metabolic engineering -...

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 (cell capability analysis) (5/12)Experimental Determination Method of Flux Distribution with Isotope Labeling(5/19)Metabolic Control Analysis (5/26)Metabolic Engineering with Bioinformatics (6/2)

April 14th – June 2nd, 2004

Lactate

rF

rLAC=85

Answer 5r1:Gluc+2ADP+2NAD=2PYR+2ATP+2NADH

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

=

⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

−−−−

−−−

70552285

100000

654321

100000010000000100000010000001200112111100

001112

rrrrrr

ArC=rm

rC=[ATA]-1ATrm

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc=55

NADH

NAD

rGluc=100

rCO2=22

r1=101.8

r2=84.1

r3=21.1

r4=100.2

r5=53.2 r6=70.0

rEtOH=70

Acetaldehyde

⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

==

⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

−

0.702.532.1001.211.848.101

)(

654321

1m

TT rAAA

rrrrrr

Fig. Metabolism from glucose in homo- and hetero-fermentativeLactococci.

Function of ExcelTRANSPOSE : transpose of matrixMINVERSE: inverse of matrixMMULTI: multiplication of matrix

2

Lactate

Acetaldehyde

Ethanol

rF

rLAC

rEtOH

Answer 5Balance　eq. of pyruvate

2r1-r2-r3-r4=0Balance eq. of AcCoA

r3+r4-r5-r6=0Balance eq. of NAD/NADH

2r1-r2+r3-2r6=0Glucose consumption rate

r1=rGluc=100r2=rL=85r3=rCO2=22r5=rAc=55r6=rEtOH=70

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc

NADH

NAD

rGluc

rCO2

r1

r2

r3 r4

r5 r6

r1:Gluc+2ADP+2NAD=2PYR+2ATP+2NADH

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

=

⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢

⎣

⎡

−−−−

−−−

70552285

100000

654321

100000010000000100000010000001200112111100

001112

rrrrrr

To be continued


Gluc

Gluc6P

Fruc6P

GAP

G3P

PEP

AcCoA

IcCyt

AKG

SucCoASuc

Fum

Mal

OxA

Ribu5P

Pyr

AcCoA

GlyOx

EtOH

AC

42

12

14

14

11

8

129

5858

91

66

79

81

94

7

0

6

13

0

0

Metabolic Flux Distribution Analysis

Analysis of distribution of reaction rates in metabolic pathway

Use of information of stoichiometry of the cell and measurements

Many applicationsUnderstanding cell physiologyProcess operationMolecular breeding

3

1. Metabolic Pathway ModelingMetabolic flux distribution is analyzed with intracellular and extracellular metabolites, based on the information of metabolic reactions.

Metabolic Pathway: Sequence of Biochemical Reactions

A(Substrate)

B C (Target Product)

D E (By-Product)

F (By-Product)

Cell

r1 r2

r3r4

r5

r1: A->B r2: B+NADPH->C r3:B->Dr4: D->E+NADPH r5:D->F

2. Material Balances in Biochemical ReactionsPseudo-Steady State Assumption

Intracellular metabolites concentrations: constant(Linear Equations)B: r1=r2+r3D: r3=r4+r5NADPH: -r2+r4=0

⎥⎥⎥

⎦

⎤

⎢⎢⎢

⎣

⎡=

⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥

⎦

⎤

⎢⎢⎢

⎣

⎡

−−−

−−

000

0101011100

00111

5

4

3

2

1

rrrrr

(Vector Form)

A(Substrate)


D E (By-Product)

F (By-Product)

Cell

r1 r2

r3 r4

r5

4

3. Observability of Metabolic Pathway(Category of System)

(1)Determined System: d=0Metabolic fluxes are uniquely determined by measured fluxes.

(2) Over-determined System: d<0Metabolic fluxes are determined with redundancy.

(3) Under-determined System: d>0Metabolic fluxes cannot be determined uniquely.

Degree of Freedom of the Metabolic Pathway:dd=k-n-mWhere k: number of unknown fluxes

n: number of balance equations (constraints)m: number of measured fluxes

(3-3) Under-Determined System (Cell Capability Analysis)Number of unknown fluxes:5, Number of balance eq.: 3Number of measured fluxe:1 (d=1)(ex.) rA: consumption rate of substrate

⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢

⎣

⎡

=

⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢

⎣

⎡

−−−

−−

Arrrrrr

000

000010101011100

00111

5

4

3

2

1

ArC=rm

Linear Programming

A(Substrate)


D E (By-Product)

F (By-Product)

Cell

r1 r2

r3 r4

r5

100 50

50

0

r4: necessary to regenerateNADPH

deleted based on copyright concern.

Palsson et al;Biotech Bioeng 42, 59-73(1993)

5

(3) Under-Determined System (2)Number of unknown fluxes:5, Number of balance eq.: 3Number of measured fluxe:1 (d=1)(ex.) rA: consumption rate of substrate

A(Substrate)


D E (By-Product)

F (By-Product)

Cell

r1 r2

r3 r4

r5

100 50

50

0

r4: necessary to regenerateNADPH

r3=r1-r2r1-r2=r4+r5r2=r1-r4-r5…(relationship ex. metabolites)

=r1-r5-r22r2=r1-r5r2=1/2r1…(r5=0)….Max Yield(r3=1/2r1, r4=1/2r1, (r1=rA))

⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢

⎣

⎡

=

⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢

⎣

⎡

−−−

−−

Arrrrrr

000

000010101011100

00111

5

4

3

2

1

Question 6In hetero-fermentative metabolism, determine all the ri fluxes so that ethanol production was maximized.


Lactate

rF

rLAC

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc

NADH

NAD

rGluc=100

rCO2

r1=100

r2

r3

r4

r5 r6

rEtOH　↓(Max)

Acetaldehyde


6

Lactate

Acetaldehyde

Ethanol

rF

rLAC

rEtOH

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc

NADH

NAD

rGluc

rCO2

r1

r2

r3 r4

r5 r6

Stoichiometric Equations


r2:PYR+NADH=LAC

r3: PYR=AceCoA+NADH+CO2

r4: PYR=FOR+AcCoA

r5:AcCoA=Ac+ATP

r6: AcCoA+2NADH=EtOHFig. Metabolism from glucose in homo- and hetero-fermentativeLactococci.



2-1 Balance　eq. of pyruvate2r1-r2-r3-r4=0

2-2 Balance eq. of AcCoAr3+r4-r5-r6=0

2-3 Balance eq. of NAD/NADH 2r1-r2+r3-2r6=0

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc

NADH

NAD

rGluc

rCO2

r1

r2

r3 r4

r5 r6

rEtOH



7

Answer 6In heterofermentative metabolism, determine all the ri fluxes such that

rEtOH->max

when rGluc=100

Balance　eq. of pyruvate2r1-r2-r3-r4=0

Balance eq. of AcCoAr3+r4-r5-r6=0

Balance eq. of NAD/NADH 2r1-r2+r3-2r6=0

Glucose consumption rater1=rGluc=100

r2=0, r4=0, r5=0

r1=100, r3=200, r6=200


Lactate

rF

rLAC

Pyruvate

Formate

NADH NAD

Acetyl-CoA

NADH

NAD

CO2NADH

NAD

Acetyl-P

Acetate

ADP

ATP

rAc

NADH

NAD

rGluc=100

rCO2

r1=100

r2=0

r3=200

r4=0

r5=0 r6=200

rEtOH=200(Max)

Acetaldehyde


Schematic Representation of Genomically Metabolic Models(Edward and Palsson, Biotechnol. Bioeng., 58, 162-169 (1998)

Application of Metabolic Flux Analysis 1


8

Application of Metabolic Flux Analysis 2

Physiological State Recognition* Application MFA to Process Operation* Comparison of MFD among several strains (MCA)

Simulation and in silico Analysis* Prediction of Cell Capability in Genetic Modification* Prediction of Redundancy and Robustness of the Cell

against Gene Deletion *Adaptation of Cell against Environmental Changes

Metabolic Pathway Analysis of a Recombinant YeastsFor Rational Strain DevelopmentR. Carison, D. Fell, F. Srienc, Biotechnol Bioeng. 79(2), 121-134 (2002)

Elementary mode analysisConvex analysis to identify all possible unique

nondecomposable biochemical pathways for a steady state-> Independent pathway in MP

Range Azero

Kernel A

k-dimension space

k-(n+m)

dim A=(n+m)n: constraintm: measurement

9

Material Balances in Biochemical ReactionsPseudo-Steady State Assumption

Intracellular metabolites concentrations: constant(Linear Equations)B: r1=r2+r3D: r3=r4+r5NADPH: -r2+r4=0

⎥⎥⎥

⎦

⎤

⎢⎢⎢

⎣

⎡=

⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢

⎣

⎡

⎥⎥⎥

⎦

⎤

⎢⎢⎢

⎣

⎡

−−−

−−

000

0101011100

00111

5

4

3

2

1

rrrrr

(Vector Form)

A(Substrate)


D E (By-Product)

F (By-Product)

Cell

r1 r2

r3 r4

r5

dim A=3

A

Dimension of biochemical space: 5

dim(Kernel A)=2

Independent Biochemical ReactionsAt Steady State Assumption

A(Substrate)


D E (By-Product)

F (By-Product)

Cell

r1 r2

r3 r4

r5

dim(Kernel A)=2

r1: A->B r2: B+NADPH->C r3:B->Dr4: D->E+NADPH r5:D->F

A B B E+NADPH (Eliminate D)

B F(Eliminate D)

(r1)

(r3, r5)

( r3, r4)

B+NADPH->C(r2)

Step 1

Step 2

A B B F(r1) (r3, r5)

2B->E+C (Eliminate NADPH)(r2, r3. r4)

Step 32A->E+C (Eliminate B) A F(Eliminate B)

Mavrobouniotis et al., BB, 36, 1119-1132 (1990)

(r1, r3, r5)(2r1, r2, r3. r4)

10

Metabolic Pathway of Saccharomyces cerevisiae

Rational Strain Development for PHB

PHB pathway (Ralstonia eutropha)

PHB pathway (Ralstonia eutropha) phbA: 2AceCOA_cyt=AcAcCoA +CoAphbB: AcAcCoA+NADPH=hBCoA+NADPpHBC:hBCoA=PHB+CoA_cyt

Requirement to enhance PHB synthesis1. Supply of AceCoA(ATP Regeneration)

Acetate_cyt+CoA_cyt+2ATP_cyt=AceCoA_cyt2. Regeneration of NADPH

AcAcCoA+NADPH=hBCoA+NADP3. Recovery of C-Source

deleted based on copyright concern.unspecified quotation.

11

1. Supply of ATPNADH produced in TCA and oxidative phosphorelation

2. Regenerate of NADPH(ICD_Cyt(NADPH dependent))AcAcCoA+NADPH=hBCoA+NADPR74: IC_cyt=AKG_cyt+NADPH (selected)IC_mit=AKG_mit +NADH

MAX Yield of PHB: 67%

1. Supply of ATPACL : CIT_cyt+ATP+CoA_cyt=AceCoA_cyt+OXA_cyt(Selected)Acetate_cyt+CoA_cyt+2ATP_cyt=AceCoA_cyt

2. Regenerate of NADPH(ICD_Cyt(NADPH dependent))AcAcCoA+NADPH=hBCoA+NADPR66:AceAde_cyt=Acetate_cyt+ NADPH (selected)




12

Supply of ATPACL : CIT_cyt+ATP+CoA_cyt=

AceCoA_cyt+OXA_cyt(Selected)Regenerate of NADPH(ICD_Cyt(NADPH

dependent))AcAcCoA+NADPH=hBCoA+NADPR66:NADH+NADP=NAD+NADPH (selected)Glycerol_P=DHAP+NADH




13

Gluc

Gluc6P

Fruc6P

GAP

G3P

PEP

AcCoA

IcCyt

AKG

SucCoASuc

Fum

Mal

OxA

Ribu5P

Pyr

AcCoA

GlyOx

EtOH

AC

0

29

25

26

29

8

179

100100

70

50

113

115

179

6

0

32

64

0

39

Gluc

Gluc6P

Fruc6P

GAP

G3P

PEP

AcCoA

IcCyt

AKG

SucCoASuc

Fum

Mal

OxA

Ribu5P

Pyr

AcCoA

GlyOx

EtOH

AC

42

12

14

14

11

8

129

5858

91

66

79

81

94

7

0

6

13

0

0

Gluc

Gluc6P

Fruc6P

GAP

G3P

PEP

AcCoA

IcCyt

AKG

SucCoASuc

Fum

Mal

OxA

Ribu5P

Pyr

AcCoA

GlyOx

EtOH

AC

100

50

66

69

63

30

64

00

53

36

35

36

38

23

10

0

0

13

(a) Glucose:100% (b)Glucose:42%, EtOH: 58% (c)EtOH:100%

338

Example 3 Flux distribution to maximize ATP formation in S. cerevisiae with different carbon sources. van Gulik, W.M. and Heijnen, J.J. Biotechnol.Bioeng., 48, 681-698 (1995)

Relationship between specific growth rate andby-product formation rate with limited oxygen supplyin E. coli fermentation (Palsson et al. AEM, 59, 2465 (1993)


14

Metabolic flux change in the case of limited oxygen supply in E. coli fermentation (Palsson et al. AEM, 59, 2465 (1993)

(a): anaerobic↓

(e): aerobic

Schematic Representation of Genomically Metabolic Models(Edward and Palsson, Biotechnol. Bioeng., 58, 162-169 (1998)



15

　　　　　　　　 Glucose　　　　　　　　　　　　　 ( extracellular)

　　　　　　　　　　　 r g　　　　　　　　 Glucose　　　　　　　　　　　　　 ( intracellular)

　　　　　　　　　　　 r 1　　　　　　 Glucose-6-phosphate　　　　　　　　　　　 r 2

　　　　　　 Fructose-6-phosphate 　　　　 r 6

　　　　　　　　　 r 3

　 r 10 　　　 Glyceraldehyde-3-phosphate　　　　　　　　　　　 r 4 3CO 2CO 2 　　　　 3-Phosphoglycerate　　　　　　　　　　　 r 5　　　　　　 PhosphoenolpyruvateGlycine　　　　　　 r 8　　 Acetate　　　　 CO 2　　　 r9 (intracellular) 　　 r 13

Histidine BiomassAcetate

　　　　 3 CO 2　　TCA　　　　　 ( extracellular)

　　　　　　　　　　r7　　　　

　　ATP 　　 ADP 　　 NADH 2　 NAD+ATP　　　　 r 11 　　　　　　　 r 12

ATP consumptionfor cell maintenance

AnabolismHistidine

production

rA

Fig.1 Simplified model of histidine production by B. flavum. H. Shimizu et al.

Acetyl-CoA

I　　 II III　 IVHigh GlucoseHigh ＡcetateHighＵｒａcil

High Ｇlucose

Low Ｕｒａcil

Time(h)

012345

Ace

tate

(g/L

)G

luco

se(g

/L)

Ura

cil(g

/L)

Bio

mas

s(g/

L)H

istid

ine(

g/L)

2 03 04 05 06 0

00 . 0 20 . 0 40 . 0 60 . 0 8

0 . 1

0

5

1 0

0 1 2 2 4 3 6 4 8012345

Acetate depletion

Uracil depletion

Phase

Fig. 2 Time course of L-histidine production in mixed substrate culture.H. Shimizu et al

16

0

2 0

4 0

6 0

0

2

4

6

0 3 0 6 0 9 0 1 2 001234

Glu

cose

(g/L

)B

iom

ass(

g/L

)H

istid

ine(

g/L

)T i m e ( h )

Fig. 3 Time course of L-histidine production in a glucose culture (a) and acetate culture (b).Hiroshi Shimizu et al.

0123456

0

0 . 5

1

1 . 5

0 1 5 3 0 4 5 6 0 7 500 . 0 10 . 0 20 . 0 30 . 0 40 . 0 5

Ace

tate

(g/L

)B

iom

ass(

g/L

)H

istid

ine(

g/L

)

T i m e ( h )

(a)

(b)

34/414.8/15

Fig. 4a Metabolic flux distribution in mixed substrate culture (Phase I/Phase III).Numbers indicate mole fluxes of metabolic pathways.Fluxes are normalized by total consumption rate of glucose and acetate as 100.Hiroshi Shimizu et al.

Glucose(extracellular)

r g

Glucose(intracellular)

r 1Glucose -6 -phosphate

r 2

Fruc tose -6 - phosphate r 6

r 3

r 10 Glyceraldehyde -3 -phosph ate

r 4 3CO 2

CO 2 3 -Phosphoglycerate

r 5Phosphoenolpyruvate

Glycine

r 8 AcetateCO 2 r 9 (intracellular) r 13

Histidine Biomass

Acetate3 CO 2 (extracellular)

r 7

ATP ADP NADH 2 NAD+ATPr 11 r 12

34/99

Phase I / III

-12/4.6

0 /20

6.4/47

1.9/605.7/13

33/ 0

66/ 0

29/84

540/1195

34/100

123/228

TCA

17

Fig. 4b Metabolic flux distribution in glucose culture. Hiroshi Shimizu et al.

Glucose(extracellular)

r g

Glucose(intracellular)

r 1Glucose -6 -phosphate

r 2

Fruc tose -6 - phosphate r 6

r 3

r 10 Glyceraldehyde -3 -phosp hate

r 4 3CO 2

CO 2 3 -Phosphoglycerate

r 5Phosphoenolpyruvate

Glycine

r 8 AcetateCO 2 r 9 (intracellular) r 13

Histidine Biomass

Acetate3 CO 2 (extracellular)

r 7

ATP ADP NADH 2 NAD+ATPr 11 r 12

Glucose cultivation

99

41

44

8997

7.4

0

103

282

0

100

1711

274.3

TCA

Fig.4 c Metabolic flux distribution in acetate culture. Hiroshi Shimizu et al.

Acetate cultivation

Glucose (extracellular)

rg Glucose (intracellular)

r1 Glucose-6-phosphate r2 Fructose-6-phosphate r6 r3 r10 Glyceraldehyde-3-phosphate r4 3CO2 CO2 3-Phosphoglycerate r5 Phosphoenolpyruvate Glycine r8 Acetate CO2 r9 (intracellular) r13 Histidine Biomass Acetate 3CO2 (extracellular) r7 ATP ADP NADH2 NAD+ATP r11 r12

-0.34

-17

-17

-34-36

1.8

110.54100

49

9.5

0

7172

TCA

18

Fig.3 Shimizu

Maintenance

Cell Histidine

Glucose (100%)

MaintenanceCell Histidine

Acetate (100%)

66x0.85%

9.7 %8.2 %57%34% 4.7%85%

Glucose (34%) Acetate (66%)

HistidineCell

Maintenance

34x0.57%34x 0.34%

66x0.047%

34x0.082%

66x0.097%

Glucose culture

Mixed substrate culture

Acetate culture

(a) (b)

(c)

metabolic engineering -...

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