Integrated Constraint-Based Modeling of E. coli Cell-Free Protein Synthesis.

Area of Science:

• Systems biology
• Synthetic biology
• Biochemistry

Background:

• Cell-free protein expression is vital for synthetic biology and biomanufacturing.
• Cell-free systems require metabolic pathways for precursor and energy regeneration.
• Understanding metabolism's role is key to optimizing cell-free protein production.

Approach:

• Developed a dynamic constraint-based simulation for the myTXTL E. coli cell-free system.
• Integrated time-resolved metabolite, mRNA, protein, and enzyme activity data.
• Simulated metabolic flux and protein production with and without electron transport chain inhibitors.

Key Points:

• myTXTL cell-free system exhibits active central carbon metabolism, with glutamate fueling the TCA cycle.
• Metabolic flux analysis indicated oxidative phosphorylation activity in the cell-free system.
• Inhibitor studies confirmed reliance on oxidative phosphorylation for sustained transcription and translation.

Conclusions:

• Cell-free systems possess active, energy-generating metabolic pathways.
• Metabolic modeling and experimental data elucidate energy regeneration mechanisms in cell-free protein synthesis.
• Findings advance the potential of cell-free technology for biomanufacturing.