1

Metabolic Engineering

Department of Bioinformatic Engineering,Graduate School of Information Science and Technology,

Osaka University

Dr. Hiroshi Shimizu Professor

SubjectsIntroduction of Metabolic Engineering (4/14)Metabolic Pathway (MP) Modeling and Observability of MP (4/21)Metabolic Flux Analysis (5/12)Experimental Determination Method of Flux Distribution with Isotope Labeling(5/19)Metabolic Control Analysis (5/26)Metabolic Control Analysis 2 Metabolic Engineering with Bioinformatics (6/2)

April 14th – June 2nd, 2004

Connection TheoremX0 S1 X3

e1 e2

v1 v2

Large FCCs: Small Elasticity(Rigid Reaction)

11

21

2

1vS

vS

Je

Je

CC

εε

−=

Small FCCs: Large Elasticity(Flexible Reaction)

v1

v2

S1

Inhibition

Time

v1

v2

S1

Inhibition

Time

DetailedReactionKineticsor Model

GroupPerturbationExperiments

Measurementof Flux Changes

GroupFlux ControlCoefficients

Elasticities

IndividualFlux ControlCoefficients

GroupFlux ControlCoefficients

IndividualFlux ControlCoefficients

Individual Reaction PerturbationExperiments

Map of Control Distribution in Metabolic Network

Top-DownApproach

Bottom-UpApproach

Two Methods in MCA

Determination of Flux Control CoefficientsDetermination of Flux Control CoefficientsBottom-up Approach

Detailed reaction kinetic modelElasticity coefficients

Critical LimitationsRequirements of each and every enzyme reaction kineticsIn vitro experimental data<->in vivo metabolic reaction control?

Top-down ApproachPerturbation experiments: use of well defined changes in enzyme

activities Measurement of flux changes: application of MFA

Bottom-up Approach

Summation and Connection Theorem

⎥⎦

⎤⎢⎣

⎡=⎥

⎦

⎤⎢⎣

⎡⎥⎦

⎤⎢⎣

⎡0111

2

121

11

Je

Je

vS

vS C

Cεε

: knownvjSiε J

eiC : to be solved

Every and all kinetics: known FCCs: to be solved

Bottom Up Approach for MCA

deleted based on copyright concern.Prof. Jens Nielson GroupTechnical Univ. Denmark

2

Fig. 11.3 The penicillin biosynthetic pathway in Penicillium chrysogenumPissala et al., Biotechnol Bioeng, 51, 168-176 (1996)

Fig. 11.3 The penicillin biosynthetic pathway in Penicillium chrysogenumPissala et al., Biotechnol Bioeng, 51, 168-176 (1996)

Fig. 11.4 Elasticity coefficients for the two enzymes ACVS and IPNS with respect to LLD-ACV during fed-batch cultivation. Note that the elasticity coefficient for ACVS is negative.

Fig. 11.4 Elasticity coefficients for the two enzymes ACVS and IPNS with respect to LLD-ACV during fed-batch cultivation. Note that the elasticity coefficient for ACVS is negative.

Fig. 11.5 Flux control coefficients for the two enzymes ACVS and IPNS during fed-batch cultivation.

Fig. 11.5 Flux control coefficients for the two enzymes ACVS and IPNS during fed-batch cultivation.

Metabolic Control Analysis in Metabolic Control Analysis in Amino Acid ProductionAmino Acid Production

Hiroshi ShimizuDepartment of Bioinformatic Engineering

Graduate School of Information Science and TechnologyOsaka University, Japan

GIM2002, Gyeongju, Korea

Top down Approach

Metabolic Control Analysis in Glu Production1. Control Mechanism of Glutamate Production2. MCA around at A Key Branch Point

deleted based on copyright concern. Nampoothiri.et.al., AMB,58,89-96(2002)Eggering and Sahm.JBB,92,201-231(2002)

deleted based on copyright concern.

deleted based on copyright concern.

deleted based on copyright concern.

3

Dr. Kimura et al. at Ajinomoto co., Inc.

For enhancement of L-glutamate production,Depetion condition of biotin which is required for cell growth.Addition of a detergent such as polyoxyethylene sorbitan

monopalmitate (Tween 40).Addition of a lactum antibiotic such as penicillin.

Back ground of Metabolic Control Analysis of L-Glutamate Production

Decrease in 2-oxoglutarate dehydrogenase complex (ODHC)activity.

Enhancement of production of L-Glutamate.

Metabolic Control Analysisaround at a Key Branch Point of 2-Oxoglutarate(αKG)

Quantification of Magnitude of Impact of (1) Enhancement of isocitrate dehydrogenase(ICDH),(2) Enhancement of glutamate dehydrogenase (GDH),

or,(3)Attenuation of 2-oxoglutarate dehydrogenase complex

(ODHC)on

Targeted Flux of L-Glutamate

Flux Control Coefficients (FCC)

Bacterial Strains and Plasmids Used in This Study

a:Bormann et al., Molecular Microbiology, 6, 317 (1992)b: Eikmanns et al., J. Bacteriol., 177, 774 (1995)

Cultivation conditionCultivation conditionpH pH 7.207.20Temperature Temperature 31.531.5℃℃DO DO > 3.0> 3.0 ppmppmAir flow rate Air flow rate 1 1 vvmvvm

Fermentation

Carbon Source: GlucoseNitrogen Source: AmmoniaTrigger of Glutamate Production : Biotin depletion

Exhaust gas CO2 analyzer

O2 analyzer

NH3

PH sensor

Thermo sensor

DO sensor

Data logger RS232C

AirFlow meter

Experimental Apparatus

RS232C

balance Laser turbidimeter

Metabolic Map of Glutamate Synthesis

IsoIso--citratecitrate

SuccinylSuccinyl--CoACoA

αα--ketoglutarateketoglutarate

LL--GlutamateGlutamate

ｒｒ77

ｒｒ88ｒｒ99

ICDHICDH

ATP

ADP

NAD+

+ATP

O2

NADH2

LacNADH2

6NADPH2

3CO2

Cell

NH3

PEP

PYR

G-6-P

G-A-P

Glucose

F-6-P

NADH2CO2

NADH2ATP

ATP

PEPPYR

CO2

NADH2

GTPGTP

CO2

5/3NADH2

NADP+

NADPH2OXA

ICIT

SucCoA

NADPH2 NADP+

NH3

L-Gluα-KG

CO2

GDHODHC

Modeling of metabolic pathwayChecking ConsistencyMetabolic Flux AnalysisMetabolic Control Analysis

deleted based on copyright concern.

4

0=Er

[ ]iC

mC

rr

rAr

=

=− 0

Material Balance Equation in the Pathway

(16 Calculated reaction rates (ri))12 Intermediate metabolites(constraints))7 Extracellular measured reaction rates rm))

Where, A: stoichiometric coefficients matrix (19x16))rc: calculated reaction rates vector (16x1)rm: measured reaction rates vector (19x1)

Degree of information redundancy = (12+7)-16=3Mole flux distribution: determined by the least squares method

(i=1..16)

rm=[rgluc,rcell,rglu,rCO2,rO2,rNH3,rlac,rG6P,rF6P,rGAP,rPEP,rpyr,ricit,rKG,rSucCoA,rOxA,rATP,rNADH,rNADPH]

[ ] mTT

c rAAAr ˆˆ1−

=

Time course of Consistency IndexTime course of Consistency Index

ΧΧ22 valuevalue（＝（＝11.311.3））(degree of freedom :3)(degree of freedom :3)

0 10 20 300

10

20

30

Time(h)

Con

sist

ency

inde

x (Method 1)

(Method 2)

Method1: addition rate of NH3 = ammonia uptake rateMethod2: addition rate of NH3 = ammonia uptake rate + addition rate for neutralization of

the produced L-glu (pKa=4.25)

G-6-P

G-A-P

Glucose

F-6-P

PEP

PYR LacNADH2

NADP+

NADPH2

CO2

ICIT

6NADPH2

OXAGTPGTP

Cell

L-Glu

SucCoA CO2

CO2

CO2

NAD

5/3NADH2

NADPH2

3CO2NADH2ATP

ATP NADH2

NH3ATP

PEPPYR

NADP+

NADH2

NAD+

+ATP

ATP

ADP

O2

NH3

α-KG

r1r1: 100100r2r2:42.4/42.4/105105

r3r3:47.1/47.1/101101

r4r4:96.6/96.6/201201r14r14:72.3/72.3/1.11.1

r5r5: 0/0/00

r12r12:2.4/2.4/00

r7r7:72.9/72.9/107107

ｒｒ99：11.1/11.1/93.593.5ｒｒ1010：61.8/61.8/13.313.3

ｒｒ1111：11.1/11.1/93.593.5

ｒｒ88：61.8/61.8/13.313.3

ｒｒ1313：0/0/00

ｒｒ1515：167/167/172172

ｒｒ1616：0/0/237237r6r6:72.9/72.9/107107

Flux Distribution Analysis

ri (growth phase/production phase)

rgluc=100

α-KG

0.267(×2.41)

0.660(×1.06)

0.9280.928((××1.27)1.27)

ICIT

L-GluSucCoA

0.7290.729

0.6180.618 0.1110.111

ICIT

L-Glu

α-KG

SucCoA

ICDHICDH

enhancedenhanced

　　riri=3.0=3.0

Flux Distribution Analysis at the Flux Distribution Analysis at the ααKG KG Branch PointBranch Point

（（Case 1: ICDH enhancedCase 1: ICDH enhanced））

0kJ

)( ki

rk

f

J

WT->AJ13679

:

:0

ri

i

e

eEnzyme Activity Amplification factor ri

0i

ri

i eer =

Flux Amplification factor kif

0,

k

rikk

i JJ

f =Enzyme activity at the original state

Enzyme activity at the perturbed state :

:

,

0

rik

k

J

J Flux at the original stateFlux at the perturbed state

0.7290.729

0.6180.618 0.1110.111

SucCoA

ICIT

L-Glu

α-KG

0.8260.826((××1.13)1.13)

0.7130.713((××1.15)1.15)

0.1130.113((××1.02)1.02)

α-KG

SucCoA L-Glu

ICIT

ＧＤＨＧＤＨ

enhancedenhanced

　　riri=3.2=3.2

Flux Distribution Analysis at theFlux Distribution Analysis at the ααKGKG Branch PointBranch Point

（（Case 2: GDH enhancedCase 2: GDH enhanced））

)( ki

rk

f

J0kJ

WT->AJ13678

Enzyme Activity Amplification factor ri

0i

ri

i eer =

Flux Amplification factor kif

0,

k

rikk

i JJ

f =Enzyme activity at the original state

Enzyme activity at the perturbed state :

:

,

0

rik

k

J

J Flux at the original stateFlux at the perturbed state

α-KG

L-Glu

ICIT

SucCoA

0.7290.729

0.6180.618 0.1110.111

SucCoA

α-KG

L-Glu

ICIT

0.6770.677((××0.93)0.93)

0.1470.147（×（×0.240.24））

0.5300.530((××4.77)4.77)

ODHCODHC

attenuatedattenuated

riri=0.52=0.52

Flux Distribution Analysis at theFlux Distribution Analysis at the ααKGKG Branch PointBranch Point

（（Case 3: ODHC attenuatedCase 3: ODHC attenuated））

)( ki

rk

f

J0kJ

By depletion of biotin

Enzyme Activity Amplification factor ri

0i

ri

i eer =

Flux Amplification factor kif

0,

k

rikk

i JJ

f =Enzyme activity at the original state

Enzyme activity at the perturbed state :

:

,

0

rik

k

J

J Flux at the original stateFlux at the perturbed state

5

Estimation of Impact of Changes in EnzymeActivities at αKG

Aim of Metabolic Control AnalysisRealization of over production of targeting metabolites

Identification of enzyme reaction(s) with the greatest impact on the overall flux

Methodology of MCA

Quantification of the magnitude of the impact of each enzyme activity on the overall flux

Flux Control Coefficients (FCC)

eJ

rJreD

k

iJki ∆

∆=

Determination of FCC by large perturbation experiments at the Branch Point

( eir , Jkr )(ei0 , Jk0 )

△J

ei

△e

J

eiJk

JeC

k

iJki ∂

∂=

)( ikDFC kJi

kJi

kJi ≠×=

ririD

ririC

FkJ

i

iJi

kJi 11

11

−−

−−

= 0/ eieirri =

Definition of FCC: infinitesimal perturbation

Real experiment: large perturbation(Deviation index)

)( ikDC iJi

iJi ==

ICD

αKG

GluSucCoA

J7

J8J9

eICDH

eODHCeGDH

k

CikICDH ODHC GDH

Ｊ7 0.32 0.33 0.19

Ｊ8 0.08 3.43 0.22

Ｊ9 1.67 -16.9 0.025

Table. FCCs around at the branch point of αKG

J7: flux through ICDHJ8: flux through ODHCJ9: flux through GDH (Glu production)

Cik : Impact of change in i-th enzyme activity

on k-th flux

ICDH(×5)

ODHC(×1/5)

GDH(×5)

f 7 1.4 0.91 1.2f 8 1.1 0.06 1.2f 9 2.8 5.6 1.0

Table. Prediction of flux change by enzyme activity enhancement and attenuation

J7: flux through ICDHJ8: flux through ODHCJ9: flux through GDH (L-Glu production)

fik : k-th flux amplification by i-th enzyme activity change

*Cki

ICDH ODHC GDH

Ｊ7 0.92 0.07 0.01

Ｊ8 0.23 0.76 0.01

Ｊ9 4.74 -3.74 0.001

Table. gFCCs around at the branch point of αKG calculated by flux amplification factor only

J7: flux through ICDHJ8: flux through ODHCJ9: flux through GDH (Glu production)

*Cik : Impact of change in i-th group enzyme activity

on k-th group flux

Corynebacterium efficiens：

・Glutamate Producing Strain

・Ability to grow at higher temperature (37oC)

than C. glutamicum (31oC)

＊Mechanism of Glutamate Production＊Effect of Temperature on Glutamate Production

in C. glutamicum and C. efficieins

6

0 10 20 30 40 500102030405060

020406080

Time(h) Time(h)

Time(h) Time(h)

OD

660

glut

amat

e(g/

L)O

D66

0gl

utam

ate(

g/L)

OD

660

glut

amat

e(g/

L)

OD

660

glut

amat

e(g/

L)

gluc

ose(

g/L)

gluc

ose(

g/L)

gluc

ose(

g/L)

gluc

ose(

g/L)

31.5℃

0 10 20 30 40 500102030405060

020406080

37.0℃

0 10 20 30 400102030405060

020406080

0 10 20 30 40 500102030405060

020406080

Time courses of OD660, gluc, and glu of C. glutamicum (a), (b) and C. efficiens (c), (d). Temperature: 31.5℃ (a), (c) and 37.0℃ (b), (d).Symbols: ●, OD660; △, glucose concentration; ○, glutamate concentration

(a)

(d)(c)

(b)

C.glutamicum

C.efficiens

Glu

Glucose 31.5℃Flux Distribiution at αKG 　

TCA cycle

Time(h) Time(h)0 10 20 30 40

00.0010.0020.003

Flux

/ dr

y ce

ll (m

ol /

h / g

-cel

l) C. glutamicum C. efficiens

0 10 20 30 400

0.0010.0020.003

α-KG

Isocitrate

GlutamateSuccinyl-CoA

r7 (ICDH)

r9 (GDH) r8 (ODHC)

Glu

Glucose 37.0℃

TCA cycle

Time(h) Time(h)Flux

/ dr

y ce

ll (m

ol /

h / g

-cel

l) C. glutamicum C. efficiens

0 10 20 30 400

0.0010.0020.003

0 10 20 30 400

0.0010.0020.003

α-KG

Isocitrate

GlutamateSuccinyl-CoA

r7 (ICDH)

r9 (GDH) r8 (ODHC)

Flux Distribiution at αKG 　

31.5℃ 37.0℃

C. g

luta

mic

umC

. effi

cien

s

ICDH and GDH Activities

　Activities of ICDH and GDH do not change significantly.

ICDH GDH

0 10 20 30 4000.5

11.5

2

Time(h)

Time(h)

Time(h)

Time(h)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

0 10 20 30 4000.5

11.5

2

0 10 20 30 4000.5

11.5

2

0 10 20 30 4000.5

11.5

2

31.5℃ 37.0℃

C. g

luta

mic

umC

. effi

cien

s

Activity of ODHC

　ODHC of C. glutamicum at 31.5 ℃ decreases to 30 %

ODHC of C. efficiens decreases 75% only.

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Spec

ific

enzy

me

activ

ity(μ

mol

/min

/mg-

prot

ein)

Time(h)Time(h)

Time(h) Time(h)

0 10 20 30 400

0.01

0.02

0 10 20 30 400

0.01

0.02

0 10 20 30 400

0.01

0.02

0 10 20 30 400

0.01

0.02

Glu

Glucose

Summary of Results of MFAAnd Enzyme Activity Measurement

TCAcycle

Flux Change Specific Activityr8 (ODHC)

r9 (GDH)

Decrease at Glu production phase

Increase at Glu production phase

Constant

α-KG

Isocitrate

GlutamateSuccinyl-CoA

r7 (ICDH)

r9 (GDH) r8 (ODHC)

Decrease at Glu production phase

7

Glu

Glucose

Km values of enzymes 　

TCAcycle

ICDH

ODHC

GDH

　　0.03 mM

　　 0.03 mM

5 mM

　 0.03 mM

　　 0.08 mM

　　 2 mM

C. glutamicum　　　 　 C. efficiens

α-KG

Isocitrate

GlutamateSuccinyl-CoA

r7 (ICDH)

r9 (GDH) r8 (ODHC)

C.glutamicum C.efficiens

Temp(℃) 31.5 37.0 31.5 37.0

∆J8/∆ODHCa 0.11 0.39 0.22 0.34

∆J9/∆ODHCa -0.082 -0.086 -0.096 -0.123

Sensitivity of J8 and J9(Glu) against ODHC Change

a: (mol/h/g-cell)/(µmol/min/mg-protein)

GDH

α-KG

Before Biotin DepletionBefore Biotin Depletion

α−KG conc is not high enough to produce Glu and Flux r8 in TCA is large, because Km of GDH is large.

　ODHC α-KG

Succinyl-CoA

ICDH

Low Km

r 8r 9

High Km

Succinyl-CoAGlutamate

Decrease in activity

After Biotin DepletionAfter Biotin DepletionDecrease in ODHC activity happens. α−KG conc is increased and flux r9 to Glu production is enhanced.

GDH

　 ODHCα-KG

ICDH

r 9

r 8

High Km

Targeting ProductProductivityProduction Yield

Specific rates ofgrowthsubstrate consumptionproduct formationCO2 formationO2 consumptionby-product formation

Metabolic fluxesenzyme reaction energy formprecursor formpolymerizationtransport

Metabolic Control

Optimal profile of Specific ratesMacroscopic modeling

Metabolic reaction modelMetabolic flux analysisMetabolic control analysis

Micro-array2-D electrophoresisLC-massData mining

Bio-informaticsgenome transcriptomeproteomemetabolome

10-103 103-105<10No. of variables

Methodology&

Tools

Cell activityinfo Metabolic pathway info Molecular level infoObjectives

SummarySummaryMetabolic flux distribution analysis and large perturbation

experiments was applied in order to analyze metabolic control.

ODHC attenuation has the greatest impact on glutamate flux.

Flux amplification of glutamate can be predicted quantitatively.

Bioinformatic data will be integrated in Metabolic Engineering.