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Optimization of L-carnitine production by enterobacteria
J.L. Iborra, M.Cánovas, A. Sevilla & V. Bernal
Department of Biochemistry &Molecular Biology “B” & Immunology
Faculty of ChemistryUniversity of Murcia
SPAIN
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Previous and actual works
L-carnitine production with immobilized Escherichia coli cellsin continuous reactors
José María Obón, Juan Ramón Maiquez, Hans-Peter Kleber, Manuel Cánovas and José Luis Iborra
Enzyme and Microbial Technology, 21: 531-536, 1997
Role of betaine:CoA ligase (CaiC) in the activaction of betaines and the transfer of coenzyme A in Escherichia coli
V. Bernal, P. Arense, V. Blatz, M.A, Mandrand-Berthelot, M. Cánovas and J.L. Iborra
Journal of Applied Microbiology 105, (2008), 42-50
31 articles
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AIMS OF CONFERENCE
• Update the knowledge on bacterialcarnitine metabolism and the potentialindustrial application of its productionmethods:
- Bioprocess development.
- Strain optimization strategies.
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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Introduction
Methodology FactorsProduction
Metabolic Link
Models
Metabolic Flux Analysis
Pulse Experiments
Cofactor Engineering
CurrentWorks
Conclusions
Strains
Results
Reactors
CH3 H│ │
CH3 - N+ - CH2 – C – CH2 – COO-
│ │ CH3 OH
PRESENTATION OVERVIEW
FuturePerspectives
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Introduction
CH3 H│ │
CH3 - N+ - CH2 – C – CH2 – COO-
│ │ CH3 OH
CH3 OH│ │
CH3 - N+ - CH2 – C – CH2 – COO-
│ │ CH3 H
CH3 H│ │
CH3 - N+ - CH2 – C – CH2 – COO-
│ │ CH3 OHCH3
│ CH3 - N+ - CH2 – CH = CH – COO-
│CH3
Osmoprotectant
ESSENTIAL NUTRIENTDaily Necessities:
•10% biosynthesis (liver, kidney)•90% dietary supply
•100% essential in neonates
L-carnitine
Metabolism of fats
Applications: clinical & nutraceutical
ChemicalBiotechnological
Production Methods
Racemic mixture Residue: D-Car
EnantioselectiveSubstrates:D-Car/Crot
Enterobacteria(Escherichia, Proteus
& Salmonella)
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Introduction Metabolism of L-Carin Enterobacteria
Biotransformationmachinery: inducible
Crot y D-Carare residues of the chemical industry
Crot
L-Car
γ-BB
CrotonobetaineReductase
D-Car
γ-BB
D-Car
L-Car
Crot
CarnitineDehydratase
CarnitineRacemase
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Introduction
caiTABCDE caiFCRPCRP -10-10 FNR
L-carnitine metabolism in E. coli y Proteus sp.:Organization of the cai operon
CaiA Crot-CoA reductaseCaiB CoA transferaseCaiC Carnitine:CoA ligaseCaiD Enoyl-CoA hydratase
CaiT Membrane Transporter CaiE Unknown
CaiF Transcriptional Activatorof Carnitine Metabolism
fixABCX
Fix Crot Reduction
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Introduction Comparison of cai operon codified proteins inEscherichia coli and Proteus sp strains
Gene Protein length (aa) Function of gene product Homology (%)
E. coli Proteus spcaiT 504 504 Transport protein* 88
caiA 380 380 Crotonobetainyl-CoA reductase 93
caiB 405 406 Betainyl-CoA transferase 86
caiC 524 518 Betainyl-CoA ligase* 69
caiD 297 261 Crotonobetainyl-CoA hydratase 82
caiE 203 197 Unknown 76
caiF 131 130 Transcriptional regulador* 51
(*) In Proteus sp strains function postulated on the basis of sequence similarities.
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Introduction
caiTABCDE caiFCRPCRP -10-10 FNR
L-carnitine metabolism in Escherichia coli:Organization of the cai operon
fixABCX
L-Car
Crot
CaiFinactive
CaiFactive
+
H-NS
RpoS
CRP
FNR
+ + +--
CRP: cAMP Receptor Protein
RpoS: sigma subunitof RNApol
H-NS: histones
O2
FNR: transcriptional factorfor anaerobiosis
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Crot
L-CarL-Car
Crot Crot-CoA
L-Car-CoA
γ-BB-CoA
γ-BB
CaiACaiC
CaiD
CaiT
CaiB CaiB
CaiC
Crot
Oxygen/Fumarate:inhibitors
Elssner et al., (2001) Biochemistry 40: 11140; Cánovas et al.,(2003) Biotecnol. Bioeng., 84: 686.
Transport/activation
ATP
Expression/performance
NADH/NAD+
Biotransformation
CoA/AcCoA
Metabolism of L-Car in Escherichia coliIntroduction
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QGincrotin
GX
μmax
GL-carCrot
METHODOLOGYMethodology
Strains
Escherichia coli & Proteus sp. strains
Wild type:
E. coli O44K74E. coli LMG194
Proteus sp.
Genetically modified:E. coli BW25113
Overexpression (caiT, caiB, caiC, caiD)Deletion (caiB, caiC, aceA, aceK, iclR, pta, acs)
pBAD24AmpR
Castellar et al., (1998) J. Appl. Microbiol., 85: 883.
Castellar et al., (2001) Enz. Microb. Techn., 28: 785.
Bernal et al., (2007) Biotech. Lett., 29: 1549.
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Continuous Cell-Recycle Reactor(HIGH CELL DENSITY)
QGincrotin
GL-carCrot
GX
μmax
Continuous Stirred Tank Reactor(CHEMOSTAT)
GL-carCrot
METHODOLOGY
Limitations:
•Low cell density(< 1 g.L-1)
•D < μmax(E. coli: 0,6 h-1)
•Contamination risk
Methodology
Reactors
Batch Stirred Tank Reactor
•Growing Cells
•Resting CellsCrotonobetaine L(-)-carnitine
Obón et al., (1999) Appl. Microbiol. Biotechnol., 51: 760. Cánovas et al., (2002) Biotechnol. Bioengin., 77: 764.
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Factorsproduction
Results Effect of inducers
• The proper biotransformation isonly induced by crotonobetaine,D-carnitine or L-carnitine.
• Maximal biotransformationcapacity is induced by 5 mMcrotonobetaine in anaerobiosis.
• The molar yield for L-carnitineproduction can reach 50-60%.
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Oxygen (%)0.00 15.00 30.00 60.00
Con
c (m
M)
0
5
10
15
20
25
30
35
DC
W (g
·L-1)
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
D-Carnitine racemization: Oxygen effect
Escherichia coli K38pT7-5KE32
0% 15% 30% 60%
Multiple effects of oxygen:
•Inhibits γ–butyrobetaine production by CaiA•Increases growth yield•Represses the expression of carnitine metabolism
CH3 OH│ │
CH3 - N+ - CH2 – C – CH2 – COO-
│ │CH3 H
Substrate:D(+)-carnitine
Factorsproduction
Results
Cánovas et al., (2005) Biochem. Engin. J., 26: 145.
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Factorsproduction
Results
Inhibits γ-butyrobetaine production by CaiA
Crotonobetaine: Fumarate effect
Escherichia coliO44K74
Enhances cell growth
Complex media
μmax(h-1) qcarmax qcrotmax
Yield carn. (%)
Conv(%)
Control 0.224 0.008 0.189 6.9 40.02 g/L fumarate 0.431 0.343 0.471 43.0 48.5
Fumarate is reduced into succinate by anaerobically
respiring bacteria
CH3│
CH3 - N+ - CH2 – CH = CH – COO-
│CH3
Substrate:Crotonobetaine
Cánovas et al., (2005) Biochem. Engin. J., 26: 145.
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Factorsproduction
ResultsL-Carnitine transport systems in E. coli
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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Factorsproduction
Results Effect of permeabilizers on cell envelopeand outer membrane
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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Factorsproduction
Results Effect of different transport engineeringstrategies on L-carnitine production
Proteus sp.
E. coli O44K74
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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Factorsproduction
Results L-Carnitine productivities for continuoussystems with growing and resting cells
Strain Productivity(g L-1 h-1)
Molar yiedl(%)
Comment
E. coli O44K74 0.3 - Immobilized in polyacrylamide
E. coli O44K74 1.8 26 Immobilized in glass beads
E. coli O44K74 6.2-12 40 Cell recycle
E. coli pT7-5KE32
1.2 24 Cell recycle
E. coli pT7-5KE32
0.71 10 Immobilized in k-carrageenan
Proteus sp. 40.5 35-50 Cell recycleProteobacteria 5.4 90-95 Cell recycle
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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Physiological state of E. coli strains duringL-carnitine production
Results
A. High density cell-recycle reactor
B. Continuous stirred tank reactor
E. coli O44K74 Immobilized E. coliK38 pT7-5KE32
Dead cells
Viable cells
Depolarized cells
Dry cell weight
Factorsproduction
Bernal et al., (2007) Microbial Cell Factories, 6: 31.
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8 h
16 h
0,0
0,2
0,4
0,6
0,8
1,0
1 2 3 4 5 6 7 8
L-ca
rniti
ne p
rodu
ctiv
ity(g
·l-1·h
-1)
No of reuse cycles
REUSE OF CELLS:100% recovery of
biocatalytical capacity
Resting Cells Reuse: physiological state
Escherichia coliO44K74
Non re-energized Cells
Re-energized
cells
Factorsproduction
Results
Cánovas et al., (2007) Process Biochem., 42: 25.
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Factors affecting thebiotransformation
Strain improvementMetabolic Engineering
Cell physiologyLink of Central
& CarnitineMetabolisms
Mathematicalmodels
Experimentaldesign
PRESENTATION OVERVIEW
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PEP
Pyruvate
Acetyl-CoA
Isocitrate
Malate
Acetyl-P
Acetyl-AMP
Acetate
Lactate
Ethanol
ICDHICL
ACS
PTA
Formate
IclR
AceK
-
-
-
Succinate
OAA
PDH/PFL
Glyoxylate Shunt:Anabolism
CENTRAL METABOLISM IN Escherichia coli
Acetate Metabolism:ATP production
TCA in anaerobiosis:biosynthetic precursors
Metabolic link
Cánovas et al. , (2003) Biotechnol. Bioeng. 84, 686.
Results
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Time (h)0 24 48 72 96
ICD
H(m
U/m
g pr
ot)
0
50
100
150
ICL
(mU
/mg
prot
)
0
4
8
12
ACS
(mU
/mg
prot
)
0
50
100
150
200
PTA
and
PD
H (m
U/m
g pr
ot)
0
10
20
30
40
50A
B
Ace
tyl-C
oA a
nd H
S-C
oA (n
M)
15
30
45
60
75
90
NA
DH
/NA
D+ ra
tio
0,0
0,1
0,2
0,3
0,4
0,5
Time (h)
0 20 40 60 80
ATP
con
tent
(mM
)
0,0
0,2
0,4
0,6
0,8
Rat
e of
tran
spor
t (n
mol
/mg
prot
. min
)
2
4
6
8
10
12
14
A
B
ATP (fmol/cell)x104
0 2 4 6 8 10
Rat
e of
tran
spor
t(n
mol
/mg.
prot
.min
)
0
4
8
12
16
C
Resting Cells: Metabolic Link
ATP linked to transport rate
Incr. Glyoxylate Shunt
Adaptation to microaerobiosis and starvation
Metabolic link
Results
Cánovas et al. , (2003) Biotechnol. Bioeng. 84, 686.
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Time (h)0 20 40 60 80A
TP (μ
Μ) a
nd B
iom
ass
(A60
0)
0
10
20
30
ICD
H/IC
L ra
tio
15
20
25
30
35
40
L-ca
rniti
ne, c
roto
nobe
tain
e an
d γ
-but
yrob
etai
ne (m
M)
0
10
20
30
40
50
60A
B
Low γ-butyrobetaine
Continuous Cultures: Metabolic Link
Escherichia coliO44K74
ATP levels decreased.Regulated ICDH/ICL ratio
Metabolic link
Results
Cánovas et al. , (2003) Biotechnol. Bioeng. 84, 686.
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0
200
400
600
800
1000
0
1
23
45
67
01
23
4Spec
ific
activ
ity (m
U/m
g pr
otei
n)
Enzy
mes
Reactor type
1. CSTR 2. Membrane 3. Batch with growing cells4. Batch with resting cells
Higher expressionof acetate metabolism
Link between Central & Secondary Metabolisms
Escherichia coliO44K74
Alteration of ICDH/ICL ratio
Metabolic link: COFACTORS(ATP and acetyl-
CoA/CoA)
Metabolic link
Results
12
3
4
5
6
Cánovas et al. , (2003) Biotechnol. Bioeng. 84, 686.
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V2, K2
crotonobetaine
d CRd T
A= ( ) ( )CR V
K CRC VK C
B−
++
+⎛⎝⎜
⎞⎠⎟
+1
1
2
2 ( )CR V
CR K−
+⎛⎝⎜
⎞⎠⎟
3
3
d Cd T
A= ( ) ( )C V
K CCR VK CR
−+
++
⎛⎝⎜
2
2
1
1
⎛⎝⎜
( )d Bud T
BCR VK CR
=+
⎛⎝⎜
3
3
⎛⎝⎜
V1, K1
L-carnitine A
OKA2e +
−Α = KA1 KA3
( ))( X
L(-)-carnitine dehydratase CaiD
A
V3, K3
γ-butyrobetaíneB
OKB2e
−B = KB1
( )X
Crotonobetaíne reductase CaiA
B
With fumarateF
KB2e−
B = KB1
( )X
Cánovas et al., (2002). Biotechnol. Bioeng. 77. 764-775.
Model of enzymatic activityModels
Results
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Macrokinetics of the high cell density reactor
G)Q·(GYμ·XV·
dtdGV· 0
xg
−+−=
·Xμ·XμdtdXV· emax −=
CR)Q·(CR·Vrdt
dCRV· 0CR −+=
LCKmVLC
CRKmVCRr
LCext
max
CRext
maxCR +
⋅+
+⋅
−=
Q·LC·Vrdt
dLCV· LC −=
CRKmVCR
LCKmVLCr
CRext
max
LCext
maxLC +
⋅+
+⋅
−=
X52
X51
X50 QGincrotin
GX
μmax
GL-car
Crot
X4
X1
X53
X3
X2
X1
High density cell recycle membrane
reactor
XX4
Cánovas et al., (2002). Biotechnol. Bioeng. 77, 764-775.
Models
Results
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Comparison between predicted and experimental L-Carnitine production rate by E .coli in a high cell density–recycle reactorModels
Results
Alvarez-Vazques et al., (2002). Biotechnol. Bioeng. 77. 895-905
• Dilution rate (Q)
• Initial crotonobetaine concentration
(Crin)
• Carnitine dehydratase
activity
CRITICAL PARAMETERS FOR
MAXIMIZING CARNITINE
PRODUCTION
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S-System Model: Mathematical frameworkResults
Models
A. Sevilla et al., (2005) Biotechonol. Prog. 21, 329-337.
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time (h)
0 2 4 6
Nor
mal
ized
Con
cent
ratio
n
1.0
1.2
1.4
1.6
1.8
2.0
LCext
CRext
CRint
LCint
LCCoA
CRCoA
time (h)
0 2 4 6
Nor
mal
ized
con
cent
ratio
n
0.8
1.0
1.2
1.4LCout
CRout
CRin
LCin
LCCoA
CRCoA
time (h)
0 2 4 6
Nor
mal
ized
con
cent
ratio
n
0.94
0.96
0.98
1.00
1.02
1.04
1.06
1.08
LCext
CRout
LCCoA
CRCoA
CRint
LCin
time (h)
0 2 4 6
Nor
mal
ized
con
cent
ratio
n
0.94
0.96
0.98
1.00
1.02
1.04
1.06
LCout
CRext
CRint
LCin
LCCoA
CRCoA
Overexpression of CaiTOverexpression of CaiBand CaiT
Overexpression of CaiB Overexpression of CaiC
S-System Model: Perturbation StudiesResults
Models
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Optimal Solution
III
IIIIV
S-System Model: Stepwise optimization.IOM approach
Results
Models
Optimum normalized values(Xi)opt/(Xi)basal
Bioreactor setting
parameters
Solutions
0 I II III IV V
X16 (Q) 2 2 2 2 2 2
X18(CBinlet) 2 2 2 2 2 2
Enzyme activity sets
Enzyme activities
0 1 2 3 4 5
X48 (CaiC) 1 5 5 5 5 5
X45 (CaiT) 1 1 5 5 5 5
X47 (CaiB) 1 1 1 5 5 5
X46 (ProU ) 1 1 1 1 0.5 0.5
X49 (CaiD) 1 1 1 1 1 5
Carnitine Production
Rate2.2 8.4 15.7 25.7 27.8 28.6
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TCAGLYCOLYSIS
PROTEIN SYNTHESISLlPIDS SYNTHESIS
ARN & ADN SYNTHESIS
PENTOSEPATHWAY
GLUCOSEUPTAKE
ELECTRONTRANSFER
Chassagnole, C., et al., (2002) Biotechnol. Bioeng. 79, 53-73
Metabolic FluxAnalysis
Results
Central metabolism in aerobic conditions
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Metabolic FluxAnalysis
Results
Metabolic Flux Analysis
A. Sevilla, et al., (2005) Metabolic Engineering, 7, 401-425
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Dynamic Evolutionof the metabolism of Escherichia coli under production conditions
Strategy METABOLIC PULSING
Short Time Window(ms - s)
Long time window(min - h)
Intracellular Metabolites
metabolic perturbationenzyme levels unnaffected
Intracellular MetabolitesExtracelullar Metabolites
EnzymesTranscription Factors
genetic perturbationenzyme levels affected
Pulseexperiments
Results
Cánovas et al. (2007) In silico Biology, 7, S3-S16
Metabolic Engineering Analysis
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QGincrotin
GX
μmax
GL-carCrot
High Cell Density
Continuous Reactor
MethodologyRapid
PulsingRapid
Sampling
t (min)
EnzymesMetabolitesCoenzymes
Cánovas et al. (2007) In silico Biology, 7, S3-S16
Pulseexperiments
ResultsDynamic pulsing of continuous steady state
E. coli cultures in production conditions
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0.0
0.5
1.0
1.5
2.0
2.5
3.0
PTA
norm
aliz
ed s
peci
fic a
ctiv
ity
0
1
2
3
4
5
ACS
norm
aliz
ed s
peci
fic a
ctiv
ity
0
20
40
60
80
100
120
140
Time (min)-20 0 20 40 60 80 100 120 140
ATP
(μM
)
10
20
30
40
50
60
CH
R n
orm
aliz
ed s
peci
fic a
ctiv
ity
0.0
0.2
0.4
0.6
0.8
1.0
L-ca
rniti
ne (m
M)
6
7
8
9
10
11
A
B
Acetate increased fast after the pulse
L-Car production increased despite lower
CDH
ATP pool increased 3-fold !!
L-Car production correlated with cellular ATP
Ace
tate
(g/L
)
Pulseexperiments
Results
Glicerol Pulse
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Improvement ofL-carnitine production
Genetically modifiedEscherichia coli strains
Strategy: COFACTOR
ENGINEERING
SecondaryMetabolism
CentralMetabolism
Overexpression of carnitine metabolism
enzymes: CaiB, CaiT & CaiC
Deletion of acetyl-CoA metabolism enzymes :
Acetate metabolism& glyoxilate shunt
Metabolic Engineering in Escherichia coliCofactor Engineering
Results
Bernal et al., (2007) J. Biotechnol. 132: 110-117.
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Effect of gene overexpression on theproduction of L-carnitine
Batch Anaerobic system
+ Fumarate 2 g.L-1
Results
3-4-fold
50-fold
Bernal et al., (2007), J. Biotechnol. 132, 110-117 Cánovas et al., (2007) In Silico Biol.,7 (S3-S16)
Cofactor Engineering
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aceAisocitrate liase
aceK ICDH phosphatase/kinase
iclR glyoxylate shunt repressor
acs acetyl-CoA sinthetase
ptaphosphotransacetylase
KO-COLLECTIONProf. Mori, Keio Univer. (Japan)
Cofactor Engineering
ResultsAcetyl-CoA metabolism single-gene deletion
KO Mutants
Bernal et al., J. Biotech., (2007), 132, 110-117.
Glyoxylateshunt
Acetatemetabolism
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Results Effect of the deletion of pta, acs, iclR, aceK and aceAon the production of L-carnitine by E. coli BW25113
pta: phosphotransferase
ENZYMES OF CENTRAL
METABOLISM
acs: AcetylCoA sinthetase
aceA: Isocitrate liase
aceK: Isocitrate deshidrogenase
iclR: Inhibition aceA
Batch anaerobic systems
Bernal et al., J. Biotech., (2007), 132, 110-117.
Without fumarateWith 2 g.L-1 fumarate
Cofactor Engineering
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CentralMetabolism
SecondaryMetabolism
Alteration of enzymes in the metabolism of acetyl‐CoA
Alteration of carnitine metabolism enzymes:
• Deletion of aceA, aceK in a double mutant• Overexpression of pta
• Overexpression of caiF by changing the promotor• Overexpression of caiTBDC by changing the promotor• Deletion of caiA in a simple and double mutant with overexpression of caiTBDC
These mutants were made by Datsenko and Wanner method
AEROBIC CONDITIONS
Time (h)
0 10 20 30 40 50
[L-C
arni
tine]
mM
0
5
10
15
20
25
E. coli BW25113 (Wt)E. coli BW25113 p37-ΔpcaiTBCDE. coli BW25113 p8-ΔpcaiF
The new mutants with theconstitutive promotor produceL‐carnitine in aerobic conditions
Central Metabolism
SecondaryMetabolism
Alteration of enzymes in the metabolism of acetyl‐CoA
Alteration of carnitine metabolism enzymes:
• Deletion of aceA, aceK in a double mutant• Overexpression of pta
• Overexpression of caiF by changing the promotor• Overexpression of caiTBDC by changing the promotor• Deletion of caiA in a simple and double mutant with overexpression of caiTBDC
These mutants were made by Datsenko and Wanner method
Current works
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CARNITINE PRODUCTIONCONCLUSIONS
• Characterize the phenotype of the new mutants.
• Analyze the effects of aerobic biotransformationsconditions.
• Determine glucose repression mechanisms.
• Integration of metabolic, genetic & signaling levels.
• Integration of central & secondary pathways.
OPTIMIZATION OF BIOPROCESS
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CONCLUSIONS
• A continuous feed-back between in silico and in vivoexperimentation is necessary for the application ofMetabolic Engineering and System Biology approachesto living systems.
• The construction of meaningful models stronglydepends on the completeness and goodness of the dataavailable.
GENERAL
• Although biotransformation processes are designed ona case by case basis, the experimental and theoreticalmethologies of Bioprocess and Metabolic Engineeringare applicable to the development de any bioprocessinvolving whole cells.
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D. Martínez C. SánchezT. De Diego A. ManjónM. Cánovas
P. ArenseC. BernalA. Sevilla
S. Revilla
J.L. Iborra
Biotechnology Group E-060-04
Dpt. Biochemistry & Molecular Biology “B” & ImmunologyFaculty of Chemistry
ACKNOWLEDGEMENTS
M. Martínez
S. Fructuoso S. Castaño J.M. Pastor M. Ferrari
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MCYT (Ref.: BIO2005-08898-C02-01).FUNDACIÓN SENECA-CARM (Ref.: 2928/PI/05).BIOCARM (Ref.: BIO2005/01-6468).
University of LeipzigHans-Peter Kleber
EXTERNAL PARTNERS
FINANCIAL SUPPORT
Universidad de La LagunaN.V. Torres
F. Álvarez-VázquezZ. Díaz
INSA-LyonM.A. Mandrand-Berthelot
University of StuttgartM. Reuss
J.W SchmidK. Mauch
ACKNOWLEDGEMENTS
University of Roma/Sigma TauMenotti Calvani
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COLLABORATORS
Ph. D.J.R. MaíquezT. Torroglosa
ProfessorsJ.M. ObónM.R. CastellarT. De DiegoC. Olivares
M. in ScienceB. BuendíaA. MarínG. EspinosaJ.L. RamírezM. GonzálezR. LealR. TeruelB. MasdemontV. García
Erasmus StudentsP. KellerS. ReimersV. Blatz
ACKNOWLEDGEMENTS
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THANK YOU VERY MUCH FOR
YOUR ATTENTION
Optimization of L-carnitine production by enterobacteria
ACKNOWLEDGEMENTS
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ORAL COMMUNICATION & POSTERS in BIOTEC’08
● “Avoiding catabolite repressIon using System Biology”• “Transcriptional regulation of the glyoxylate shunt in E.coli”
• “The fundamental role of acetate in bioprocessoptimization in E. coli”
• “Salt stress effects on the central and carnitineproduction metabolisms of E. coli”
• “Engineering E. coli to improve L-carnitine production”
• “In silico model of the mitochondrial metabolism incardiac cell undergoing metabolic alteration in carnitinesystem”
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Optimization of L-carnitine production by enterobacteria
J.L. Iborra, M.Cánovas, A. Sevilla & V. Bernal
Department of Biochemistry &Molecular Biology “B” & Immunology
Faculty of ChemistryUniversity of Murcia
SPAIN