plasmid
arabinose
Chromosome
A A
A A
araC
Green Fluorescent Protein
inside
outside
arabinose
Expression of gfp from the
arabinose-inducible promoter
Fluorescence/OD600
100000
10000
1000
100
0.00001
0.0001
0.001
0.01
0.1
Arabinose (wt %)
1
10
Average
gene expression
Varying gene
expression levels by
varying induction in
individual cells
Inducer concentration
Average
gene expression
Varying gene
expression levels by
varying the number
Inducer concentration of induced cells
Flow cytometric analysis
Laser
Fluorescence
detector
Frequency
FALS sensor
Fluorescence
Frequency
Frequency
Frequency
Varying gene expression levels by
varying the number of induced cells
Fluorescence
Fluorescence
Fluorescence
Frequency
Frequency
Frequency
Varying gene expression levels by
varying induction in individual cells
Fluorescence
Fluorescence
Fluorescence
Native arabinose-inducible system
gives rise to two populations
Increasing
inducer
concentration
Fluorescence intensity
All-or-None Pathway Control
Pyruvate
IPP
DMAPP
Pyruvate
IPP
DMAPP
GPP
GPP
FPP
FPP
Amorphadiene
Amorphadiene
Artemisinin
Artemisinin
The arabinose-inducible PBAD
promoter
arabinose
inside
outside
arabinose
arabinose arabinose
arabinose
GFP
Population analysis of engineerined
E. coli expressing gfp
Increasing
inducer
concentration
Fluorescence intensity
Regulated Pathway Control
Pyruvate
IPP
DMAPP
Pyruvate
IPP
DMAPP
GPP
GPP
FPP
FPP
Amorphadiene
Amorphadiene
Artemisinin
Artemisinin
Gene expression tools for metabolic
Expression
engineering
of multiple
genes
Balancing enzymatic reactions
in the cell
gene 3
gene 2
gene 1
gene 4
A Enzyme 1
X
Enzyme 4
Enzyme 3
B
Enzyme 2
C
Y2
Y1
Z
Using individual control elements
P4
P1
gene 4
P3
gene 1
P2
gene 3
gene 2
A
Enzyme 1
X
Enzyme 4
Enzyme 3
B
Enzyme 2
C
Y2
Y1
Z
Synthetic operons
P
DNA
gene 3
gene 4
gene 2 gene 1
mRNA
A
Enzyme 1
X
Enzyme 4
Enzyme 3
B
Enzyme 2
C
Y2
Y1
Z
RNase
mRNA
A
B
C
Enzyme 1
X
Enzyme 2
Enzyme 3
Y2
Y1
Enzyme 4
Z
Secondary structures in the mRNA
protect natural mRNAs against
nucleases
ribosome
RNase E
endonuclease
RBS
exonuclease
ggagtcgacttatctcgagtgagatattgttgacggtaccccg
cctcagctgaatagagctcactctataacaactgccatggggc
Sal I
Asp718
A cassette
system to design
mRNA stability
tccatacgtcgacggtaccgtattttggatgataacgaggcgcaaaaaatg
aggtatgcagctgccatggcataaaacctactattgctccgcgttttttac
Sal I Asp718
Insertion of hairpin cassette
lacZ
tccatacgtcgacttatctcgagtgagatattgttgacggtaccgtattttggatgataacgaggcgcaaaaaatg
aggtatgcagctgaatagagctcactctataacaactgccatggcataaaacctactattgctccgcgttttttac
ag
g u
c g
u a
c g
u a
a u
u au
u u
c g
a u
g u
c g
u a
g c
gguaccguauuuuggaugauaacgaggcgcaaaaaug
ac
Transcription
A family of
synthetic
hairpins
pHP9
pHP14
pHP15
pTC40
t1/2 = 2.1 min
acgucgacagguaccguauuuu
t1/2 = 2.6 min
ga gu
c g
u a
c g
u a
a u
u a
u
u a
c g
t1/2 = 6.1
a u
g u
c g
u a
g c
ac gguaccguauuuu
ga gu
c g
u a
c g
u au
c gu
a u
t1/2 = 5.5
g u
c g
u a
g c
ac gguaccguauuuu
ga g u u
c
c
u
a
c
a
u
u
a
u
ua
ua
c g
t1/2 =
a u
g u
g
c
u a
g c
ac gguaccguauuuu
ag
g u pHP8
c g
u a
c g
u a
c g
u a
a u
u a
u uu
c g
a u
g u
c g
u a
g c
ac gguaccguauuuu
4.9 min
min
pHP17
gc a g u
u
u
c
pHP16 ac
a
g
a
gc
a
u a
g
at
c g
cg
u a
ua
c g
c g
u a
u a
g u
a u
a u
u a
g c
u u
c gu
g
c
u a
a u
g u
min
c gu
c g
pHP4 ua au
u a
g c
u au
c gguaccguauuuu
a
a
g
u u
g u
c g
t1/2 = 19.8 min
c g
a u
u a
g
u
c g
c g
u a
pHP10 ua au
g c
u au
c gguaccguauuuu
a
g
a
u
g u
c gu t = 12.5 min
c g
1/2
a u
u a
g u
c g
c g
u a
u a
a u
g c
u a
u a
ac gguaccguauuuu
c g
t1/2 = 8.3 min
a u
g u
c g
u a
g c
ac gguaccguauuuu
t1/2 = 6.8 min
A synthetic operon for carotenoid
production
CrtE
CrtI
Phytoene
CrtY
Lycopene
HP
p70yHPxi
crtY
b-Carotene
HPx
crtI
RNase E site
3'
HP
5'
crtY
HP16
crtI
3'
5'
3'
5'
3'
CrtE
CrtI
Phytoene
CrtY
Lycopene
b-Carotene
Variation in hairpins
RNase E site
p70yi
5'
crtY
crtI
3'
HP17
p70yHP17i
5'
crtY
crtI
3'
HP4
p70yHP4i
5'
crtY
crtI
3'
HP16
p70yHP16i
5'
crtY
crtI
3'
Relative levels of carotenoids
CrtE
CrtI
b-carotene/lycopene
Phytoene
CrtY
Lycopene
b-Carotene
400
300
200
100
0
p70yi
p70yHP4i p70yHP16ip70yHP17i
Synthesis of
artemisinin in cells
Clone the genes
Artem.
FPP
Poor performance
of plant sesquiterpene cyclases
2
0.08
0.8
0.04
0.4
0
5-epi-aristolochene
Cadinene
Low yields:
0.05 to 0.7
ng/mL/OD
Vetispiradiene
Expression of
rare E. coli codon
tRNA did not
much help
0
1.6
12
10
1.2
8
0.8
6
4
Cadinene (µg/l)
Cell growth (OD600nm)
5-epi-aristolochene (µg/l)
0.12
1.2
0.4
2
0
0
Cell growth (OD600nm)
1.6
7
6
1.2
5
4
0.8
3
2
0.4
1
0
0
0
10
20
30
Time (hrs)
40
50
Vetispiradiene (µg/l)
Cell growth (OD600nm)
0.16
1.6
Martin et al., Biotech.
Bioeng. 2001
Amorphadiene and artemisinin
biosynthetic pathway
OPP
Amorphadiene
Synthase
Farnesyl diphosphate
(FPP)
Amorphadiene
Cytochrome
P450
Chemical Synthesis
Y=40%
O O
O
HO
O
O
Artemisinin
O
Artemisinic Acid
Assembly of rcAmorphadiene Cyclase
• Take gene sequence from patent
• Optimize sequence for expression in desired
host
• Synthesize 84 oligonucleotides of ~40
basepairs each
• Assemble into complete gene using the
polymerase chain reaction (PCR)
Amorphadiene production by the
synthetic amorphadiene cyclase
Amorphadiene production
(ug/ml/OD600)
Native DXP pathway
0.1
0.08
0.06
0.04
0.02
0
0
2
4
6
8
10
12
14
Time (Hours)
142-fold improvement over other native cyclases
(100 ng/mL/OD)
Synthesis of
artemisinin in cells
Supply of
intracellular
precursors
DXP pathway
pyridoxine
thiamine
Pyruvate
4-diphospho-2Cmethyl-D-erythritol
O
Dxs
CO2H
H3C
OH
O
P
O-
D-glyceraldehyde3-phosphate
(G3P)
HO
O
O
O-
OH
IspD
O
-
P
OH
O-
Dxr
O
O
O
OHC
OH
O
P
O-
O-
OH
N
HO
2C-methyl-Derythritol-4phosphate (MEP)
1-deoxy-Dxylulose-5phosphate (DXP)
NH2
O
O
OH
O
P
O
O-
OH
P
O
O
N
O
O-
OH OH
IspE
O
O
Isopentenyl
Pyrophosphate
(IPP)
P
O
O-
P
IspF
O
OO
IspH
O
OH
O
O
P
-
Dimethylallyl
Pyrophosphate
(DMAPP)
O
O
O
O-
P
-
O
NH2
O
O
P
O-
IspG
O
O
P
O
O OO
P
O-
1-hydroxy-2-methyl2-(E)-butenyl 4diphosphate
O
O
P
N
O
O
O-
O
OH
P
O-
-
OH
P
O-
O
O
P
O
O
N
O
O-
O
OOH OH
OH
OH
2C-methyl-Derythritol
2,4-cyclodiphosphate
4-diphosphocytidyl-2C-methylD-erythritol-2-phosphate
Expression of genes known to
limit production
Pyruvate
DXS
IPP
DMAPP
IdI
IspA
FPP
Amorphadiene
G3P
Amorphadiene production
(ug/ml/OD600)
Amorphadiene production by the
synthetic amorphadiene cyclase
0.4
Native DXP pathway
0.35
0.3
Engineered DXP
pathway
0.25
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
Time (Hours)
12
14
Additional 3-fold
(300 ng/mL/OD)
Intermediates in the DXP pathway
are necessary for growth
Pyruvate
DXP Pathway
G3P
pyridoxine
thiamine
IPP
DMAPP
GPP
Monoterpenes
FPP
Sesquiterpenes
GGPP
Carotenoids
Diterpenes
Mevalonate pathway
O
H3C
O
Acetyl-CoA HSCoA
SCoA
acetyl-CoA
Acetyl-CoA
thiolase
O
Acetyl-CoA HSCoA
SCoA
H3C
acetoacetyl-CoA
HMG-CoA
synthase
CH3 OH O
HO2C
S-CoA
3-hydroxy-3-methylglutaryl-CoA
2 NADPH
HMG-CoA
reductase
2 NADP
HSCoA
CH3 OH
HO2C
AT P
AD P
AT P
OP(OH) 2O
Mevalonate
mevalonate 5-phosphate kinase
AD P
CH3 OH
HO2C
OH
mevalonate
Phosphomevalonate
kinase
CH3 OH
HO2C
AT P
OPO(OH)OP(OH)2O
AD P
Pi CO2
OPO(OH)OP(OH)2O
Mevalonate
mevalonate 5-diphosphate
pyrophosphate decarboxylaseIsopentenyl diphosphate (IPP)
Construction of synthetic
mevalonate pathway operons
MevT
Acetyl-CoA
atoB
P
MBI
P
Mevalonate
(1.2kb)
HMGS
tHMGR
(1.5kb)
(1.6kb)
MK
PMK
MPD
idi
(1.2kb)
(1.3kb)
(1.3kb)
(0.5kb)
Mevalonate
IPP
DMAPP
Amorphadiene production
(ug/ml/OD600)
Amorphadiene from the full
mevalonate pathway
3.5
Mevalonate pathway
3
2.5
2
1.5
1
0.5
0
0
2
DXP pathway
4
6
8
Time (Hours)
10
12
14
30-fold improvement
(3 mg/L/OD)
Amorphadiene production
in a two-phase fermentation
Amaorphadiene
concentration (mg/L)
500
450
400
350
300
250
200
150
100
50
0
0:00
12:00
24:00
36:00
48:00
60:00
Time after induction (hr)
72:00
Expression of plant mono-, sesqui-, and
di-terpenes cyclases in E. coli
FPP
Sesquiterpenes
5-epi-aristolochene
Tobacco
GPP
Monoterpene
GGPP
Diterpene
Myrcene synthase
Arabidopsis thaliana
d-cadinene
cotton
Vetispiradiene
Hyoscyamus muticus
ent-Kaurene cyclase
fungi
Casbene cyclase
Castor bean
Design Rules
for Doing Chemistry in Bacteria
• Low copy number is generally better for
reconstituting metabolic pathways
• Consistent promoter control is essential for
product and pathway homogeneity
• Construction of operons and the use of mRNA
stability is an efficient way to coordinate
expression of multiple genes
• Imbalances in gene expression can cause
accumulation of intermediates and can be
toxic to cells
Acknowledgements
Graduate Students
Trent A. Carrier
Kristala Jones
Christina Smolke
Doug Pitera
Sydnor Withers
Brian Pfleger
Yasuo Yoshikuni
Post-docs
Artem Khlebnikov
Seon-Won Kim
Vincent Martin
Jack Newman
Kinkead Reiling
Funding
National Science Foundation
Office of Naval Research
Maxygen
Diversa
University of California Discovery Grant