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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