application of enzymes and microorganisms for organic ......metabolic engineering: introducing new...
TRANSCRIPT
1
Application of Enzymes and Microorganisms for Organic
Synthesis
Jay Keasling
TaxolHO
O
OHCH3
O
O O
CH3 CH3O
O
CH3
OCH3O
H3CHN
O
HO
O O
2
Taxol is extracted from the Pacific Yew•The Pacific yew tree is an environmentally protected species and one of the slowest growing trees in the world.
•Isolation of the compound, which is contained in the bark, involves killing the tree.
•One 100-year old tree results in approximately 350 mg of taxol, just enough for one dose for a single cancer patient.
Total synthesis of taxol
3
Metabolic engineering: introducing new pathways in cells
Synthesis of complex molecules
• May require several enzymes from one or more organisms
• Expression of genes must be balanced– Underexpression of any one gene may limit flux
through the pathway and therefore product yields– Overexpression will lead to inefficiencies
• Precursors (from inside the cell or supplied from outside the cell) should not severely limit production of the desired product
4
Balancing enzymatic reactions in the cell
C
A
BX Y1 Z
Enzyme 1 Enzyme 3
Enzyme 2
Enzyme 4
Y2
Over-produced enzyme
Under-produced enzyme
What is metabolic engineering?• Metabolic engineering is a redirection of
enzymatically-catalyzed reactions for the production of a new compound or the degradation of a compound
– genetic modification of a single organism
– engineering a consortium of organisms
5
Why do metabolic engineering?• Production of novel compounds
– new biopolymers– antibiotics
• Production of existing compounds in better ways
• Bioremediation of recalcitrant compounds– pesticides/nerve agents– PCBs
HOO
OHCH3
O
O O
CH3 CH3O
O
CH3
OCH3O
H3CHN
O
HO
O
12
34 5
614
13
1211
109
8 715 16
17
18
19
20
O
Taxol has a diterpene
core
6
OPO(OH)OP(OH)2O
Geranylgeranyl diphosphate (GGPP)
Terpene metabolic pathways
OPO(OH)OP(OH)2O
Farnesyl diphosphate (FPP)
OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
Dimethylallyl diphosphate(DMAPP)
Geranyl diphosphate (GPP)
OPO(OH)OP(OH)2O
Isopentenyl diphosphate (IPP)
Diterpenes
Monoterpenes
Sesquiterpenes
Carotenoids
In bacteria, IPP is produced via the non-mevalonate pathway
H3C CO2H
O
OHC
OH O OH
H3C
OH
OH OH
HO
1-deoxyxylulose-5-P (DXP)
OP(OH)2O OP(OH)2O
OP(OH)2O
Pyruvate glyceraldehyde-3-P
2-C-methyl-D-erythritol-4-P (MEP)
DXP synthase
DXP reductoisomerase
OPO(OH)OP(OH)2O
Isopentenyl diphosphate (IPP)
7
Metabolic engineering of carotenoid production
Pyr + G3P IPP
FPP
dxs dxrDMAPP
idi
0
5
1015
20
25
3035
Car
oten
oid
prod
uctio
n(μ
g/m
L/O
D60
0)
crtEBIdsx,idi
crtEBIdxs Carotenoids
crtEBI
crtEBI
dxs and dxr under Ptac control on high-copy cloning vectors
Pconst crtE crtI crtY
High-copyplasmid
Medium-copyplasmid
Ptac dxs
8
Overexpression of dxs from a high-copy plasmid with a strong promoter
01234567
0
Car
oten
oid
(μg/
ml)
Cell G
rowth (O
D570 )0
1234567
0.3 0.6 0.9IPTG concentration (mM)
Overexpression of dxs is a
metabolic burden on cell growth
Glc
G6P
Pyr
AcCoA
CIT
ICT
OGT
SUC
MAL
OAA
GOX
G3PDHAPDXP
Secretion ??
IPP GGPP
Carotenoids
(-)
9
Needs for Metabolic Engineering
• Optimal fluxes through the heterologous metabolic pathways
• Strict control over gene expression
• Consistent control of gene expression in all cells
• Minimal burden of the heterologous genes on the host
Some key problems in gene expression control
• Many expression vectors are unstable and have variable copy number in the host
• Many promoters do not allow tight and consistent control of gene expression
• There are few techniques to regulate expression of multiple heterologous genes
• Can we predict the levels of gene expression needed for flux redistribution?
10
Gene expression tools for metabolic engineering:
A Metabolic Engineering Toolbox
Gene expression tools for metabolic engineeringExpression
vector
11
Bacterial Artificial Chromosome (BAC)
oriV oriS parTn1000 flmccd
rep FIB rep FIA5 34 43 45 48 64 kb
Native F plasmid of Escherichia coli
EEE B P H
F plasmid
0
25
50
75
A partition element ensures that each daughter cell will receive a BAC
oriV oriS parccd
EE B P H43 45 48
Cell
Division
12
Specific replication origins time BAC replication with the cell cycle
oriV oriS
EE B P H43 45 48
parccd
A “Kill” element ensures that BAC-free daughter cells do not survive
oriV oriS parccd
EE B P H43 45 48
Cell
Division
13
BACs are stable indefinitely in the absence of selection pressure
0 40 80 120 1600
0.2
0.4
0.6
0.8
1
Culture time (generations)
Frac
tion
plas
mid
-bea
ring
ce
llsGene expression
induced Not induced
The auxiliary chromosomes have improved control of gene expression
Uninduced
Expres
sion
Induced
Expres
sion
Growth R
ate
of Host
BAC
High-CopyPlasmid
0.69 hr-1
0.53 hr-1
15 units
200 units
4,000 units
12,500 units
14
dxs and dxr under PBAD control on bacterial artificial chromosome
Pconst crtE crtI crtY
Bacterialartificial
chromosome
Medium-copyplasmid
PBAD
araC
dxs
Production of lycopene: dxs under control of PBAD on low-copy plasmid
Cel
l gro
wth
(OD
600n
m)
Lyc
open
e (m
g/l)
Arabinose concentration (mM)
0.0
2.0
4.0
6.0
8.0
10.0
0 0.013 0.133 1.33 13.3
15
Gene expression tools for metabolic engineeringReproducible
promotercontrol
inside
outside
P BADgfp
P Cara
C
araE
P araE
Green fluorescent protein
Experimental system
Chromosome
Plasmid
arabinose
arabinose
16
Expression of gfp from the arabinose-inducible promoter
100
1000
10000
100000
0.00001 0.0001 0.001 0.01 0.1 1 10
Arabinose (wt %)
Fluo
resc
ence
/OD
600
Desired gene expression in population
Induction/cell
Freq
uenc
yin
pop
ulat
ion
Induction/cellInduction/cell
100% of cells
are half induced
Gen
eex
pres
sion
Inducer concentration
17
‘All-or-None’ Gene Expression
Induction/cell
Freq
uenc
yin
pop
ulat
ion
Induction/cellInduction/cell
50% are fully induced
50% are uninduced
Gen
eex
pres
sion
Inducer concentration
Flow cytometric analysis
Fluorescence
Freq
uenc
y
Fluorescencedetector
FALS sensor
Laser
18
Native arabinose-inducible system gives rise to two populations
Increasing inducer concentration
Fluorescence intensity
Native arabinose-inducible system gives rise to two populations
Fluorescence intensity
Uninduced cellsInduced cells Tim
e afte
r inducti
on [h
]
01
23
46
Cel
l num
ber
19
Population dynamics as a function of arabinose concentration
0
20
40
60
80
100
0 2 4 6
Time [h]
Indu
ced
cells
[%]
0.0002%
0.002%
0.02%
0.2%
Experiment:steady-state induction levels as a function of
inducer concentration
0
20
40
60
80
100
Indu
ced
cells
[%]
0
2000
4000
6000
8000
-5 -4 -3 -2 -1 0 1Arabinose concentration [log %]
Fluo
rese
nce/
OD
20
inside
outside
P BADgfp
P Cara
C
araE
P araE
Stimulation of inducer transport gene expression
arabinose
arabinose
Green fluorescent protein
arabinose
inside
outside
P BADgfp P C
araC
araE
P con
arabinose
Decoupled transporter/reporter
system
21
Population analysis of E. coliexpressing gfp
Time a
fter i
nduction
[h]
01
34
6
Fluorescence intensity
Uninduced cellsInduced cells
2
Cel
l num
ber
Population analysis of E. coliexpressing gfp
Increasing inducer concentration
Fluorescence intensity
22
Experiment: Steady-state
induction levels as a function of
inducer concentration
0
20
40
60
80
100
Indu
ced
cells
[%]
0
2000
4000
6000
8000
-5 -4 -3 -2 -1 0 1Arabinose concentration [log %]
Fluo
rese
nce/
OD
Native system
Engineered system
inside
outside
P BADGen
e of
interest
P Cara
C
araE
P araE
Native system
Chromosome
Plasmid
Protein of interest
arabinose
arabinose
ON
OFF
23
inside
outside
P BAD
P Cara
Cara
EP con
Engineered system
Protein of interest
Gene o
f
interest
arabinose
arabinose
Low
arabinose
Medium
arabinosearabinose
High
Gene expression tools for metabolic engineering
Expressionof multiplegenes
24
Balancing enzymatic reactions in the cell
C
A
BX Y1 Z
Enzyme 1 Enzyme 3
Enzyme 2
Enzyme 4
Y2
gene 3
gene 4
gene 2
gene 1
Using individual control elements
C
A
BX Y1 Z
Enzyme 1 Enzyme 3
Enzyme 2
Enzyme 4
Y2
gene 4
P4
P1
gene 1P3
gene 3 P2
gene 2
25
Synthetic operons
C
A
BX Y1 Z
Enzyme 1 Enzyme 3
Enzyme 2
Enzyme 4
Y2
gene 3 gene 4
P
gene 2 gene 1
mRNA
DNA
C
A
BX Y1 Z
Enzyme 1 Enzyme 3
Enzyme 2
Enzyme 4
Y2
mRNA
RNase
26
The puf operon in Rhodobacterencodes a multi-subunit enzyme
Q B A L M X
? ?
RC
LHIMulti-subunitenzyme
The production of enzyme subunits is controlled by mRNA stability
DNA
mRNA
Black arrows indicate RNase E cleavages
27
mRNA is inactivated by a cleavage inside the coding region
endoribonuclease
5’ RBSmRNA
ribosome
cleavage site
exonuclease
Secondary structures in the mRNA protect natural mRNAs against
nucleases
RNase Eendonuclease
exonucleaseRBS
ribosome
28
tccatacgtcgacggtaccgtattttggatgataacgaggcgcaaaaaatgaggtatgcagctgccatggcataaaacctactattgctccgcgttttttac
A cassette system to design mRNA stability
lacZSal I Asp718Insertion of hairpin cassette
tccatacgtcgacttatctcgagtgagatattgttgacggtaccgtattttggatgataacgaggcgcaaaaaatgaggtatgcagctgaatagagctcactctataacaactgccatggcataaaacctactattgctccgcgttttttac
Transcription
gguaccguauuuuggaugauaacgaggcgcaaaaaug
ga
ac
c
c
c
c
c
u
uuu
uu
g
gg
g
g
g
a
a
a
a
a
u
u
u
u
u
ga
ggagtcgacttatctcgagtgagatattgttgacggtaccccgcctcagctgaatagagctcactctataacaactgccatggggc
Sal I Asp718
A family of synthetic hairpins
acgucgacagguaccguauuuut1/2 = 2.6 min
pTC40
cc
c
c
uu
uu
g
g
g
a
a
u
gaccu
gggau
u
a
ga
gguaccguauuuu
t1/2 = 4.9 min
pHP14
cc
c
c
auu
g
g
gau
u
a
ga
gguaccguauuuu
u
u
ua
g
cc
a
a
g
u
cu u
a
u a
t1/2 = 2.1 min
pHP15t1/2 = 6.1 min
c
c
c
c
c
c
u
uuu
uu
g
gg
g
g
a
aaa
a
uu
u
u
a
ga
ggau
gguaccguauuuu
c gu a
pHP8
c
c
c
c
c
c
u
uauu
g
g ggg
g
g
a
a
aaa
a
uu
u
u
u
a
ga
gguaccguauuuu
t1/2 = 6.8 min
pHP10
cc
c
c
ua
uu
g
g
g
a
a
u
ccu
u
g ggg
a
a
aau
u
u
u
a
ga
gguaccguauuuu
t1/2 = 5.5 min
pHP9
c
c
c
c
c
c
u
uuu
uu
g
g ggg
g
g
a
a
aaa
a
uu
u
u
u
a
ga
gguaccguauuuu
t1/2 = 8.3 min
pHP4
t1/2 = 19.8 min
pHP17
ac
c
c
c
a
uu
g
g
gau
u
ga
gguaccguauuuu
g
c
ga
g
au
c
c
a
a
ug u
a
a t
uuu
uau acu ga
gc
t1/2 = 12.5 min
pHP16
c
c
c
c
c
c
u
uuu
uu
g
gg
g
g
g
g
a
a
a
aa
a
uu
c
u
u
a
ga
gguaccguauuuu
cuucu
g cagag
uu
u
a
29
A strategy to design mRNA stability?
cc
c
c
auu
g
g
gau
u
a
ga
gguaccguauuuu
u
uua
g
cc
aa
g
u
cuu
aua
c
c
c
c
c
c
u
uuu
uu
g
gg
g
g
a
aaa
a
uu
u
u
a
ga
ggau
gguaccguauuuu
c gu a
c
c
c
c
c
c
u
uuu
uu
g
g
g
g
a
gaa
a
uu
u
u
a
ga
ggcc
gguaccguauuuu
aa
g
caau
uac
aucgcag
u
0
5
10
15
20
5 10 15 20 25
mR
NA
hal
f-lif
e (m
in)
-ΔGfolding
(kcal/mol)
Degradation of multicistronic mRNA
30
Degradation of multicistronic mRNA
Constructs to test operons
pGL
pGH1E1H4L
pGH1E2H4L
gfp lacZ3'5'
gfp lacZ3'5'
RNase E site 1
gfp lacZ3'5'
RNase E site 2
H4H1
31
Relative protein levels/mRNA stabilities
0
1
2
3
4
pGL
pGH1E1H4LpGH1E2H4L
β-G
al/G
fp
Relative enzyme activities
Reversing gene order
pLG
pLH1E1H4G
pLH1E2H4G
lacZ gfp3'5'
lacZ gfp3'5'
RNase E site 2
lacZ gfp3'5'
RNase E site 1
H4H1
32
Relative protein levels/mRNA stabilities
0.1
1
10
100
1000
pLG
pLH1E1H4GpLH1E2H4G
β-G
al/G
fp
Relative enzyme activities
A synthetic operon for carotenoid production
Phytoene Lycopene β-Carotene
p70yHPxicrtY crtI 3'5'
RNase E site
HPxHP
CrtE CrtI CrtY
33
No hairpin 5’ of crtI
Phytoene Lycopene β-Carotene
p70yicrtY crtI
3'5'
RNase E site
HP
CrtE CrtI CrtY
Carotenoid Production
0
100
200
300
p70yi + pAC-PHYT
Rel
ativ
e Pr
oduc
tion
(Pea
k A
rea/
OD
600)
Phytoene
β-Carotene
Lycopene
34
crtY crtI3'5'
HP
Phytoene Lycopene β-CaroteneCrtE CrtI CrtY
3'5'
3'5'
A strong hairpin 5’ of crtI
Phytoene Lycopene β-Carotene
p70yHP4icrtY crtI 3'5'
RNase E site
HP4HP
CrtE CrtI CrtY
35
Carotenoid Production
0
100
200
300
p70yi + pAC-PHYT
p70yHP4i +pAC-PHYT
Rel
ativ
e Pr
oduc
tion
(Pea
k A
rea/
OD
600)
Phytoene
β-Carotene
Lycopene
crtY crtI3'5'
HP
Phytoene Lycopene β-CaroteneCrtE CrtI CrtY
3'5'
5' 3'
HP4
36
A stronger hairpin 5’ of crtI
Phytoene Lycopene β-Carotene
p70yHP16icrtY crtI 3'5'
RNase E site
HP16HP
CrtE CrtI CrtY
Carotenoid Production
0
100
200
300
p70yi + pAC-PHYT
p70yHP4i +pAC-PHYT
p70yHP16i +pAC-PHYT
Rel
ativ
e Pr
oduc
tion
(Pea
k A
rea/
OD
600)
Phytoene
β-Carotene
Lycopene
37
crtY crtI3'5'
HP
Phytoene Lycopene β-Carotene
CrtE CrtI CrtY
3'5'
HP16
5' 3'
A weak hairpin 5’ of crtI
Phytoene Lycopene β-Carotene
p70yHP17icrtY crtI 3'5'
RNase E site
HP17HP
CrtE CrtI CrtY
38
Carotenoid Production
0
100
200
300
p70yi + pAC-PHYT
p70yHP4i +pAC-PHYT
p70yHP16i +pAC-PHYT
p70yHP17i +pAC-PHYT
Rel
ativ
e Pr
oduc
tion
(Pea
k A
rea/
OD
600)
Phytoene
β-Carotene
Lycopene
crtY crtI3'5'
HP
Phytoene Lycopene β-CaroteneCrtE CrtI CrtY
3'5'
HP17
5' 3'
39
Gene expression tools for metabolic engineering
Metabolic models toquantify
flux
How do you coordinate
the expression of multiple
genes?
40
Mass Balance on Cellular MetabolitesdXdt
= S ν - b where
X = Concentration of metabolitesS = Stoichiometric matrix
-> known enzymatic reactionsb = Uptake, secretion, and biomass synthesis
-> known from cell compositionν = Reaction flux vector
-> unknown
Mass Balance on Cellular Metabolites
dXdt
= S ν - b
GenomicsProteomicsPhysiomics
41
=S ν bnumber of
fluxes(495)
number of metabolites
(289)>
Objective Function: Z = ΣConstraints:
LowerBound
UpperBound< νi < i = 1, 2, ...
S ν = b1.2.
Solve for fluxes using linear optimization
At steady-state:
Linear Optimization:
ci νii
Predicted fluxes for growth on glucose and acetate
Experimental DataWalsh & Koshland (1985)
Model Predictions
7.260.0
2.91
CELL CONSTITUENTS
0.92
4.62
4.62
0.0
4.62
1.14
3.77
4.64
4.62
1.75
1.73
FATTY ACIDS
ACETATE
PYR
CIT
CELLCONSTITUENTS
GLX
OA
ISOCIT
SUCC
7.00GLUCOSE
0.81
5.00
5.00
1.13
3.87
0.00.0
3.87
3.87
1.78 0.81
5.0
5.00
0.81
MAL
AcCoA
PEP
-KGαFUM
42
Some environmental examples of metabolic engineering
Application of metabolic engineering to biodesulfurization of fossil fuels
• Dibenzothiophene (DBT) is typical of the organic sulfur compounds found in fossil fuels.
• DBT is recalcitrant to hydrodesulfurization.• Used extensively in biodesulfurization studies
S
DBT (dibenzothiophene)
43
Rhodococcus erythropolis IGTS8 desulfurization pathway
S S
O
O2 O2
DBT (dibenzothiophene)
DszC,FMNH2
Dsz C,FMNH2 S
O O
O2 DszA,FMNH2
S
O
O
HOHO
Dsz B
HBP (2-hydroxybiphenyl)
+SO32-
DBT-5-oxide DBT-5,5-dioxide
2-(2' hydroxyphenol)benzene sulfinate
Role of reducing equivalents
• DszA & DszC require reduced flavin (FMNH2).
NADH NAD
FMN FMNH2
O2 H2O
DBT DBTO
N
N
OHOH
OH
H O
NH
ON
H
OPO3 DSZ
FMN reductase
44
Comparison of degradation rates
Organism Rate(mg/hr/g dcw)
Source
E. coli pRED/pDSZ
51 This work
Rhodococcus erythropolis IGTS8
3 Gallardo (1997)
Rhodococcus erythropolis H-2
5 Oshiro (1996)
Relieving bottlenecks in the desulfurization pathway
S S
O
O2 O2
DBT (dibenzothiophene)
Dsz C,FMNH2
Dsz C,FMNH2 S
O O
O2 Dsz A,FMNH2
S
O
O
HOHO
Dsz B
HBP (2-hydroxybiphenyl)
+SO32-
DBT-5-oxide DBT-5,5-dioxide
2-(2' hydroxyphenol)benzene sulfinate
45
Overlapping reading frames in dszABmay limit flux through the pathway
dszB dszCdszA
DBT DBTO2HBPS HBP
dszB dszCdszA
DBT DBTO2 HBPS HBP
Aerobic precipitation of cadmium
Cys
Cysteinedesulfhydrase
NH3+ pyruvate+
Cd2+ CdS
S2-
46
Native cysteine biosynthesis pathway
SO42-
Serineacetyltransferase
(SAT)
Cysteine
(-)
Serine +Acetyl-CoA
O-Acetyl-Serine
(+)
S(II)
Deregulated cysteine biosynthesis pathway
SO42-
Serineacetyltransferase
(SAT)
Cysteine
Serine +Acetyl-CoA
O-Acetyl-Serine
(+)
S(II)
47
Cysteine production is inhibited by cadmium
0
100
200
300
400
500
0 50 100 150
Cd (μM)
Cys
tein
e ( μ
M)
Control
Cys production(SAT*)
Expression of cysE* and cysDfor aerobic cadmium precipitation
CysCysE*
S2-
Cd2+
CdS
cysE*Pcon
PyruvateNH3
CysD
cysDPtrclacIQ
48
Precipitation of CdS by the engineered strain
Control Engineered strain
CdS
Cadmium precipitates on the cell wall as cadmium sulfide
50 10
Energy(keV)
Cou
nts
200
150
100
50
0
Cu
Os
Os
Cl
Cl
S
Cou
nts
200
150
100
50
0
S
S
Cu
Cu
Os
Os
Cd
Cd
Cd
Ca
CaCa
Cl
Cl
Cu
0 5 10
Energy (keV)
a b
c d
CuCu
49
Acknowledgements
Funding– National Science Foundation– Office of Naval Research– DARPA– Department of Energy– National Institutes of Health– Maxygen– Merck
Graduate StudentsJaya PramanikDoug BoleschRobert PapeNatalya EliashbergStephen Van DienSalomeh KeyhaniTrent A. Carrier
Kristala JonesR. Brent NielsenPiper TrelstadClifford WangIlana AldorDavid ReichmuthJessica Hittle
Andrew WalkerNichole GoedenNeil RenningerChristina SmolkeSundiep TeharaStacie CowanCynthia Gong
Michel MaharbizDavid Lubertozzi Katherine McMahonDoug PiteraKai WangSydnor WithersLance KizerBrian FrushourBrian Pfleger
Post-docsSusan T. SharfsteinWubin PanSang-Weon BangEric GilbertArtem KhlebnikovYet-Pole ISeon-Won KimVincent MartinGuangyi Wang