CARD Computer-Aided Rational Design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli. http://www.cadlive.jp
Objectives Propose CAD-based rational design of a biochemical network for an engineering purpose cell Substrate Product Substrate
Computer simulation Biological experiment Network design CADLIVE System Concept of CAD
Robustness Perturbation Parameter Time Biological systems maintain their homeostasis against environmental stress, genetic changes and noises. Feedback Feedforward Pathway redundancy How do you change such a robust system?
Architecture check Mathematical check Design strategy for rational design of biochemical networks
Modular decomposition Define an engineering purpose. Function of sub-networks is assigned in analogous to control engineering architecture Check our paper: Heat shock response PLoS Comp Biol, 2006 PNAS, 2005
Perturbation analysis for finding critical genes Cell substrate Product vs vp E: enzyme
Plant FB Sensor Accelerator Actuator Computer Brake Actuator Glucose PTS network map
Plant FB Sensor Accelerator Actuator Computer Brake Actuator Accelerator Flux Module Brake Flux Module
cAMP IIA-P IIA ptsI ptsH ptsG Dynamic simulation reproduces the experimental behaviors Glucose depletion on 500 min
Model validation by experiments cAMP Experimental data are reproduced by our dynamic model IIA-P Mlc knockout ptsG
Plant FB Sensor Accelerator Actuator Computer Brake Actuator Critical genes are explored for enhanced glucose uptake PTS proteins Accelerator Flux Module crp mlc Brake Flux Module
Recombinant strategy • Brake flux module Negative feedback DELETE • Accelerator flux module Positive feedbackENHANCE
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.
Dynamic simulations PtsI overexpression is effective for enhanced PLANT and increases cAMP
EXPERIMENT Enhanced specific glucose uptake by ptsI overexpression in an mlc knockout mutant as predicted.
PERTURBATION EXPERIMENT Experimental results of growth, glucose uptake, specific glucose uptake, and cAMP concentration in growing cells. 11.08 (prediction)
MODEL IMPROVEMENT FB Sensor Plant Accelerator Actuator Computer Brake Actuator
PERTURBATION ANALYSIS (SIMULATION) In the improved model
Conclusion 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.