An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli

Area of Science:

• Synthetic Biology
• Molecular Biology
• Protein Engineering

Background:

• Accelerating protein evolution is crucial for developing novel functions.
• Existing methods for directed evolution face limitations in speed and efficiency.
• Maintaining host genome integrity during hypermutation is a key challenge.

Purpose of the Study:

• To engineer an orthogonal DNA replication system for controlled gene hypermutation in Escherichia coli.
• To enhance protein functions through accelerated evolution.
• To demonstrate the system's utility in rapidly improving enzyme activity.

Main Methods:

• Constructed an orthogonal DNA replication system (T7-ORACLE) in Escherichia coli using bacteriophage T7 replisome components.
• Engineered T7 DNA polymerase variants to achieve significantly elevated in vivo mutation rates.
• Utilized circular plasmids for high transformation efficiency and integration into standard molecular biology workflows.

Main Results:

• Achieved in vivo mutation rates of 1.7 × 10^-5 substitutions per base, ~100,000-fold higher than the genomic mutation rate.
• Demonstrated continuous evolution of TEM-1 β-lactamase, increasing its activity 5000-fold against specific antibiotics within one week.
• The system maintained host genome integrity during hypermutation.

Conclusions:

• The T7-ORACLE system provides a powerful platform for rapid, targeted protein evolution.
• This approach significantly accelerates the discovery and optimization of protein functions.
• The system has broad applications in protein engineering and synthetic biology.