BIOTEC'2004OVIEDO 19-23 DE JULIO

Design of metabolic engineering strategies Design of metabolic engineering strategies for maximizing L(for maximizing L(--))--carnitinecarnitine production by production by

Escherichia coliEscherichia coli. Integration of the . Integration of the metabolic and bioreactor levelsmetabolic and bioreactor levels

M. Cánovas, A. Sevilla, V. Bernal, N. Torres y J.L. Iborra

Dpto. Bioquímica y Biología Molecular B e Inmunología.Facultad de Química. Universidad de Murcia.

L(-)-CARNITINE INTEREST

CH3 H

CH3 - N+ - CH2 – C – CH2 – COO-

CH3 O H

Function: Fatty acids MetabolismUse: Medicine

Nutraceutic

Energetic Drinks

Bacteria and yeast growth

BIOTRANSFORMATIOND-carnitine

Crotonobetaine L-carnitineEnterobacteria

Intracellular

L-carnitine

Crotonobetaine

CaiT

IM

PepG layerPhospholípids of OM

LPS de la OM

Porins Membrane

proteíns

OM

Transporter of theTrimethylamonium

compounds

CH3│

CH3 - N+ - CH2 – CH ═ CH – COO-

│CH3

CH3│

CH3 - N+ - CH2 – CH - CH2 – COO-

│ I CH3 OH

CH3│

CH3 - N+ - CH2 – CH - CH2 – COO-

│ I CH3 OH CH3│

CH3 - N+ - CH2 – CH ═ CH – COO-

│CH3

γ-butyrobetaine

Carnitinedehydratase

Activity

Crotonobetainereductase

Activity

PLSac of the OM

Crotonobetaine

ProU

Cai D

Cai A

Extracellular o Reactor Bulk Líquid

L-carnitine

X

E.coli L(-)-CARNITINEGENETIC REGULATION

inactiveCai F Protein

activeCai F Protein

-10-10

PromotercaiF

Promotercai/fix

L(-)-carnitine

crotonobetaine

+5’

fixABCX

caiTABCDE

caiF- H-NS

RpoS-

CRP

CRPCRP

+ +

FNR

FNR

+

T7 RNApolymerase

pGP1-2

KanR

λ pL

pT7-5

T7 ø10AmpR

cai F

cai DE

CaiT CarrierCaiA Crotonobetaine reductaseCaiB CoA-transferaseCaiC Crotonobetaine-CoA ligaseCaiD Enoyl-CoA hydrataseCaiE e- carrier/transferase

Groups of Profs. Mandrand-Berthelot andH-P. Kleber

J. Bacteriol., 178, 1248-1257. (1996).

HIGH CELL-DENSITY REACTORSHIGH CELL-DENSITY REACTORS

Advantages

High cell concentration.

Possibility of growing/resting cycles

Possible problems

Clogging of the membranes

Cell death

Medio

Efluente

O2 CO2 N2

pH y pO2

Air N2

SensorsStirring

Substrate

L-carnitine

MEMBRANE REACTORS FOR CELL RETENTION

Membranes ofmicrofiltration

Tangencial filtration

Software Controlled

IMMOBILIZATION WITH CELL GROWTH

0

1

2

3

4

5

6

7

0,5 1 1,5 2

Dilution rate (h-1)

Prod

uctiv

ityg

L-1

h-1

Bio

mas

sgL

-1

Time (h)0 50 100 150 200 250

0

5

10

15

20

25

30

0,2 h-1

1,0 h-1

2,0 h-1

Maximum Productivity : 6,2 g L-1 h-1

Conversión: 40%

MEMBRANEREACTOR

0,5 h-1

( 1 )

( 2 )

Time (h)

L(-

)-ca

rniti

nem

M

0 25 50 75 1000

2

4

6

8

10

Appl. Microb. BioTechnol. 51, 760-764. 1999.

Acetyl-AMP

Acetyl-P

Acetate

Isocytrate

α-cetoglutarate

Succinate

Fumarate

Malate

Oxalacetate

Pyruvate

PEP

FormiateEthanol

Lactate

Glyoxilate ICL

ICDH

MDH

PDH

PK

ACS ACS

PTA ACK

MS

Biosynthesis

PEPCXPEPCK

Fatty acidsGlycerol

NADH

NADPH

FADH2

ATPO2

CTe--

NAD+

2NAD+

ADP

ATP

CO2

H2

PolysacharidesMonosacharides

CO2

KREBS CYCLE

ANAEROBIC

METABOLISMGLYOXILATE CYCLE

ANAPLERÓTICS REACTIÓNS

H2OREDUCING POWER

PFL

Acetyl-CoA

E. coliCENTRAL

METABOLISM

ATPPpi

- EMBO J. 10, 675-679. (1991). -http://biocyc.org-http://ecocyc.PangeaSystems.com/ecocyc/ecocyc. (2003)

- J. Bacteriol. 182: 4173-4179. (2000)

LípidsATP

NADH

ATP

ATP

ATP

L(-)-carnitine

Crotonobetaine Crotonobetaine

L(-)-carnitine

γ-butyrobetaine γ-butyrobetaine

L(-)-carnitinyl-CoA

Crotonobetainyl-CoA

Crotonobetainyl-CoA

γ-butyrobetainyl-CoA

Tioesterase

L(-)-carnitinedehydratase

Ligase

Ligasa

Crotonobetainereductase

Tioesterase

ATP

ATP

ATP

ADP

ADP

ADP

γ-butirobetaíne

Crotonobetaine

CoA transferase

CoA transferaseCrotonobetaine

L(-)-carnitineH2O

A: Enoyl-CoAhidratasa

H2

CoA-SH

A

CoA-SH

INTRACELLULAR

EXTRACELLULAR

METABOLISM OF CARNITINE IN

E. coli

Leipzig groupBiochemistry. 39, 10761-10769. (2000). Biochemistry. 40, 11140-11148. (2001).

Murcia group.J. Basic Microbiol. 43. 259-268.(2003).Proyecto de InvestigaciónProyecto de Investigación

CENTRAL METABOLISM ENZYMES EVOLUTION

0

200

400

600

800

1000

01

23

45

67

01

23

4Spec

ífic

activ

ity (m

U/m

g pr

otei

na)

Enzy

mes

Reactor type

1. CSTR 2. Membrane 3. Batch. cell growth4. Batch with resting cells

Time (h)0 20 40 60 80A

TP ( µ

Μ)

and

Bio

mas

s (A

600)

0

10

20

30

ICD

H/IC

L ra

tio

15

20

25

30

35

40ATP A600 CDH/ICL

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

ICL and ACS showed the

highest levels

Glyoxilate Cycleand production ofthe Ac-CoA

ATP/ intermediaries

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

Proyecto de InvestigaciónProyecto de Investigación

ProU is also involved in theprocess

J. Basic Microbiol. 43. 259-268. 2003.

Intracellular

L-carnitine

Crotonobetaine

CaiT

IM

PepG layerPhospholípids of the

OM

LPS of the OM

Porins proteins ofMembrane

ME

Trimethylammoniumcompounds carrier

CH3│

CH3 - N+ - CH2 – CH ═ CH – COO-

│CH3

CH3│

CH3 - N+ - CH2 – CH - CH2 – COO-

│ I CH3 OH

CH3│

CH3 - N+ - CH2 – CH - CH2 – COO-

│ I CH3 OH CH3│

CH3 - N+ - CH2 – CH ═ CH – COO-

│CH3

γ-butyrobetaine

Activitycarnitine

dehydratase

ActivityCrotonobetaine

reductase

PLSac of the OM

Crotonobetaine

ProU

Cai D

Cai A

Extracellular or Reactor bulk Líquid

L-carnitine

L-carnitine orcrotonobetaine

L-carnitine orcrotonobetaine

0 50 100 150 2000

10

20

30

Tiempo (s)

Tran

spor

te L

(-)c

arni

tina

(nm

ol/m

g pr

otei

na)

ATP

ADP

L(-)-carnitine

Crotonobetaine Crotonobetaine

L(-)-carnitine

γ-butyrobetaineγ-butyrobetaíne

L(-)-carnitinyl-CoA

Crotonobetainil-CoA

Crotonobetainyl-CoA

γ-butyrobetainyl-CoA

Tioesterase

L(-)-carnitinedeshidratase

Ligase

Ligase

Crotonobetainereductase

Tioesterase

ATP

ATP

ATP

ADP

ADP

ADP

γ-butyrobetaine

Crotonobetaine

CoA transferase

CoA transferaseCrotonobetaíne

L(-)-carnitineH2O

A: Enoyl-CoAhydratase

H2

CoA-SH

A

CoA-SH

INTRACELLULAR

AcCoA/CoA

EXTRACELLULAR

LINK BETWEEN THE PRIMARY AND SECONDARY METABOLISM.

CARNITINE METABOLISMIN E. coli

ATP

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

crotonobetainyl-CoA

CARNITINE METABOLISM

L(-)-carnitinyl-CoA

γ-butyrobetainyl-CoA

CaiA

CaiD

γ-butyrobetainyl-CoA

L(-)-carnitine

crotonobetaine crotonobetaine

L(-)-carnitine

γ−butyrobetaíne

γ-butyrobetaine

INTRACELLULAREXTRACELLULAR

CoA

ATP

ATP

ADP

ADP

CaiC

CaiB CaiB

CaiT

CaiT

γ-butyrobetaine

CaiC

CaiBCoA

crotonobetainyl-CoA

L(-)-carnitinyl-CoA

CaiD

γ-butyrobetaineCaiC

HH2OO HH2OO

CoA

CoA CaiB

ATP?

ATP?ATP?

CoA

γ-butyrobetainyl-CoA

Crotonobetainyl-CoA

L-carnitinyl-CoA

γ-butyrobetainyl-CoA

CaiD

CaiA

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

crotonobetainyl-CoA

CARNITINE METABOLISM

L(-)-carnitinyl-CoA

γ-butyrobetainyl-CoA

CaiA

CaiD

γ-butyrobetainyl-CoA

L(-)-carnitine

crotonobetaine crotonobetaine

L(-)-carnitine

INTRACELLULAREXTRACELLULAR

CoA

ATP

ATP

ADP

ADP

CaiB CaiB

CaiT

CaiT

crotonobetainyl-CoA

L(-)-carnitinyl-CoA

CaiDHH2OO HH2OO

Crotonobetainyl-CoA

L-carnitinyl-CoA

CaiDProU

ATP

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

H2O

NADH

NADPH

FADH2

ETC

Acetyl-CoA

Acetyl-AMP

Acetyl-P

Acetate

Isocitrate

α-ketoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Pyruvate

PEP

Formate

Ethanol

Lactate

GlyoxylateICL

ICDH

CSMDH

PDH

PK

ACS ACS

PTA ACK

MS

Biosynthesis

PEPCXPEPCK

Fatty AcidsGlycerol

ATPO2

NAD+

2NAD+

ADP

ATP

ATP

ATP

CO2

H2

PolysacharydesMonosacharydes

CO2

PFL

CoA

ADP

NADH

2NADHADPATP

Ppi

CoA

Citrate

Glycerol-3P

ATPADP Fats

NAD+

NADHADP

ATP

G3P

NADH

NAD+

Pi

AMP

H2O

NADH

NADPH

FADH2

ETC

Acetyl-CoA

Acetyl-AMP

Acetyl-P

Acetate

Isocitrate

α-ketoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Pyruvate

PEP

Formate

Ethanol

Lactate

GlyoxylateICL

ICDH

CSMDH

PDH

PK

ACS ACS

PTA ACK

MS

Biosynthesis

PEPCXPEPCK

Fatty AcidsGlycerol

ATPO2

NAD+

2NAD+

ADP

ATP

ATP

ATP

CO2

H2

PolysacharydesMonosacharydes

CO2

PFL

CoA

ADP

NADH

2NADHADPATP

Ppi

CoA

Citrate

Glycerol-3P

ATPADP Fats

NAD+

NADHADP

ATP

G3P

NADH

NAD+

Pi

AMPAcetyl-CoA

Acetyl-AMP

Acetyl-P

Acetate

Isocitrate

α-ketoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Pyruvate

PEP

Formate

Ethanol

Lactate

GlyoxylateICL

ICDH

CSMDH

PDH

PK

ACS ACS

PTA ACK

MS

Biosynthesis

PEPCXPEPCK

Fatty AcidsGlycerol

ATPO2

NAD+

2NAD+

ADP

ATP

ATP

ATP

CO2

H2

PolysacharydesMonosacharydes

CO2

PFL

CoA

ADP

NADH

2NADHADPATP

Ppi

CoA

Citrate

Glycerol-3P

ATPADP Fats

NAD+

NADHADP

ATP

G3P

NADH

NAD+

Pi

AMP

L(-)-carnitine

crotobetaine crotonobetaine

L(-)-carnitine

γ−butyrobetaine

γ-butyrobetaine

L(-)-carnitinyl-CoA

crotonobetainyl-CoA

γ-butyrobetainyl-CoA

INTRACELLULAREXTRACELULAR

CoA

CaiA

ATP

ATP

ADP

ADP

CaiC

CaiDCaiB CaiB

CaiT

CaiT

γ-butyrobetaine γ-butyrobetainyl-CoA

CaiC

CaiBCoA

crotonobetainyl-CoA

L(-)-carnitinyl-CoA

CaiD

γ-butyrobetaine

CaiC

HH2OOHH2OO

CoA

CoA CaiB

ATP?

ATP?

ATP?

CoA

L(-)-carnitine

crotobetaine crotonobetaine

L(-)-carnitine

γ−butyrobetaine

γ-butyrobetaine

L(-)-carnitinyl-CoA

crotonobetainyl-CoA

γ-butyrobetainyl-CoA

INTRACELLULAREXTRACELULAR

CoA

CaiA

ATP

ATP

ADP

ADP

CaiC

CaiDCaiB CaiB

CaiTCaiT

CaiTCaiT

γ-butyrobetaine γ-butyrobetainyl-CoA

CaiC

CaiBCoA

crotonobetainyl-CoA

L(-)-carnitinyl-CoA

CaiD

γ-butyrobetaine

CaiC

HH2OOHH2OO

CoA

CoA CaiB

ATP?

ATP?

ATP?

CoA

Glycerol

Glycerol

PentosePhosphate

shunt

ATP

AcCoA/

CoA

ProUProP

Biosynthesis

aminoacids

Fatty acidsAminoacids

LipidsNucleic acids

ATPATP

FAD

NAD+

NADP++

Sugars

Biosynthesis ofproteins

Fumarate

SO4-2

NH4+

Acetate

Lactate

Formate

Ethanol

E. coliMETABOLISM

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

The controllingenzymes are:

ACS, ICL, PDH

Xµµ XTX

ex −==d

r d

µ (Go) =( )

µ γmax G

K Gg i

+g

Oµ (O) = ( )K O+o

CELLULAR GROWTH MODEL

( )OKioe +

−

= f(O) =Kg Kgo1 Kgo1

GoKige +

−

γi = f(Go) = Ki1 Ki2( )

gxgY

µ X-TG ==

ddr

µ = f(G,O) = µ (Go) (1 + µ(O))

Biom

ass(A600)

Gly

cero

l(m

M)

Time (h)

0 25 50 75 1000

25

50

75

100

0

2

4

6

8

10

V2, K2

crotonobetaine

MODELO ACTIVIDADES ENZIMÁTICAS

V1, K1

L-carnitine A

OKA2e +

−Α = KA1 KA3

( ))( X

L(-)-carnitine dehydrataseCaiD

A

V3, K3

γ-butyrobetaíneB

OKB2e

−B = KB1

( )X

Crotonobetaíne reductaseCaiA

B

With fumarateF

KB2e−

B = KB1( )

X

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

⎛⎝⎜

Biotechnol. Bioeng. 77. 764-775, (2002a)

EXPERIMENTAL AND SIMULATED DATA Glycerol 75 mM, Oxygen variable

Time (h)

Gly

cero

l(m

M)

Biom

asa (A600)

Time (h)

Gly

cero

l(m

M)

Time (h)

Gly

cero

l(m

M)

Biom

ass(A600)

Time (h)

Gly

cero

l(m

M)

Biom

ass(A

600)

0 5 10 15 20 250

25

50

75

100

0

2

4

6

8

10

0 5 10 15 20 250

25

50

75

100

0

2

4

6

8

10

0 5 10 15 20 250

25

50

75

100

Biom

asa (A600)

0

2

4

6

8

10

0 25 50 75 1000

25

50

75

100

0

2

4

6

8

10

0 % 15 %

30 % 60 %

( )Vd CRd T r V Q CR CRcr in= + −

Vd Cd T

r V Q Cc= −

V d Bud T

r V QBubut= −

crotonobetaíne

L(-)-carnitine

γ-butyrobetaíne( )V d G

d TV X

YxgQ G G= − + −

µin

Glycerol

MODEL AND RESULTS FROM THE MEMBRANE REACTOR

-1

0,0 0,5 1,0 1,5 2,0 2,50

10

20

30

Dilution rate ( h -1 )

Bio

mas

s(g

L)

µe=0,15 h-1

Biotechnol. Bioeng. 77. 764-775, (2002a)

0.0 0.5 1.0 1.5 2.0 2.50

10

20

30

40

50

0,0 0,5 1,0 1,5 2,0 2,50

10

20

30

40

50

Con

vers

ión

(%)

Dilution rate ( h -1 )

0.0

2.5

5.0

7.5

0,0

2,5

5,0

7,5 Productivity(g

L-1

h-1)

Stephanopoulos and cols. -Science, 252, 1675-1681. (1991).-Metabolic Engineering. Principles and Methodologies. Academic. Press. (1998).

¿How the metabolic pathways can be optimized to produce higher levels of L-carnitine?.

¿How the whole process can be optimized to produce higher levels of L-carnitine?

METABOLICENGINEERING

-Metabolic Flux Analysis

-Biochemical System Theory

CARNITINE METABOLISM IN E. coli

CRext CRint CRCoA

LCint LCCoALCext

ATP

ADP+ Pi

CaiB CaiDCaiT

ProU ATPCoA

CaiCATPCoA

CaiC

Biotechnol. Bioeng. Doi:10. 1002/bit. 10822 (2003).

PREVIOUS KNOWLEDGE

SystemsBatchContinuous

System OptimizationNeed to find the CONTROL POINTS

PCT/ST-21/BS0P000341 y P200301217Appl. Microbiol. Biotechnol. 51, 760-764 (1999).

INTEGRATION

Microkynetics ofbiotransformation of carnitine

Macrokinetics of a highdensity cell reactor

Microkynetics ofCentral Metabolism

MICROKINETICS OF THEL(-)-CARNITINE

PRODUCTIÓN PROCESS

Types of kinetics

ii XkV ⋅=

j

jmaxi XKm

XVV

+⋅

=

P

P

S

S

P

Pmaxr

S

Smaxf

i

KX

KX1

KXV

KX·V

V++

−=

• Lineal

• Michaelis-Menten

• Michaelis-Mentenreversible

Comp. Appl. Biotechnol. CAB 9 (In press). (2004).

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 −=

X52

X51

X50 QGincrotin

GX

µmax

GL-car

Crot

X4

X1

X53

X3

X2

X1

XReactor de alta

densidad celular

X4

CRKmVCR

LCKmVLCr

CRext

max

LCext

maxLC +

⋅+

+⋅

−=Biotechnol. Bioeng. 77. 764-775. (2002a)

Biotechnol. Bioeng. 80. 794-805. (2002b)

Acetyl-AMP

Acetyl-P

Acetate

Isocitrato

α-cetoglutarate

Succinate

Fumarato

Malate

Oxalacetate

Pyruvate

PEP

FormiateEthanol

Lactate

Glyoxilate ICL

ICDH

MDH

PDH

PK

ACS ACS

PTA ACK

MS

Biosynthesis

PEPCXPEPCK

LípidsGlycerol

NADH

NADPH

FADH2

ATPO2

H2O

CTe--

NAD+

2NAD+

ADP

ATP

ATPCO2

H2

PolysacharidesMonosacharides

CO2

REDUCINGPOWER

PFL

Acetyl-CoA

E. coliCentral

Metabolism

ATPPpi

NADH

Fatty acidsATP

NADH

ATP

ATP

S-SYSTEMMODEL

∏∏+

=

+

=

−=mn

j

hji

mn

j

gjii

jiji XXX11

·,, βα

FOR i = 1, 2, …, n.

ATP

S-SYSTEM MODEL

0

2

4

6

8

12

34

56

78

12

34

56

78

|S(Xi,α i

)|

α i

Xi

αi

1 2 3 4 5 6 7 8

Σ(S(Xi, αi))

-4

-2

0

2

4

6

8

10

12

B

Xi

1 2 3 4 5 6 7 8

Σ(S(Xi, αi))

-2

0

2

4

6

8

10

12

C

∏∏+

=

+

=

−=mn

j

hji

mn

j

gjii

jiji XXX11

·,, βα

FOR i = 1, 2, …, n. X1

' α1X50g1.50⋅ X51

g1.51⋅ β1X1h1.1⋅ X4

h1.4⋅ X50h1.50⋅ X53

h1.53⋅ X54h1.54⋅−=

X2' α2X3

g2.3⋅ X4g2.4⋅ X6

g2.6⋅ X45g2.45⋅ X50

g2.50⋅ X52g2.52⋅ β2X2

h2.2⋅ X4h2.4⋅ X22

h2.22⋅ X45h2.45⋅ X46

h2.46⋅ X50h2.50⋅−=

X3' α3X2

g3.2⋅ X4g3.4X5

g3.5⋅ X45g3.45⋅ β3X3

h3.3⋅ X4h3.4⋅ X45

h3.45⋅ X50h3.50⋅−=

X4' α4X1

g4.1⋅ X4g4.4⋅ X53

g4.53⋅ β4X4h4.4⋅−=

X5' α5X3

g5.3⋅ X7g5.7⋅ X45

g5.45⋅ X47g5.47⋅ β5X5

h5.5⋅ X45h5.45⋅ X47

h5.47⋅−=

X6' α6X2

g6.2⋅ X8g6.8⋅ X45

g6.45⋅ X46g6.46⋅ X47

g6.47⋅ β6X3h6.3⋅ X6

h6.6⋅ X21h6.21⋅ X22

h6.22⋅ X45h6.45⋅ X47

h6.47⋅ X48h6.4⋅−=

X7' α7X5

g7.5⋅ X7g7.7⋅ X8

g7.8⋅ X47g7.47⋅ X49

g7.49⋅ β7X7h7.7⋅ X8

h7.8⋅ X47h7.47⋅ X49

h7.49⋅−=

X8' α8X6

g8.6⋅ X7g8.7⋅ X8

g8.8⋅ X47g8.47⋅ X48

g8.48⋅ X49g8.49⋅ β8X7

h8.7⋅ X8h8.8⋅ X47

h8.47⋅ X49h8.49⋅−=

Perturbation studies.– Glycerol in (X1).– Crotonobetaine out

(X2).– Cell Concentration (X4).– Crotonobetaine in. (X6).– ATP (X22).– CaiT (X45).– CaiB (X47).– Cai D (X49).– CaiT (X45) y CaiB (X47).– Q (X50).– Reactor Crotonobetaine

Concentration (X52).

20% Metabolites, 50% enzymes Cellular Homeostasis

S-SYSTEM MODEL

tim e (h)

0 20 40 60

Nor

mal

ized

con

cent

ratio

n

0 ,99

1,00

1,01

1,02

1,03

1,04

CR ext

Cel

G lycerolext

Glycerol in. (X1).

tim e (h )

0 1 2 3 4 5 6 7

Nor

rmal

ized

con

cent

ratio

n

0 ,99

1,00

1,01

1,02

1,03

1,04

C R int

LC C oALC int

LC ext

C R C oA

C R ext

•Crotonobetaineout (X2)

S-SYSTEM MODEL

• ATP (X22)

time (h)

0 2 4 6

Con

cent

raci

ón n

orm

aliz

ada

0,97

0,98

0,99

1,00

1,01

LCext

CRext

LCCoA, LCint

CRCoA, CRint

• CaiB (X47)

time (h)

0 2 4 6

Con

cent

raci

ón n

orm

aliz

ada

0,94

0,96

0,98

1,00

1,02

1,04

1,06

LCext

CRext

CRint

LCint

LCCoA

CRCoA

• Cai D (X49)

tiempo (h)

0 2 4 6

Con

cent

raci

ón n

orm

aliz

ada

0.90

0.92

0.94

0.96

0.98

1.00

1.02

1.04

1.06

LCext

CRext

CRint

LCint

LCCoA

CRCoA

• Cai T (X45) andCaiB (X47)

tiempo (h)

0 2 4 6

Con

cent

raci

ón n

orm

aliz

ada

0.8

1.0

1.2

1.4LCext

CRext

CRint

LCint

LCCoA

CRCoA

MODELO S-SYSTEM Comp. Appl. Biotechnol. CAB 9 (en prensa). 2004.

Biotechnol. Progress. (en revisión)

METABOLIC FLUX ANALYSIS MODEL

Collaboration:Prof. M. Reuss (Univ. Stuttgart, Germany)

Entrada Fumarato

TCA

GLUCONEOGENESIS

SINTESIS PROTEINAS

SINTESIS LlPIDOS

ARN & ADN SINTESIS

SHUNTPENTOSAS

ASIMILACION DE GLICEROL

REDUCCION FUMARATO

PRODUCTOS DEFEMENTATION

GLUCÓLISISCélulas

Glicerol

NH4+

SO4-2

Salida Fumarato

H+ +H2O

METABOLIC FLUX ANALYSISATP utilized in:Futil Cycle 57.24 %L-carnitine Synthesis 24.83 %Biomass 15.17 %Other Processes 2.76 %

100.00 %

CARNITINE YIELD

LactateLactateCOCO22

YLC/GLIC= 2 YLC/GLIC= 1

CARNITINE YIELD

• Theoretical yield YLC/GLICEROL = 2.00 ~ 1.00 mol/mol

• Experimental yieldYLC/GLYCEROL = 0.36 mol/mol

• STRATEGYImprovement Factor

Use of resting cells 1.52

Overexpression of CaiT and CaiB 3.00

CaiT knock out mutants 6.00

L-Carnitineext

Crotonobetaíneext

L-Carnitineint

Crotonobetaíneint

L-CarnitinylCoA

CrotonobetainylCoA

CrotonobetainylCoA

CaiT

CrotonobetaíneextCrotonobetaíneint

L-CarnitinylCoAL-Carnitineext L-Carnitineint

CoACaiTCaiB

CaiB

CaiC

CaiD

CaiDCoA

CARNITINE METABOLISM OPTIMIZATION

Comp. Appl. Biotechnol. CAB 9 (en prensa). 2004.

GENETIC MODIFICATION WIT PLASMIDS IN E. coli CELLS

PBAD

NacI

cai Tcai F

cai B E. coli REACTOR

araC

M13blarrnBT12

pKLJ12

12.4Kb

MCSPBAD

pML31

EcoRI

EcoRI Sal1

araC

M13 AmpR

rrnBT12pBAD24

4.5Kb

MCS

pBRori

Metabolic and genetic engineering in de producción of L-carnitine with Escherichia

coli and Proteus mirabilis strains.

.

• Prof. H.P. Kleber (Univ.of Leipzig, Leipzig, Alemania)

• Prof. N. Torres Darias (Univ. de La Laguna, España)

• Prof. M. Reuss (Univ. of Stuttgart, Stuttgart, Alemania)

• Prof. M. Calvani (Sigma-Tau S.p.A, Roma, Italia)

• Prof. N. Lindley (CNRS-INSA-Toulouse, Francia)

Collaborations

FUTURE PERSPECTIVES

- Dynamic Model- Signalome analysis: o2, crotonobetaine, glycerol

-OverexpresiónCaiT, CaiB, CaiD y

CaiF

- Strain improvement by Metabolic and Genetic

Engineering

Studies ofMetábolic

and GenéticEngineering

withtransformed

strains

OPTIMIZATION

Proyecto de Proyecto de InvestigaciónInvestigación