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Metabolic Engineering for Fuels and Chemicals · Metabolic Engineering for Fuels and Chemicals K.T....
Transcript of Metabolic Engineering for Fuels and Chemicals · Metabolic Engineering for Fuels and Chemicals K.T....
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Metabolic Engineering for Fuels and Chemicals
K.T. Shanmugam and Lonnie O. Ingram
Dept. of Microbiology and Cell ScienceUniversity of FloridaGainesville, Florida
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Florida Center for Renewable Chemicals and Fuels
Dr. Lonnie O’Neal Ingram, Director
Metabolic Metabolic EngineeringEngineering
Renewable Renewable Biomass Biomass
to to Chemicals Chemicals
& & FuelsFuels
http://fcrc.ifas.ufl.edu
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Below ground
RENEWABLE FUELS AND CHEMICALS
Displacement of oil–– Commodity Commodity
chemicalschemicals•• polylactic acidpolylactic acid•• solventssolvents•• acidsacids
–– FuelsFuels•• ethanolethanol•• biodieselbiodiesel•• powerpower
–– Rural EmploymentRural Employment
Carbon Carbon Sequestration Sequestration
in soilin soil
Above ground
Older carbonspecies
Newer carbonspecies
CO2
CO2CO2
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EthanolEthanol
Lactic acidLactic acid
Succinic Succinic acidacid
1,21,2--PropandiolPropandiol
1,31,3--PropandiolPropandiol
PolyhydroxybutyratePolyhydroxybutyrate
Reduced compounds produced under anaerobic conditions
Reduced compounds produced Reduced compounds produced under anaerobic conditionsunder anaerobic conditions
PROPOSED BIOMASS-DERIVED COMPOUNDS
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FEEDSTOCK PROCESS ETHANOL (CHEMICALS)
1. Choice2. Availability3. Cost4. Quality
1. H+
2. Cellulases3. Hemicellulases4. Inhibitors
1. Recovery2. Waste Disposal
SolidLiquid
BiocatalystDepolymerization
CONVERSION OF LIGNOCELLULOSICS TO ETHANOL
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Depolymerization
1. CelluloseCellulasesOptimize with the Biocatalyst
2. XyloseXylanases, Xylosidases
3. Glucuronoxylanα-Glucuronidase; Xylosidase
4. Acid Hydrolysis
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BIOCATALYST
1. High Growth Rate2. High Cell Yield3. High Product Yield
Volumetric ProductivitySpecific Productivity
4. Purity of the ProductOpticalChemical
5. Minimal Growth Requirements6. Metabolic Versatility7. Co-utilization of Various Sugars8. Tolerate High Sugar Concentration9. Resistance to Inhibitors10. Insensitive to Product Inhibition11. High-value Co-products12. Amenable to Genetic Engineering13. Robust14. Cellulases15. Xylan degradation
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E. coliE. coli: Potential Industrial Platform : Potential Industrial Platform for Renewable Fuels and Chemicalsfor Renewable Fuels and Chemicals
1. Safety, reliability, and industrial experience.
2. Uses broad range of sugars derived from biomass (hexose, pentose, sugar alcohol and sugar acid; expanded to – cellobiose and xylobiose).
3. Simple nutrient requirements.
4. Well understood physiology and established tools for genetic manipulation.
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HEXOSES + PENTOSES
Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate
Succinate
Lactate
Acetate
Acetyl-CoA + Formate
COCO22 H2EthanolEthanol
Lactate Dehydrogenase7.2 mM (ldhA)
PyruvateFormate-Lyase2 mM (pfl)
Pyruvate Decarboxylase0.4mM (pdc)
Acetaldehyde + COCO22
Alcohol Dehydrogenase(adhB)
Ethanol >95% of Ethanol >95% of TheorTheor. . YieldYield
(Zymomonas mobilis)Zymomonas mobilis)PYRUVATEPYRUVATE
Microbial PlatformMicrobial Platform
PEPX
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0 12 24 36 48 60 72 84 96
0
20
40
60
80
100
Organic Acids
Biomass (g/L)
Xylose (g/L)
0
2
4
6
8
10
Time (h)
Xylo
se a
nd E
than
ol (g
/lite
r)
Cell M
ass(g/liter)
0 12 24 36 48 60 72 84 96
0
20
40
60
80
100Xylose (g/L)
Biomass (g/L)
Ethanol (g/L)
0
2
4
6
8
10
Time (h)Xy
lose
and
Eth
anol
(g/li
ter)
Cell M
ass(g/liter)E. coli BE. coli B (organic acids) and KO11 (ethanol)(organic acids) and KO11 (ethanol)
(10% Xylose, pH 6.5, 35C) (10% Xylose, pH 6.5, 35C)
Yield Yield –– 0.50 g ethanol and 0.49 g CO0.50 g ethanol and 0.49 g CO22 per g per g xylose xylose
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PRODUCTIVITY IS RELATED TO CELL MASS
0
0.5
1
1.5
2
0 1 2 3 4 5 6
Cell Mass (g)
Rat
e of
Eth
anol
Pro
duct
ion
(g/li
ter.
h)
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SUGAR UTILIZATION and SSCF
CELLULOSE: GLUCOSE
HEMICELLULOSE: XYLOSE
SEQUENTIAL – Catabolite RepressionSIMULTANEOUS
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Culture was grown with 13C1- glucose and 13C1- xylose at 37C in the NMR withour pH control.
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TOLERANCE TO HIGHER LEVEL OF ETHANOL
Higher Product Yield
Lower Product Cost
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Ethanol Tolerance: Mutants reach over 6.5% w/v ethanolEthanol Tolerance: Mutants reach over 6.5% w/v ethanol(14% (14% xylosexylose, 35C, pH 6.5, 100 rpm, , 35C, pH 6.5, 100 rpm, Luria Luria Broth)Broth)
0 24 48 72 96 1200
250
500
750
1000
1250
1500
K011
Mut 1
Mut 2
Time (h)
Etha
nol (
mM
)
> 65 g ethanol/liter> 65 g ethanol/liter
50 g ethanol/liter50 g ethanol/liter
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FERMENTATIONS AT HIGH SUGAR CONCENTRATIONS
Observed: Lower Growth Rate and Cell Yield of KO11
Cause: Osmotic Effect
Expect: Higher Product Yield
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Limiting Acetyl-CoA Pool
Acetaldehyde Acetyl-CoA
Citrate
Isocitrate
2-Ketoglutarate
Succinate
Fumarate
Malate
Oxaloacetate
X
Pyruvate
Acetyl-P AcetateEthanoladhB
pflpdc
pta
gltAcitZ
NADHNAD+
Glycolysis
adhE
ATP
AMP+ PPi
acs
Glutamate
ackA
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Genetic solution
AcetylAcetyl--CoACoA
CitrateCitrate
IsocitrateIsocitrate
22--KetoglutarateKetoglutarateFumarate
Malate
Oxaloacetate
Succinate
Acetaldehyde Ethanol
Acetyl~P AcetateadhE
adhEadhBpdc
pta ackA
CitZ
Glucose
Pyruvate
Glutamate(Osmoprotectant)
(B. subtilis)
X
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Fermentations with ∆ackA and ∆adhE
0 12 24 36 48 60 72 84 96
0.0
0.5
1.0
1.5
2.0
KO11∆ adhE
∆ ackAAcetate
Time (h)
Cel
l Mas
s (g
/L)
0 12 24 36 48 60 72 84 96
0
10
20
30
40
50
KO11∆ adhE
∆ ackAAcetate
Time (h)
Eth
anol
(g/L
)
• Deletion of ackA eliminates conversion of acetyl-CoA to acetate.
• This resulted in a stimulation of growth and ethanol production similar to acetate supplementation.
• Ethanol yield by ∆ackA, 0.47 g/g totalxylose (92%).
• Average volumetric productivity for ∆ackA increased (0.57 g/L/h), compared to KO11 (0.33 g/L/h).
• Average specific productivity for ∆ackA(0.38 g/g/h), similar to KO11 (0.36 g/g/h).
• The combination (∆ackA ∆adhE) was no better than the ∆ackA.
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(Weitzman, PDJ. 1966. Biochim. Biophys. Acta 128:213-215)
Inhibited by NADH & 2-ketoglutarate
70% inhibition at 50µM NADH and 0.16mM Acetyl-CoA
B. subtilis Citrate Synthase
Inhibited by ATP 2 mM NADH – No effect
E. coli Citrate Synthase
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Expression of B. subtilis citZ in KO11
0 12 24 36 48 60 72 84 96
0.0
0.5
1.0
1.5
2.0
2.5
KO11 (TOPO)
Bs citZ (pLOI2514)2 g/L Acetate
Time (h)
Cell
Mas
s (g
/L)
0 12 24 36 48 60 72 84 960
10
20
30
40
50
KO11 (TOPO)
Bs citZ (pLOI2514)2 g/L Acetate
Time (h)Et
hano
l (g/
L)
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Microbial Zoo(E. coli)
Erwinia Klebsiella Bacillus Zymomonas~33kb secretion genes 2 PTS cellobiose genes citrate synthase PDC+ADH
2 cellulases 2 xylobiose genes pectate lyase
Sugars, Sugars, OligosaccharidesOligosaccharidesEthanolEthanol& other& otherproductsproducts
Pseudomonasesterase for ethyl acetate
Who knows what the future will bring?
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PRODUCTION OF OXIDIZED COMPOUNDS
Anaerobic: Redox Neutral or Reduced Compounds
C6H12O6 2 C3H6O3 or 2 C2H6O + 2 CO2Glucose Lactic acid Ethanol
Aerobic: Oxidized Compounds
C6H12O6 2 C2H4O2 + 2 CO2 + 4HGlucose Acetic Acid
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Overview of Metabolism in E. coliOverview of Metabolism in Overview of Metabolism in E. coliE. coli
Up to 95 % of carbon converted to products (low CO2 production)
2.5 ATP produced
Low growth rate
Internal electron acceptor
Cell MassCell Mass
5% of Carbon5% of Carbon
AnaerobicAnaerobicGlucose, C6H12O6
50% of carbon converted to CO2
33 ATP (calc.) produced
High growth rate
External electron acceptor
AerobicAerobic
Cell Mass
50% of Carbon
Glucose, C6H12O6
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Goal: Combine the Attributes of Aerobic & Anaerobic Metabolism
Goal: Combine the Attributes of Aerobic Goal: Combine the Attributes of Aerobic & Anaerobic Metabolism& Anaerobic Metabolism
High product yield High product yield
Low cell yield Low cell yield
High growth rateHigh growth rate
External eExternal e-- acceptoracceptor
++
Anaerobic
Aerobic
Single Single Biocatalyst Biocatalyst
Neutral or Oxidized ProductsNeutral or Oxidized ProductsNeutral or Oxidized Products
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NADH
out
F0
atpIBEFHAGDC
F1
H+ O2 + e-
in
Electron TransportElectron TransportSystemSystem
Pi
+ADP ATP
H+
NAD+
F0
F1
Cytb1(ox)
Glyoxylate
frdABCD
Glucose
gltA
~P
Isocitrate
CO2
PyruvatePyruvate
adhE
LactateLactateLactate
NADHlpdA NAD+
HCOOH
pflB
FADH2
FAD+
NADH
AcetateAcetateAcetatepta
Triose-P
PEP
ackA
~P
poxBCO2 Acetyl-CoAAcetyl-CoA
~P
NAD+
~P
Cytb1(red)
Fumarate
NADH
Malate
NAD+
Citrate
Oxaloacetate
Succinyl-CoA
glcB aceB
Acetyl-CoAmdh
acnB
icdA
sucDC
fumABC
NADP+
CO2
ppc
pykA
~P
pykF
NAD+CO2
EthanolEthanolEthanol
2 NAD+2 NADH
NAD+
NADHldhA
Pi
+ADP ATP
F1H+
~P
sdhABCD
UQH2
UQ
Glucose Metabolism Glucose Metabolism
CO2
sucAB
H2O
NADPH
aceA
SuccinateSuccinateSuccinate
aceEF lpdA
NADH
2-Oxoglutarate22--OxoglutarateOxoglutarate
Yield:>85%
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0 6 12 18 24 30 36
0
100
200
300
400
500
0
1
2
3
4
5
Glucose Added
GlucoseAcetateTC36
Time (h)
Glu
cose
& A
ceta
te (m
M)
Cell M
ass (g. L
-1)
NEW RESEARCH AREASLimits for Glycolytic Flux?Control of Carbon
Partitioning?Limits for Growth Rate?Maximum Cell Density?
Isogenic Strains:
(Mixed acid, ethanol, lactate, acetate, pyruvate, glutamate,succinate, alanine, citrate)
ATP/ADP?NADH/NAD?MetabolomicsProteomicsTranscriptome Analysis
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Acetyl-CoA
Glucose
Triose 3-P PEP Pyruvate~P
~P
HCO3-
CO2-
AcetateAcetate
Engineered Engineered E. coli E. coli TC44 MetabolismTC44 Metabolisme- transport chain No ox-phos
NADH + H~P
Acetyl-P~P
NADH + H
UQ
UQH2 AcetateAcetate
CO2-
2H2H++
H2O½O2e- +
Pi
+ADP ATP
F1
ptapta
ackAackA
poxBpoxB
Fumarate
Malate
Oxaloacetate
Citrate
Isocitrate
2-Ketoglutarate
CO2
NADPH2
NADH
IncompleteTCA
IncompleteIncompleteTCATCA
NADH oxidized by electron transport system.
~ 2 ATP per glucose.
~ 5-10% of glucose carbon is converted to cell mass.
NADH oxidized by NADH oxidized by electron transport electron transport system.system.
~ 2 ATP per glucose.~ 2 ATP per glucose.
~ 5~ 5--10% of glucose 10% of glucose carbon is converted to carbon is converted to cell mass.cell mass.
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BIOCATALYST
1. High Growth Rate2. High Cell Yield3. High Product Yield
Volumetric ProductivitySpecific Productivity
4. Purity of the ProductOpticalChemical
5. Minimal Growth Requirements6. Metabolic Versatility7. Co-utilization of Various Sugars8. Tolerate High Sugar Concentration9. Resistance to Inhibitors10. Insensitive to Product Inhibition11. High-value Co-products12. Amenable to Genetic Engineering13. Robust14. Cellulases15. Xylan degradation
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Future StudiesFuture Studies
Gene Array Investigations:Global regulators for carbon metabolism
(mutations in mlc, crp, csrA)Global regulators for redox control
(mutations in fnr, arcA)Prolonging the growth phase and metabolism
(comparing ethanol/lactic acid)
Improvements for Ethanol and Other Chemicals:Ethanol tolerance, Process simplificationCarbon partitioning/production costsRates and yieldsCellulases, cellobiose/triose; Xylanases, xylobiose/triose
Metabolic Engineering for Higher Value Products:L(+)-lactic acid and D(-)-lactic acidAcetic acid, pyruvic acid, succinate, glutamate, citrate
BioRefineryBioRefinery
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Dependence on petroleum Dependence on petroleum remains as the remains as the
single most important factor single most important factor affecting the world distributionaffecting the world distributionof wealth, global conflict, of wealth, global conflict, human health, and human health, and environmental quality.environmental quality.
Reversing this dependence Reversing this dependence would increase employment, would increase employment, preserve our environment, and preserve our environment, and facilitate investments that facilitate investments that improve the health and living improve the health and living conditions for all. conditions for all.
Professor Ohta conductingfermentation studies at the University of Florida