Download Metabolic Engineering for Fuels and Chemicals

Metabolic Engineering for
Fuels and Chemicals
K.T. Shanmugam and Lonnie O. Ingram
Dept. of Microbiology and Cell Science
University of Florida
Gainesville, Florida
Florida Center for Renewable Chemicals and Fuels
Metabolic
Engineering
Renewable
Biomass
to
Chemicals
&
Fuels
Dr. Lonnie O’Neal Ingram, Director
http://fcrc.ifas.ufl.edu
RENEWABLE FUELS AND CHEMICALS
CO2
CO2
CO2
™ Displacement of oil
ov
b
A
n
u
o
r
eg
d
– Commodity
chemicals
• polylactic acid
• solvents
• acids
– Fuels
•
•
•
ethanol
biodiesel
power
– Rural Employment
Newer carbon
species
Older carbon
species
Below ground
Carbon
Sequestration
in soil
PROPOSED BIOMASS-DERIVED COMPOUNDS
™
Ethanol
™
Lactic acid
™
Succinic acid
™ 1,2-Propandiol
™ 1,3-Propandiol
™ Polyhydroxybutyrate
Reduced
Reduced compounds
compounds produced
produced
under
under anaerobic
anaerobic conditions
conditions
CONVERSION OF LIGNOCELLULOSICS TO ETHANOL
FEEDSTOCK
1.
2.
3.
4.
PROCESS
Choice
Availability
Cost
Quality
Depolymerization
1.
2.
3.
4.
H+
Cellulases
Hemicellulases
Inhibitors
ETHANOL (CHEMICALS)
1. Recovery
2. Waste Disposal
Solid
Liquid
Biocatalyst
1. Cellulose
Cellulases
Optimize with the Biocatalyst
Depolymerization
2. Xylose
Xylanases, Xylosidases
3. Glucuronoxylan
α-Glucuronidase; Xylosidase
4. Acid Hydrolysis
1.
2.
3.
4.
BIOCATALYST
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
High Growth Rate
High Cell Yield
High Product Yield
Volumetric Productivity
Specific Productivity
Purity of the Product
Optical
Chemical
Minimal Growth Requirements
Metabolic Versatility
Co-utilization of Various Sugars
Tolerate High Sugar Concentration
Resistance to Inhibitors
Insensitive to Product Inhibition
High-value Co-products
Amenable to Genetic Engineering
Robust
Cellulases
Xylan degradation
E. coli: Potential Industrial Platform
for 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.
+
HEXOSES
PENTOSES
Microbial Platform
Embden-Meyerhof-Parnas Entner-Doudoroff
Succinate
X
PYRUVATE
PEP
Lactate Dehydrogenase
7.2 mM (ldhA)
Pyruvate
Formate-Lyase
2 mM (pfl)
Lactate
Acetyl-CoA
Acetate
Ethanol
+
Formate
Pentose Phosphate
(Zymomonas mobilis)
Pyruvate Decarboxylase
0.4mM (pdc)
Acetaldehyde + CO2
Alcohol Dehydrogenase
(adhB)
CO2
H2
Ethanol >95% of Theor.
Yield
Xylose (g/L)
8
6
60
4
40
Biomass (g/L)
2
20
Organic Acids
0
0
0
12
24
36
48
60
Time (h)
72
84
96
10
100
Xylose (g/L)
8
80
60
6
Ethanol (g/L)
4
40
20
Biomass (g/L)
2
0
0
0
12
24
36
48
60
72
84
96
Time (h)
Yield – 0.50 g ethanol and 0.49 g CO2 per g xylose
(10% Xylose, pH 6.5, 35C)
Cell Mass(g/liter)
80
Xylose and Ethanol (g/liter)
10
100
Cell Mass(g/liter)
Xylose and Ethanol (g/liter)
E. coli B (organic acids) and KO11 (ethanol)
PRODUCTIVITY IS RELATED TO CELL MASS
1.5
(g/liter.h)
Rate of Ethanol Production
2
1
0.5
0
0
1
2
3
4
Cell Mass (g)
5
6
SUGAR UTILIZATION and SSCF
CELLULOSE:
GLUCOSE
HEMICELLULOSE:
XYLOSE
SEQUENTIAL – Catabolite Repression
SIMULTANEOUS
Culture was grown with 13C1- glucose and 13C1- xylose at 37C in the NMR withour pH control.
TOLERANCE TO HIGHER LEVEL OF ETHANOL
Higher Product Yield
Lower Product Cost
Ethanol Tolerance: Mutants reach over 6.5% w/v ethanol
(14% xylose, 35C, pH 6.5, 100 rpm, Luria Broth)
Ethanol (mM)
1500
> 65 g ethanol/liter
1250
1000
K011
750
Mut 1
50 g ethanol/liter
500
Mut 2
250
0
0
24
48
72
Time (h)
96
120
FERMENTATIONS AT HIGH SUGAR CONCENTRATIONS
Expect: Higher Product Yield
Observed: Lower Growth Rate and Cell Yield of KO11
Cause: Osmotic Effect
Limiting Acetyl-CoA Pool
NAD+
NADH
adhE
Acetaldehyde
Ethanol
adhB
is
s
y
l
o
c
Gly
Acetyl-CoA
Acetyl-P
Acetate
pfl
pdc
ackA
pta
acs
Pyruvate
glt
A
citZ
Oxaloacetate
Citrate
ATP
AMP
+ PPi
Isocitrate
Malate
Fumarate
2-Ketoglutarate
X
Succinate
Glutamate
Glucose
Genetic solution
Pyruvate
pdc
Acetaldehyde
adhB
adhE
Ethanol
adhE
Acetyl-CoA
pta
Acetyl~P
X
ackA
Acetate
CitZ
Oxaloacetate
(B. subtilis)
Citrate
Isocitrate
Malate
Fumarate
2-Ketoglutarate
Glutamate
(Osmoprotectant)
Succinate
Fermentations with ∆ackA and ∆adhE
Cell Mass (g/L)
• Deletion of ackA eliminates conversion
of acetyl-CoA to acetate.
Acetate
∆ ackA
2.0
1.5
• This resulted in a stimulation of growth
and ethanol production similar to acetate
supplementation.
1.0
KO11
0.5
∆ adhE
0.0
0
12
24
36
48
60
72
84
96
Time (h)
• Average volumetric productivity for
∆ackA increased (0.57 g/L/h), compared
to KO11 (0.33 g/L/h).
Ethanol (g/L)
50
Acetate
∆ ackA
40
30
• Average specific productivity for ∆ackA
(0.38 g/g/h), similar to KO11 (0.36 g/g/h).
20
10
∆ adhE
KO11
0
0
12
24
• Ethanol yield by ∆ackA, 0.47 g/g total
xylose (92%).
36
48
60
Time (h)
72
84
96
• The combination (∆ackA ∆adhE) was no
better than the ∆ackA.
E. coli Citrate Synthase
Inhibited by NADH & 2-ketoglutarate
70% inhibition at 50µM NADH and 0.16mM Acetyl-CoA
(Weitzman, PDJ. 1966. Biochim. Biophys. Acta 128:213-215)
B. subtilis Citrate Synthase
Inhibited by ATP
2 mM NADH – No effect
Expression of B. subtilis citZ in KO11
50
2 g/L Acetate
Bs citZ (pLOI2514)
2.0
2 g/L Acetate
Ethanol (g/L)
Cell Mass (g/L)
2.5
1.5
1.0
0.5
40
Bs citZ (pLOI2514)
30
20
10
KO11 (TOPO)
KO11 (TOPO)
0.0
0
12
24
36
48
60
Time (h)
72
84
0
96
0
12
24
36
48
60
Time (h)
72
84
96
Sugars, Oligosaccharides
Microbial Zoo
(E. coli)
Erwinia
Klebsiella
~33kb secretion genes 2 PTS cellobiose genes
2 cellulases
2 xylobiose genes
pectate lyase
Pseudomonas
esterase for
ethyl acetate
Ethanol
& other
products
Bacillus
Zymomonas
citrate synthase
PDC+ADH
Who knows what
the future will bring?
PRODUCTION OF OXIDIZED COMPOUNDS
Anaerobic:
Redox Neutral or Reduced Compounds
C6H12O6
2 C3H6O3
Glucose
Lactic acid
Aerobic:
or 2 C2H6O + 2 CO2
Ethanol
Oxidized Compounds
C6H12O6
Glucose
2 C2H4O2 + 2 CO2 + 4H
Acetic Acid
Overview of Metabolism in E. coli
Anaerobic
Glucose, C6H12O6
Aerobic
Glucose, C6H12O6
Cell Mass
Cell Mass
5% of Carbon
50% of Carbon
¾ Up to 95 % of carbon converted to
products (low CO2 production)
¾ 2.5 ATP produced
¾ Low growth rate
¾ Internal electron acceptor
¾ 50% of carbon converted to CO2
¾ 33 ATP (calc.) produced
¾ High growth rate
¾ External electron acceptor
Goal: Combine the Attributes of Aerobic
& Anaerobic Metabolism
Anaerobic
High product yield
Low cell yield
Single
Biocatalyst
+
High growth rate
External e- acceptor
Aerobic
Neutral or Oxidized Products
Glucose
Lactate
~P
~P
Triose-P
NAD+
NADH
~P
+
NAD
PEP
Yield:
>85%
CO2
ldhA
NADH
CO2
NADH
NAD+
pykA
pykF
Pyruvate
Cytb1(red)
Cytb1(ox)
CO2
Glucose Metabolism
poxB
ppc
aceEF
lpdA
pflB
2 NADH 2 NAD+
Acetyl-CoA
pta
ackA
HCOOH
Ethanol
adhE
Acetate
~P
NADH
NAD+
Oxaloacetate
H+
gltA
Malate
glcB aceB
F0
Citrate
Glyoxylate
Fumarate
aceA
UQH2
Electron Transport
System
atpIBEFHAGDC
F1
acnB
NAD+ NADH
Isocitrate
NADP+
sdhABCD
icdA
Succinate
CO2
22-Oxoglutarate
-Oxoglutarate
sucAB
lpdA
sucDC
NAD+
~P
~P
NADH
Succinyl-CoA
CO2
F1
+
NADPH ADP H ATP
UQ
FAD+
F0
in
fumABC
frdABCD
H2O
out
mdh
Acetyl-CoA
FADH2
H+ O2 + e-
+
Pi
ADP
+
Pi
ATP
NEW RESEARCH AREAS
500
4
Glucose Added
400
3
300
200
2
100
1
Cell Mass (g . L-1)
Glucose & Acetate (mM)
5
Glucose
Acetate
TC36
¾Limits for Glycolytic Flux?
¾Control of Carbon
Partitioning?
¾Limits for Growth Rate?
¾Maximum Cell Density?
Isogenic Strains:
(Mixed acid, ethanol, lactate,
acetate, pyruvate, glutamate,
succinate, alanine, citrate)
¾ATP/ADP?
0
0
0
6
12
18
Time (h)
24
30
36
¾NADH/NAD?
¾Metabolomics
¾Proteomics
¾Transcriptome Analysis
Engineered E. coli TC44 Metabolism
e- transport chain No ox-phos e- + ½O2
Glucose
UQH2
NADH + H
~P
~P
Triose 3-P
~P
PEP
poxB
CO2NADH + H
Acetyl-CoA
NADH
Malate
Fumarate
Incomplete
TCA
Citrate
Isocitrate
NADPH2
CO2
2-Ketoglutarate
F1
CO2-
Pyruvate
HCO3Oxaloacetate
2H+
Acetate
UQ
H2O
pta
Acetyl-P
ackA
~P
Acetate
ADP
+
Pi
ATP
¾ NADH
NADH oxidized
oxidized by
by
electron transport
system.
¾ ~ 2 ATP per glucose.
¾ ~ 55-10%
-10% of glucose
carbon is converted to
cell mass.
1.
2.
3.
4.
BIOCATALYST
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
High Growth Rate
High Cell Yield
High Product Yield
Volumetric Productivity
Specific Productivity
Purity of the Product
Optical
Chemical
Minimal Growth Requirements
Metabolic Versatility
Co-utilization of Various Sugars
Tolerate High Sugar Concentration
Resistance to Inhibitors
Insensitive to Product Inhibition
High-value Co-products
Amenable to Genetic Engineering
Robust
Cellulases
Xylan degradation
Future 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)
BioRefinery
Improvements for Ethanol and Other Chemicals:
Ethanol tolerance, Process simplification
Carbon partitioning/production costs
Rates and yields
Cellulases, cellobiose/triose; Xylanases, xylobiose/triose
Metabolic Engineering for Higher Value Products:
L(+)-lactic acid and D(-)-lactic acid
Acetic acid, pyruvic acid, succinate, glutamate, citrate
Dependence on petroleum
remains as the
single most important factor
affecting the world distribution
of wealth, global conflict,
human health, and
environmental quality.
Reversing this dependence
would increase employment,
preserve our environment, and
facilitate investments that
improve the health and living
conditions for all.
Professor Ohta conducting
fermentation studies at
the University of Florida