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Alfredo Martínez Jiménez Dpto. Ingeniería Celular y Biocatálisis
Instituto de Biotecnología – UNAM
1
Escherichia coli as Microbial Cell Factory for the
Biorefinery Concept
Escherichia coli como Fábrica Celular
Microbiana para el Concepto de Biorrefinería
Jorge Hilbert
INTA - Argentina
Francisco Girio
LNEG - Portugal
Buenos Aires,
Argentina.
23/Nov/2016
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Campus Morelos - Universidad Nacional Autónoma de México
www.ibt.unam.mx
Where we are:
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Diversas areas agrupadas en 5 Departamentos
Universidad Nacional Autónoma de México Campus Morelos
•Biología Molecular de Plantas
•Genética del Desarrollo y Fisiología Molecular
•Medicina Molecular y Bioprocesos
•Microbiología Molecular
•Ingeniería Celular y Biocatálisis
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What we do:
Alfredo Martinez: Fermentative Pathways, Biofuels and Biochemicals.
Metabolic engineering, synthetic biology and bioprocess development with Escherichia coli for biofuels (fuel ethanol, butanol and long chain alcohols), lactate, butyrate and R-3-hydroxybutirate (biopolymer precursor) production.
Physiological studies with oleaginous microalgae under heterotrophic conditions.
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Who is Escherichia coli?
What does E. coli do for humans?
Approximately 33% of the therapeutic proteins for human use are currently produced using E. coli using industrial fermenters.
Human growth hormones; interferons; interleukins; erythropoietin; among others
L-fenilalanina, PHB, and Propanediol, among others
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Saccharomyces cerevisiae
Yeast: Produce Ethanol
E. coli
Bacteria Sanchez-Garcia et al. Microbial Cell Factories, 201615:33
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(Future!) Global Challenge: New fuels & materials are needed to substitute fossil fuels and oil derivatives
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Energías Alternas Eólica
Hidráulica Mareomotriz Geotérmica
Nuclear Solar:
Electricidad
Martínez 2009
Se requieren Combustibles:
Sólidos Gaseosos Líquidos:
Bio-Etanol Bio-Diesel
Bio-Turbosina Bio-Gasolina
Bio-Plásticos Bio-degradables
& Bio-Químicos, otros
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Generation of Ethanol (Agro-Fuels) and (Agro-) Chemicals from Lignocellulose
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Chemicals Ethanol (Fuel)
Lignocellulose - Biomass
Agricultural Residues
Bagasses and Stovers
Energy Crops
Fermentation
Hydrolysis
CO2
Sugars + Acetic &
Glucuronic Acids
Furans
Artificial CO2 cycle
The Sun
Purpose: Design microorganism and process to transform ALL the SUGARS contained into lignocellulose (cellulose: glucose & hemicellulose: pentoses, hexoses, disaccharides) to ethanol (or other chemicals)
Carreón Rodríguez et al., 2009
1 kg of Sugar Yields 0.51 kg of Ethanol
0.5 USD / L
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Fermentation Products Escherichia coli
D-Lactato
NAD+ NADH
ldhA D-Lactate
Acetaldehyde
Ethanol
NAD+
NAD+
NADH NADH
adhE
adhE
AcetylCoA
frdABCD
Oxalacetate Fumarate
Succinate
ppc
Citrate
Cell Mass
Formate CO2
H2
Glucose Xylose
PEP
ADP
Pyruvate
ATP
Pdh pflB
Acetato
ATP
ADP
ackA
Acetate
ATP
ADP
ackA
Acetyl-P
pta
CO2
CO2
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Second Generation Bio-Plastics
Purpose: Design microorganism and process to transform Lignocellulose (cellulose & hemicellulose: pentoses, hexoses, disaccharides) to optically pure lactates (D&L): Biopolymer Precursors
Lactate
Lignocellulose - Biomass
Agricultural Residues
Sugar Cane Bagasse Fermentation
Hydrolysis
CO2
Xylose, Cellobiose Glucose,
etc. Cellulose,
Hemicellulose
Artificial CO2 cycle
The Sun 1 kg of Sugar Yield 1 kg of Lactic Acid >1 USD / kg
PLLA: > 4 USA dol/kg
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Fermentation Products Escherichia coli
D-Lactato
NAD+ NADH
ldhA D-Lactate
Acetaldehyde
Ethanol
NAD+
NAD+
NADH NADH
adhE
adhE
AcetylCoA
frdABCD
Oxalacetate Fumarate
Succinate
ppc
Citrate
Cell Mass
Formate CO2
H2
Glucose Xylose
PEP
ADP
Pyruvate
ATP
Pdh pflB
Acetato
ATP
ADP
ackA
Acetate
ATP
ADP
ackA
Acetyl-P
pta
CO2
CO2
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CH2OH
OH
OHO
OH
OH
OH
O
OH
O
O
O
O
O
O
OH
HO
CH3
CH3
CH3
O
O
On
L-Láctico Dímero PLA: Poly Lactic Acid Ópticamente puro Glucosa
Lactic Acid PLA, and other uses
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L-Lactato D-Lactato
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Embden-Meyerhof-Parnas Entner-Doudoroff Vïa de las Pentosas
PIRUVATO Lactato
Acetato
Acetil-CoA
Etanol
Formato
HEXOSAS (Glucosa) + PENTOSAS (Xilosa)
PEP
ADH
Succinato X X
X
E. coli Lactogénicas
Yomano et al., 2007 Martínez et al., 2007 Utrilla et al., 2008
Fernández et al., 2008 Orencio et al., 2008 1
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Sugar Cane Bagasse Hydrolysate 60 g/L total sugars, acetic acid
Aprox. 1 g/L Cells
0 12 24 36 48 60 720
100
200
300
400
500
600
700
Tiempo (h)
Ácid
o p
rod
ucid
o (
mM
)Hydrolysate + 0.9 g/L (NH4)2HPO4 – NH4H2PO4
1 mM Betaine 0.1 g/L Citric acid
D-Lactate Berenice Trujillo 2008
P-D-LA
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Sugar Cane Bagasse Hydrolysate JU15 Sequential sugar consumption
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0
1 0
2 0
3 0
4 0
5 0
0
1
2
3
4
5
6
7
Glu
co
se
(g/L
)Xy
los
e (
g/L
)
Ara
bin
os
e (g
/L)
0 1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6
0
2 0
4 0
6 0
0
2 0
4 0
6 0
D-L
A (g
/L)A
ce
tate
(g
/L)
T im e (h )
Y D -L A (g D -L A /g S u g a r s ) = 1 .3 0 0 .0 0 9
Q D -L A (g D -L A /L h ) = 0 .5 1 0 .0 1 1
Utrilla – Vargas-Tah et al. 2016
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Stover from White Corn Sequential: Thermochemical Hydrolysis,
Enzymatic Saccharification and Fermentation
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Agave Bagasse Ethanol and Lactic Acid
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Corn Stover Hydrolysate AV03: Simultaneous sugar consumption
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Utrilla – Vargas-Tah et al. 2016
0
1 0
2 0
3 0
4 0
5 0
0
1
2
3
4
5
6
7
Glu
co
se
(g
/L)
Xy
los
e (
g/L
)
Ara
bin
os
e (g
/L)
0
1 0
2 0
3 0
4 0
5 0
0
1
2
3
4
5
6
7
Glu
co
se
(g
/L)
Xy
los
e (
g/L
)
Ara
bin
os
e (g
/L)
Y D -L A (g D -L A /g S u g a r s ) = 0 .9 5 0 .0 1 0
Q D -L A (g D -L A /L h ) = 1 .3 2 0 .0 2 5
Y D -L A (g D -L A /g S u g a r s ) = 1 .1 1 0 .0 6 4
Q D -L A (g D -L A /L h ) = 1 .2 1 0 .0 5 0
A )
0 1 2 2 4 3 6 4 8
0
2
4
6
0
2 0
4 0
6 0
D-L
A (g
/L)
Ce
ll m
as
s (
g/L
)
Ac
eta
te (
g/L
)
T im e (h )
0 1 2 2 4 3 6 4 8
0
2
4
6
0
2 0
4 0
6 0
D-L
A (g
/L)
Ac
eta
te (
g/L
)
T im e (h )
B )
Y > 1 !!!
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Scale-Up
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Aire
Glicerol Agar LB Medio AM2 Fermentador de 85 y 110 L Mini-fermentador de 200 mL
Fermentador de 11 L
Aire
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Base case Study - 1 Liter
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Scale-Up: 1 110 Liters
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1 L11
L85
L
110
L1
L11
L85
L
110
L1
L11
L85
L
110
L
110
L*
0.0
0.1
0.2
0.3
0.4
0.0
0.5
1.0
1.5
2.0
2.5
Yp/s Qp (glactato/Lh) (h-1)
(
h-1
)Y
p/s
(gla
cta
to/g
glu
co
sa )
QP(g
lac
tato
/L.h
)
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Lactic acid (D and L) production with Metabolic Engineered E. coli strains
D-Lactato
NAD+ NADH
ldhA
ltcEBs
D-Lactic acid
L-Lactic acid
Acetaldehyde
Ethanol
NAD+
NAD+
NADH NADH
adhE
adhE
AcetylCoA
frdABCD
Oxalacetate Fumarate
Succinate
ppc
Citrate
Cell Mass
Formate CO2
H2
Glucose Xylose
PEP
ADP
Piruvate
ATP
pflB
Acetato
ATP
ADP
ackA
Acetate
ATP
ADP
ackA
Acetyl-P
pta
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Utrill Carreri et al., 2009; Leal Reyes 2010; Martinez et al. WO 2011 PCT 016706 A2
ldhA from E. coli was chromosomally substituted by ltcE from B. subtilis
No Plasmids
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Lactato Como materia prima para la
Industria Química
Polipropileno: 80 millones ton (2014)
Mercado PLA
2005 220 000 ton
2010 500 000 ton
2020 >1000 000 ton Chemical Market Report 2001
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Fermentation Products Escherichia coli: Biochemical Platform: Homo-Fermentative
D-Lactato
NAD+ NADH
ldhA D-Lactate
Acetaldehyde
Ethanol
NAD+
NAD+
NADH NADH
adhE
adhE
AcetylCoA
frdABCD
Oxalacetate Fumarate
Succinate
ppc
Citrate
Cell Mass
Formate CO2
H2
Glucose Xylose
PEP
ADP
Pyruvate
ATP
pflB
Acetato
ATP
ADP
ackA
Acetate
ATP
ADP
ackA
Acetyl-P
pta
L-Lactate
Ethanol
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1.00 0.51
0.51 0.65
> 1.00 - 1.30 1.00 CO2
CO2
CO2
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Ácido Succínico
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El ácido succínico polímeros, surfactantes, solventes,
detergentes, saborizantes y fragancias
Mercado global potencial de $15 billones de dólares.
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Bio-Refinería
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• Plásticos
• Polímeros
• Solventes
• Fenólicos
• Resinas (furfural)
• Ac. grasos
• Ac. orgánicos
• Pigmentos
• Detergentes
• Etanol
• Butanol
• Propanol
• Metano
• Bio-Diesel
• Bio-Gasolina
•Isopentenol
• Bio-Queroseno
• Bio-Electricidad
Integra los procesos de conversión de biomasa para obtener combustibles, electricidad y productos químicos (plásticos, resinas, intermediarios, etc.). Base de una nueva industria cimentada en materiales biológicos biodegradables, renovables, sustentables, menos contaminantes.
Biomasa
Plataforma
Bioquímica
Plataforma
Termoquímica
Combustibles,
Productos y
Materiales
Químicos
Calor y
Electricidad
Residuos
Gas
Gases
Azúcares
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Con E. coli se ha investigado la producción de
Ácido Succínico Ácido Láctico Propanodiol PHB
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Gracias CONACyT
ERAnet-LAC 2015-2018
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