Optimization of L-carnitine production by enterobacteria

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

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

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.

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

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)

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

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

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.

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

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

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.

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.

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%.

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.

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.

Factorsproduction

ResultsL-Carnitine transport systems in E. coli


Factorsproduction

Results Effect of permeabilizers on cell envelopeand outer membrane


Factorsproduction

Results Effect of different transport engineeringstrategies on L-carnitine production

Proteus sp.

E. coli O44K74


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


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


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


Non re-energized Cells

Re-energized

cells

Factorsproduction

Results

Cánovas et al., (2007) Process Biochem., 42: 25.

Factors affecting thebiotransformation

Strain improvementMetabolic Engineering

Cell physiologyLink of Central

& CarnitineMetabolisms

Mathematicalmodels

Experimentaldesign

PRESENTATION OVERVIEW

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

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


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


ATP levels decreased.Regulated ICDH/ICL ratio

Metabolic link

Results


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


Alteration of ICDH/ICL ratio

Metabolic link: COFACTORS(ATP and acetyl-

CoA/CoA)

Metabolic link

Results

12

3

4

5

6


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

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

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

S-System Model: Mathematical frameworkResults

Models

A. Sevilla et al., (2005) Biotechonol. Prog. 21, 329-337.

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

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

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

Metabolic FluxAnalysis

Results

Metabolic Flux Analysis

A. Sevilla, et al., (2005) Metabolic Engineering, 7, 401-425

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

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

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

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.

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)


aceAisocitrate liase

aceK ICDH phosphatase/kinase

iclR glyoxylate shunt repressor

acs acetyl-CoA sinthetase

ptaphosphotransacetylase

KO-COLLECTIONProf. Mori, Keio Univer. (Japan)


ResultsAcetyl-CoA metabolism single-gene deletion

KO Mutants

Bernal et al., J. Biotech., (2007), 132, 110-117.

Glyoxylateshunt

Acetatemetabolism

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


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

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

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.

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

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

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

THANK YOU VERY MUCH FOR

YOUR ATTENTION


ACKNOWLEDGEMENTS

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”


J.L. Iborra, M.Cánovas, A. Sevilla & V. Bernal

Department of Biochemistry &Molecular Biology “B” & Immunology

Faculty of ChemistryUniversity of Murcia

SPAIN

Documents

Optimization of L-carnitine production by enterobacteria