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Metabolic Engineering

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

Osaka University

Dr. Hiroshi Shimizu and Dr. Keisuke NagahisaProfessor Assist Professor

No.1Introduction of Metabolic Engineering

April 13th – July 27th, 2005

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Subjects1. Introduction of Metabolic Engineering 2. Metabolic Engineering for Bioproduction3. Metabolic Pathway (MP) Modeling and Observability of MP 4. Metabolic Flux Analysis (Cell Capability Analysis)5. Metabolic Flux Analysis (Genome Scale Flux Analysis) 6. Metabolic Control Analysis 17. Metabolic Control Analysis 28. Metabolic Control Analysis 39. Molecular Metabolic Engineering 110. Molecular Metabolic Engineering 211. Experimental Determination Method of Flux Distribution with Isotope Labeling12. Metabolic Engineering with Bioinformatics 1 Report

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Metabolic Network

Genetic network

Protein network

Physiological Change

Hierarchical networks in microorganisms

Adjustment to change in environmental conditions Metabolome

Product Formation by microorganisms

Quantitative analysis of Metabolic pathway: Metabolic Engineering

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Process Systems Engineering and Metabolic Engineering

Process Systems Engineering is defined as:An engineering field and methodology for making engineeringdecisions in a system which is composed of many subsystems.Engineering Decision Making:Planning, Reactor Design, Operation, Optimal Strategy, ControlTakeichiro Takamatsu, Proceeding of International Symposium on PSE, Kyoto, pp. 3-18 (1982)

Metabolic Engineering is defined as:A targeted improvement methodology of the product formation or cellular properties through the modification of the specific bio-chemical reactions:Greg Stephanopoulos and J. Vallino, 252, 1675-1681 (1991)J.E. Bailey, Science, 252, 1668-1674 (1991)S.Y. Lee and E.T. Papoutsakis, Preface, in “Metabolic Engineering”, 7-9 (1999)

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MicroorganismsSubstrate

FermentationProcess Data*pH, DO, CO2, O2Cell conc. Sub.conc

Metabolic Pathways

Amino Acids

Fatty Acids

Estimation ofSpecific ActivitiesGrowthSubstrate consumptionProduct formation

ObjectivesProductivityYield

Metabolic Flux Data

Process operationOptimizationControlManagement

Metabolic Engineering

Gene Expression

Genome

Transcriptome

Proteome

Metabolome

Metabolites

Metabolic Control

Systems and Elements in Bioprocesses

Alcohols

Gene

Protein

Bioinformatics

Molecular Level Info

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Systems and elements in the cell (Network in the cell)

Physiological Change, Adaptation

Change in EnvironmentalCondition

Metabolic Network

Genetic Network

Protein NetworkProteome Analysis

Transcriptome Analysis

Control(Cy5)

NaCl添加(Cy3)

重ね合わせ(Cy5+Cy3)

Transcriptome

Proteome

Metabolome

Analysis and Integration of Each Network Information

Model Building of Cell

Applications to Molecular Breeding and Process Optimization

Flux Analysis

Deleted based on copyright concern.

Deleted based on copyright concern.

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Concept of Cyclic Modification of Metabolic Pathway

Synthesis・Modification of Metabolic Pathway

DesignSystematic Presentation Metabolic Modification

Process OptimizationDevelopment of OperationStrategy of Bioreactor

Bioinfromatic DataTranscriptomeProteomeMetabolic Flux Analysis

AnalysisCharacterization of Metabolic

Pathway

Molecular BiologyGenetic EngineeringCloningTransformation

Analysis of Control Mechanism of Metabolic Pathway based on Metabolic Control Analysis

Genome Information

Model

Evaluation

Engineering Objectives

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Table 1. Methodologies in metabolic engineering

a. Metabolic flux analysis (MFA) and observability metabolicpathway

b. Cell capability analysisc. On-line metabolic flux analysisd. Metabolic control analysis (MCA) in complicated bionetworkse. Experimental determination of flux distribution by isotope labeling

and trace experiment f. Kinetic analysisg. Integration of Bioinformatic data

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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

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Table 2 Metabolic reaction model of lysine producing Corynebacterium glutamicumPEP: glucose phospho-transferase system(1) Glc+PEP => Glc6P+pyrStorage compound: treharose(2) Glc6P+0.5ATP => 0.5TreharoseEmbden-Meyerhof-Parnas pathway(3) Glc6P => Frc6P(4) Fruc6P+ATP => 2GAP+ADP(5) GAP => G3P+ATP+NADH(6) G3P => PEP(7) PEP => pyr+ATP(8) pyr+NADH => Lac Anaplerotic reaction: PEPcarboxylase(9) PEP+CO2 => OAATorycarboxylic acid cycle(10) pyr => AcCoA +CO2 +NADH(11) AcCoA+OAA => IsoCit(12) IsoCit => AKG+NADPH+CO2(13) AKG => SucCoA+CO2+NADH(14) SucCoA => Suc +ATP(15) Suc => Mal+FADH(16) Mal => OAA+NADHAcetate production and consumption(17) AcCoA => Ac +ATP

Glutamate, glutamine, alanine, valine production(18) NH3+AKG+NADPH => Glu(19) Glu+NH3+ATP => Gln(20) pyr+Glu => Ala+AKG(21) 2pyr+NADPH+Glu => Val+CO2+AKGPentose phosphate pathway(22) Glc6P => Ribu5P+CO2+2NADPH(23) Ribu5P => Rib5P(24) Ribu5p => Xyl5P(25) Xyl5P+Rib5P => Sed7P+GAP(26) Sed7P+GAP => Fruc6P+E4P(27) Xyl5P+E4P => Fruc6P+GAPOxidative phosphorylation: P/O(28) 2NADH+O2 => 4ATP(29) 2FADH+O2 => 2ATPAspartate amino acid family(30) OAA+Glut => Asp+AKG(31) Asp+pyr +2NADPH+SucCoA+Glut+ATP =>Suc+AKG+CO2+Lys(I)(32) Lys(I) => Lys(O)Biomass synthesis: C1.97H6.46O1.94N0.345, 3.0%Ash(33)0.021Glc6P+0.007Fruc6P+0.09Rib5P+0.036E4P+0.013GAP+0.15G3P+0.052PEP+0.03pyr+0.332AcCoA+0.08Asp+3.82ATP+0.476NADPH =>Biomas+0.364AKG+0.312NADH+0.143CO2Excess ATP(34)ATP => ADP +Pi

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Metabolic Flux Distribution in Lysine Production by Corynebacterium glutamicum(Stephanopoulos et al., BB, 41, 633 (1993))

Deleted based on copyright concern.

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Simple Network

Measurement：V1（Consumption）

Experimental Determination of Metabolic Flux with Isotope Labeling

Assumption

No accumulation of B and C

0==dtdC

dtdB

(ii)

(i)

0)()(

0)()(

2213

41

4232

3221

･･

･･→←

←→

→←

−+=

=∴

=+−+=

=+−+=

VVVV

VV

VVVVdtdC

VVVVdtdB

Balance Eqs of B and C

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Combination of two13C Atoms incorporated

：１２C ：１３C

∑ ∑ ∑ ∑= = = =

====3

0

3

0

3

0

3

01)()()()(

i i i iDCBA iIiIiIiI

IA(0)

IA(1)

IA(2)

IA(3)

1- 2-

IA(1): ratio of 1-13C and 2-12C in

Metabolite A

① ②

Use of １３C

I A (1)Ratio Met Pattern of 13C

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Material Balance of B and C in Atomic Level

（Assumption ）

( )

( )

( )

( )

( ) ( )

( )

( )

( ) ( ) ･･⑩

･･⑨

･･⑧

･･⑦

･･⑥　

･･⑤

･･④

③･･

0)3()3()3(

0)2()1()2()2(

0)1()2()1()1(

0)0()0()0(

0)3()3()3()3(

0)2()2()2()2(

0)1()1()1()1(

0)0()0()0()0(

4232

4232

4232

4232

3221

3221

3221

3221

=+⋅−+⋅=

=+⋅−⋅+⋅=

=+⋅−⋅+⋅=

=+⋅−+⋅=

=+⋅−⋅+⋅=

=+⋅−⋅+⋅=

=+⋅−⋅+⋅=

=+⋅−⋅+⋅=

←→

←→

←→

←→

→←

→←

→←

→←

VVIVVIdt

dI

VVIVIVIdt

dI

VVIVIVIdt

dI

VVIVVIdt

dI

VVIVIVIdt

dI

VVIVIVIdt

dI

VVIVIVIdt

dI

VVIVIVIdt

dI

CBC

CBBC

CBBC

CBC

BCAB

BCAB

BCAB

BCAB

0==dtdC

dtdB

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( )( )

( )( )

⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜

⎝

⎛

⋅⋅

⋅⋅

=

⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜

⎝

⎛

⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜

⎝

⎛

+−+−

−+−+

←→

←→

←→

←→

1

1

1

1

4

4

4223

4232

232

232

)0()3(

00)2()1(11

)3()2()1()0()3()2()1()0(

00000000000000

00000000000000000000001111000000001111

VIVI

VIVI

IIIIIIII

VV

VVVVVVVV

VVVVVV

A

A

A

A

C

C

C

C

B

B

B

B

Subsequently

Rank＝8

IA(0)～IA(3)：Measure (GCMS or NMR)V1（=V4 ∵(i)）: Determined

Unknown Vars：V2→、 V2← 、V3、IB(0)～IB(3)、IC(0)～IC(3)

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lac I lac P lac O lac Z lac Y lac A

mRNA

Lactose Absent

lac repressor (active)

lac I lac P lac O lac Z lac Y lac A

mRNA

Lactose Present

Repressor-lactosecomplex (inactive)lactose

RNAPolymerase

mRNA mRNA mRNA

Transcription

No Transcription

Molecular Regulation in lacoperon

Lactose Absent

Lac operon is repressed by lac repressor

Lactose present→repressor inactivated

Β-galactosidase、lactose permiase transcripted

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Metabolic Control Analysis

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

Problem: which enzyme has the greatest impact on target product in complicated network ?

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J

Definition of FCC: infinitesimal perturbation at original point

Fig.6 Shimizu

X1 X2 X3 X4 Xn Xn+1

e1 e2 e3 en

J1 J2 J3 Jn

( eir , Jk

r )(ei

0 , Jk0 )

△J

ei

△e

i

k

eJ

JeC

k

iJki ∂

∂=

k

Quantification of impact of change in enzyme act on flux

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Today’s Home Work

What is essence of Metabolic Engineering?