1. Computer-Aided Rational Design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli. http://www.cadlive.jp CARD

2. Objectives Propose CAD-based rational design of a biochemical network for an engineering purpose Product Substrate cell

3. Network design Computer simulation Biological experiment CADLIVE System Concept of CAD

4. Robustness Feedback Feedforward Pathway redundancy How do you change such a robust system? Time Parameter Perturbation Biological systems maintain their homeostasis against environmental stress, genetic changes and noises.

5. Methods

6. Design strategy for rational design of biochemical networks Mathematical check Architecture check

7. Modular decomposition Define an engineering purpose. Function of sub-networks is assigned in analogous to control engineering architecture PLoS Comp Biol, 2006 PNAS, 2005 Check our paper: Heat shock response

8. Perturbation analysis for finding critical genes substrate Product vpvp vsvs Cell E: enzyme

9. Results and Discussion

10. PlantFB Sensor Computer Accelerator Actuator Brake Actuator Glucose PTS network map

11. PlantFB Sensor Computer Accelerator Actuator Brake Actuator Brake Flux Module Accelerator Flux Module

12. Dynamic simulation reproduces the experimental behaviors IIA-P IIA cAMP Glucose depletion on 500 min ptsI ptsH ptsG

13. Model validation by experiments Experimental data are reproduced by our dynamic model Mlc knockout ptsG IIA-P cAMP

14. Critical genes are explored for enhanced glucose uptake PlantFB Sensor Computer Accelerator Actuator Brake Actuator Brake Flux Module Accelerator Flux Module mlc PTS proteins crp

15. Recombinant strategy Brake flux module Negative feedback DELETE Accelerator flux module Positive feedback ENHANCE

16. 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.

17. PtsI overexpression is effective for enhanced PLANT and increases cAMP Dynamic simulations

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

19. PERTURBATION EXPERIMENT Experimental results of growth, glucose uptake, specific glucose uptake, and cAMP concentration in growing cells. 11.08 (prediction)

20. Plant FB Sensor Computer Accelerator Actuator Brake Actuator MODEL IMPROVEMENT

21. PERTURBATION ANALYSIS (SIMULATION) In the improved model

22. 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