Download ATP driving force

代謝工程學
metabolic engineering
生質能源 (酒精等)
chemicals
1
Using metabolically engineered cyanobacteria to produce
commodity chemicals from CO2
ETHAN LAN
20160511
Department of Biological Science and Technology
National Chiao Tung University
Renewable energy and chemical production can alleviate dependence on fossil fuels
World Petroleum Consumption
(Million barrels per Day)
Alleviate dependence on fossil resources
with renewable biological resources
Sunlight
Biofuel
CO2
Biochemical
Source: EIA Monthly Energy Review (Feb 2012)
Transportation fuel
Petrochemical - plastics
3
Bioplastics/Biopolymers & precursors
Lee et al. 2011 Current Opinion in Biotechnology, 22:758–767
Pharmaceuticals, Nutrachemical, drug precursor, and proteins
Supplements
Chiral drug precursor
Pharmaceuticals
lycopene
Artemisinin – anti-malarial
Resverstrol
Zhang et al., 2010
Taxol – anti-cancer
Reduces cost and negative
environmental impacts
Pacific yew tree
glycolysis
A metabolic pathway
6
TCA cycle
7
Biological
feedstocks
CO2, waste
proteins,
Lignocellulose,
Sugars, natural
gas, etc
Whole cell Catalysis
Modify and regulate microbial metabolism
Using native
metabolic capability
to utilize different
biological feedstock
Remove native
competing pathways
Fuel & Chemical
Bioenergy,
chemical feedstock,
pharmaceuticals,
fragrance,
nutraceuticals,
polymers, plastics,
etc
Design and express
synthetic pathways for
production of
desirable compounds
代謝工程學
A
B
C
B (product)
A
A
C (byproduct)
B (product)
C (byproduct)
Outline
An example for metablic engineering
OH
1-butanol
We (ETHAN LAN’s group) aim to study
the engineering of cyanobacteria for the synthesis of chemicals using CO2
12
Importance and market of n-butanol
fuels
plasticizers
Gasoline fuel
Butyl phthalates
2-Ethyl hexanol
Butane
OH
n-Butene
n-Butanol
butadiene
Polybutylene
Butyraldehyde
Polyvinyl butyral
Butyl acetate
Butyl glycol ethers
Butyrate
Butyrate esters
Butyl acylate
Butyl methacrylate
solvents
polymers
Annual production :2.9 million metric tons,
(not accounting for use as fuel)
$5.7 billion Market
grows 4.7% a year.
As a fuel: Better than ethanol because 1) Low hygroscopicity less corrosive,
2) higher energy density
3) compatible with current gasoline engine
13
History & on-going progress of Biobutanol production
•
1861 – Louis Pasteur observed biological production of butanol
•
1912 - 1914 – Chaim Weizmann isolated Clostridium acetobutylicum and
discovered ABE fermentation (Acetone:Butanol:Ethanol)
•
1916 – (World War I) – ABE was commercialized in UK
for making cordite (smokeless gun powder).
•
1950 - 1960s – Development of ABE became non-economical in
Western countries due to competition from cheap petroleum sources.
•
1970s – Renewed interest because increase in petroleum price.
(Energy crisis in 1970s)
•
•
1980s – increased research on improving ABE process
1990s – Advances in genetic research enabled strain improvement
•
2000s – Advances in genomics (and other “omics”) – C. acetobutylicum
sequenced & enabled transfer of butanol pathway to other organisms –
however poor expression
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Clostridium n-Butanol pathway was poorly expressed in heterologous hosts
Clostridium pathway
Acetyl-CoA
O
CoA
thl
O
O
Acetoacetyl-CoA
NADH
CoA
hbd
OH
O
3-Hydroxybutyryl-CoA
CoA
crt
1-Butanol titer (g/L)
Acetyl-CoA
25
20
15
10
5
0
10 to 20
Recombinant producer
Native producer
1.2
0.0025
0.3
0.58
0.12
Escherichia
coli
Saccharomyc
es cerevisiae
Lactobacill
us brevis
Pseudomon
as putida
Bacillus
subtilis
Atsumi et al., 2008
Inui et al., 2008
Steen et al.,
2008
Nielson et al.,
2009
Nielson et al.,
2009
Berezina et al.,
2010
Clostridium
beijerickii
BA101
Chen and
Blaschek,
1999
O
Fdred
Fdox
2 NADH
Crotonyl-CoA
FADH2
FAD
NAD+
NADH
bcd/
etfAB
CoA
1. Pathway was reversible and lack significant driving force
O
Butyryl-CoA
CoA
2. Bcd/Etf protein may require Clostridium ferredoxin for
optimal function
adhE2
O
n-Butyraldehyde
NADH
H
adhE2
n-Butanol
Several features of the Clostridium pathway was noted:
OH
Synthetic driving forces increase butanol production
To increase the driving force for
butanol production:
Glycolysis
Glucose
NAD+
NADH
NADH
Lactate
NADH
Succinate
1. Replaced Bcd/Etf withTransenoyl-CoA reductase (Ter),
which irreversibly reduces
crotonyl-CoA with NADH,
effectively increaseing butanol
production.
NADH
Ethanol
Acetyl-CoA
Acetate
Acetoacetyl-CoA
NADH
2. Knocked out the major
pathways consuming NADH
and acetyl-CoA, increasing
substrate pool
Fdred
Fdox
Crotonyl-CoA
FADH2
NADH
FAD
2 NADH
NAD+
Butyryl-CoA
NADH
NADH
Butyraldehyde
n-Butanol
Shen et al. (2011). Appl Environ Microbiol
Butanol production in cyanobacteria is difficult under photosynthetic condition
Lan and Liao. (2011). Metabolic Engineering
Ribulose5P
Ribose5P
G3P
ATP
Ribulose1,5BP CO
2
Xylulose5P
Cytoplasm
NADPH
S7P
3PGA
ATP
S1,7BP
E4P
Xylulose5P
G3P
F6P
Glycogen
hν
hν
Fdox
1,3BPGA
PQ
PQH2
NADPH
F1,6BP
Cytochrome
b6f complex
G3P
Pc-Cu+
Photosystem II
DHAP
Challenge:
1. Increase Driving force
2. Oxygen sensitivity
• Less than 1 mg/L of
n-butanol observed in
culture medium under
photosynthetic condition
H2O
G3P
O2
Pc-Cu2+
Photosystem I
Lumen
Acetyl-CoA
Unfavorable reaction Keq = 10-5
Acetoacetyl-CoA
NADH
Crotonyl-CoA
NADH
Butyryl-CoA
NADH
• Butanol was observed
only under anoxic
incubation
Fdred
NADH
Butyraldehyde
n-Butanol
Oxygen sensitivity prohibits
enzyme functions under oxygenic
photosynthetic conditions
Learning from nature: similarity to fatty acid synthesis and degradation
Claisen condensation of two acetyl-CoA is thermodynamically unfavorable…
cyanobacteria are more difficult to manipulate acetyl-CoA pool (as opposed to E. coli)
CoA n-butanol pathway
O
Acc
O
-O
CoA
O
Ketoacyl-ACP synthase III
(KASIII)
O
O
ACP
R
ACP
CoA
O
ATP CO2
O
O
R
CoA
O H
A metabolic
pathway very
similar to fatty
acid degradation
in reverse
O
HO
R
C o A
R
C oA
O
C o A
R
A
C
P
O
O
Fatty Acids
O
O
CoA
CoA
O
ACP
O
+
+
CoA
A
C
P
O
O
O
O
ACP
O
OH
ATP
CO2
O
+
H2 O
+ ATP + H2O
CoA
ADP
O
+
O
+ HS-CoA
CoA
Pi
R
OH
ΔGo’ ≈ 6.8 kcal/mol
Keq ≈ 1.1 x 10-5
ΔGo’ ≈-7.3 kcal/mol
Keq ≈ 5.6 x 105
+ HS-CoA + ADP + Pi
CoA
ΔGo’ < 0
Keq > 1
ATP hydrolysis is the
energy input! for
chain elongation!
18
Redesigning butanol pathway with ATP driving force
We also expressed NADPH dependent
dehydrogenase instead of NADH dependent ones
Ribulose5P
Ribose5P
G3P
Xylulose5P
ATP
Ribulose1,5BP CO
2
Cytoplasm
NADPH
S7P
3PGA
ATP
S1,7BP
E4P
Xylulose5P
G3P
F6P
hν
hν
Fdox
1,3BPGA
PQ
PQH2
NADPH
F1,6BP
n-Butanol Titer (mg/L)
DHAP
Cytochrome
b6f complex
G3P
Pc-Cu+
ATP
G3P
Acetyl-CoA
Photosystem II
H2O
O2
Malonyl-CoA
CO2
Acetoacetyl-CoA
35
30
25
20
15
10
5
0
NADPH
Fdred
Fdox
Crotonyl-CoA
FADH2
NADH
FAD
2 NADH
NAD+
Butyryl-CoA
O2
5
10
15
20
Time since induction (days)
NADPH utilizing only
ATP driving force only
ATP driving force + NADPH utilization
Fdred
NADPH
0
Butyraldehyde
Lan and Liao (2012) PNAS
NADPH
n-Butanol
Pc-Cu2+
Photosystem I
Lumen
In vitro validation of PduP oxygen tolerance and butyryl-CoA specificity
Leal et al. Arch Microbiol (2003) 180 : 353–361
NADH
O
Specific activity
(umol min-1 mg-1)
CoA
NAD+, CoA
PduP under aerobic condition
Assay condition:
His-tag purified
30 °C, aerobic assay
100 mM Tris-HCl pH 7.0
500 µM NADH
600 µM acyl-CoA
O
H
70
60
50
40
30
20
10
0
S.ent
L.mon
K.pne
L.bre
P.gin
A.hyd
2
3
4
6
8
Acyl-CoA chain length
Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681
10
12
20
Expression of PduP & YqhD in cyanobacteria achieved first demonstration
of acetyl-CoA based ethanol production
Plasmid DNA
O
NADH
CoA
NADPH
O
5’-NSI
H
pduP
pduP yqhD TrrnB specR 3’-NSI
OH
Acetaldehyde
Acetyl-CoA
Ptrc
lacIq
Ethanol
yqhD
Recombination
Neutral Site I
6
All strains expressing PduP
homologues with YqhD achieved
ethanol production under
photosynthetic conditions.
5
150
OD730
ethanol titer (mg L-1)
200
100
50
4
3
2
1
0
0
0
2 4 6 8 10 12
Days post induction
Strain (PduP expressed)
ETOH-KP (PduP_K.pneumoniae)
ETOH-LM (PduP_L.monocytogenes)
S. elongatus 7942 genomic DNA
-2
3
8
13
Days since induction
ETOH-LB (PduP_L.brevis)
ETOH-SE (PduP_S.enterica)
Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681
This result indicated that PduP is
functionally expressed in
cyanobacteria for conversion of
acyl-CoA to aldehyde.
21
Redesigning butanol pathway with ATP driving force
Cumulative accounts for dilution made
to culture due to nutrient feeding
Ribulose5P
Ribose5P
ATP
Ribulose1,5BP CO
2
Xylulose5P
G3P
Effective n-butanol titer
(mg L-1)
500
S7P
3PGA
ATP
S1,7BP
E4P
Xylulose5P
G3P
F6P
1,3BPGA
400
300
200
100
0
NADPH
F1,6BP
DHAP
n-butanol production titer
(mg L-1)
0
G3P
ATP
G3P
Acetyl-CoA
NADPH
Fdred
Fdox
Crotonyl-CoA
200
FADH2
150
NADH
FAD
2 NADH
100
NAD+
50
Butyryl-CoA
NADH
O2
0
0
5
10
Days post induction
15
Butyraldehyde
Summary:
ATP driving force and oxygen
tolerance are important
factors for achieving direct
photosynthetic n-butanol
production.
NADPH
n-Butanol
BUOH-LB (PduP_L.brevis)
BUOH-SE (PduP_S.enterica)
BUOH-LM (PduP_L. monocytogenes)
15
10-fold increase
CO2
300
5
10
Days after induction
300 mg/L
Malonyl-CoA
Acetoacetyl-CoA
350
250
In-Flask
Cumulative
Lan and Liao. (2013) Energy Environ Sci. 6, 2672-2681
Clostridium pathway
Functional expression
E. coli
Acetyl-CoA
Acetyl-CoA
ATP driving force – direct
photosynthetic production
ATP driving force
Acetyl-CoA
accABCD
Acetyl-CoA
thl
Acetyl-CoA
atoB
Acetoacetyl-CoA
NADH
hbd
3-Hydroxybutyryl-CoA
crt
Fdred
Fdox
2 NADH
Crotonyl-CoA
FADH2
FAD
bcd*/
etfAB
Acetoacetyl-CoA
NADH
3-Hydroxybutyryl-CoA
Universal e- donor crt
& Irreversible trap
Crotonyl-CoA
NADH
NADH
adhE2*
n-Butyraldehyde
NADH
adhE2*
n-Butanol
* Indicates oxygen sensitivity
ter
Butyryl-CoA
Butyryl-CoA
NAD+
hbd
NADH
adhE2*
n-Butyraldehyde
NADH
adhE2*
n-Butanol
CO2
Acetyl-CoA
CO2
Acetoacetyl-CoA
phaB
Acetyl-CoA
ATP
Malonyl-CoA
nphT7
Elimination of oxygen
sensitivity
accABCD
CO2
Malonyl-CoA
nphT7
Acetyl-CoA
CO2
Acetoacetyl-CoA
NADPH
phaB
3-Hydroxybutyryl-CoA
ATP
NADPH
3-Hydroxybutyryl-CoA
phaJ
phaJ
Crotonyl-CoA
Crotonyl-CoA
NADH
ter
Butyryl-CoA
Bldh*
Butyryl-CoA
NADH
n-Butyraldehyde
yqhD
n-Butanol
NADH
ter
NADPH
pduP
NADH
n-Butyraldehyde
yqhD
NADPH
n-Butanol
23
Oxygen tolerance