Computer-Aided Rational Design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli.

http://www.cadlive.jp

CARD

Objectives

Propose CAD-based rational design of a biochemical network for an engineering purpose

Product

Substrate

Substrate

cell

Network design

Computer simulationBiological experiment

CADLIVE System

Concept of CAD

Robustness

FeedbackFeedforwardPathway redundancy

How do you change such a robust system?

Time

Par

amet

er Perturbation

Biological systems maintain their homeostasis against environmental stress, genetic changes and noises.

Methods

Network map constructionDynamic modeling

Module decomposition analogous to control engineering

Finding critical genes by perturbation analysis

Design completed

Design start

ConflictNo conflict

Experimental verification

Design strategy for rational design of biochemical networks

Mathematical check

Architecture check

Modular decomposition

Define an engineering purpose.Function of sub-networks is assigned in analogous to control engineering architecture

PLoS Comp Biol, 2006PNAS, 2005

Check our paper: Heat shock response

Perturbation analysis for finding critical genes

E

E

E

E

E

E

E

E

E

E

substrateProduct

vp

vs

pp

vS

E

s

s

vS

E

Cell

E: enzyme

Results and Discussion

G6P PEP

IICB IIA HPr EIGlucose

PYR

Mlc

mRNA(ptsG)

mRNA(crr) mRNA(ptsH)mRNA(ptsI)ATPcAMP

CRP

CYA

mRNA(cyaA)

mRNA(mlc)

mRNA(crp)

Mlc(ptsGp2)

Mlc(ptsGp1)

CRP(ptsGp2)

CRP(ptsGp1)

ptsGcrp

CRP(crpp1)

CRP(crpp2)

Mlc(mlcp2)

CRP(mlcp2)

Mlc(mlcp1)

CRP(mlcp1)

Mlc

CRP(cyaAp)

cyaAMlc(ptsHp0)

CRP(ptsHp1)

CRP(ptsHp0)

ptsH

crr

IICB-P IIA-P HPr-P EI-P

Mlc(ptsIp0)

CRP(ptsIp1)

CRP(ptsIp0)

ptsI

Plant FBSensor

Computer

Accelerator Actuator

Brake ActuatorGlucose PTS network map

Plant FBSensor

Computer

Accelerator Actuator

Brake ActuatorBrake Flux Module

Accelerator Flux Module

Dynamic simulation reproduces the experimental behaviors

IIA-P

IIA

cAMP

Glucose depletion on 500 min

ptsI

ptsHptsG

Model validation by experiments

Experimental data are reproduced by our dynamic model

Mlc knockoutptsG

IIA-P

cAMP

Critical genes are explored for enhanced glucose uptake

Plant FBSensor

Computer

Accelerator Actuator

Brake ActuatorBrake Flux Module

Accelerator Flux Modulemlc

PTS proteins

crp

Recombinant strategy

• Brake flux module

Negative feedback DELETE

• Accelerator flux module

Positive feedback ENHANCE

Geneamplification

none mlc knockoutcrp

overexpression

Wild 1 1.02 2.91

ptsG 0.81 0.81 2.91

ptsH 7.95 8.05 8.23

ptsI 10.83 11.08 9.53

crr 3.48 3.51 3.51

Genetic background

PERTURBATION ANALYSIS (SIMULATION)Prediction of changes in the specific glucose uptake rate for mathematical mutants. The values are the ratios of the specific glucose uptake rate for a mutant to that for wild type.

PtsI overexpression is effective for enhanced PLANT and increases cAMP

Dynamic simulations

Enhanced specific glucose uptake by ptsI overexpression in an mlc knockout mutant as predicted.

EXPERIMENT

Strain GrowhSpecific glucose

uptakecAMP

concentration

Wild 1 1 10.88

ptsI overexpression 1.2 1.2 7.54

mlc knockout 0.83 1.1

mlc knockout +ptsI overexpression

0.14 1.7

PERTURBATION EXPERIMENTExperimental results of growth, glucose uptake, specific glucose uptake, and cAMP concentration in growing cells.

11.08 (prediction)

G6P PEP

IICB IIA HPr EIGlucose

PYR

Mlc

mRNA(ptsG)

mRNA(crr) mRNA(ptsH)mRNA(ptsI)ATPcAMP

CRP

CYA

mRNA(cyaA)

mRNA(mlc)

mRNA(crp)

Mlc(ptsGp2)

Mlc(ptsGp1)

CRP(ptsGp2)

CRP(ptsGp1)

ptsGcrp

CRP(crpp1)

CRP(crpp2)

Mlc(mlcp2)

CRP(mlcp2)

Mlc(mlcp1)

CRP(mlcp1)

Mlc

CRP(cyaAp)

cyaAMlc(ptsHp0)

CRP(ptsHp1)

CRP(ptsHp0)

ptsH

crr

IICB-P IIA-P HPr-P EI-P

Mlc(ptsIp0)

CRP(ptsIp1)

CRP(ptsIp0)

ptsI

Plant FBSensor

Computer

Accelerator Actuator

Brake Actuator

MODEL IMPROVEMENT

Geneamplification

none mlc knockoutcrp

overexpression

Wild 1 1.21 3.85

ptsG 1.25 1.27 4.55

ptsH 3.49 4.55 9.86

ptsI 3.87 5.7 10.87

crr 0.86 1.04 3.25

Genetic background

PERTURBATION ANALYSIS (SIMULATION)In the improved model

A computer-aided rational design approach was successfully applied to the Escherichia coli glucose PTS to increase the specific glucose uptake rate.

The proposed method predicted that the mlc knockout mutant with ptsI gene overexpression greatly increases the specific glucose uptake rate and verified it by biological experiments.

Conclusion