An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli

Area of Science:

• Molecular Biology
• Protein Engineering
• Synthetic Biology

Background:

• Accelerating protein evolution is crucial for developing new functions.
• Existing methods for directed evolution often face limitations in speed and efficiency.
• The integrity of the host genome must be maintained during hypermutation.

Purpose of the Study:

• To develop an orthogonal DNA replication system for accelerated gene evolution.
• To engineer bacteriophage T7 DNA polymerase for high-fidelity hypermutation.
• To demonstrate the system's efficacy in enhancing protein function.

Main Methods:

• Constructed an orthogonal DNA replication system in E. coli using bacteriophage T7 replisome components.
• Engineered T7 DNA polymerase variants to achieve high in vivo mutation rates.
• Utilized circular plasmids for efficient transformation and integration into molecular biology workflows.
• Applied the system for continuous evolution of TEM-1 beta-lactamase.

Main Results:

• Achieved mutation rates 100,000-fold higher than the genomic mutation rate (1.7 × 10^-5 substitutions/base).
• Demonstrated rapid protein evolution, increasing TEM-1 beta-lactamase activity 1,000-fold.
• Enhanced enzyme activity against clinically relevant monobactam and cephalosporin antibiotics within one week.

Conclusions:

• The engineered T7 replisome system enables rapid and efficient hypermutation without compromising host genome integrity.
• This platform significantly accelerates the evolution of novel or enhanced protein functions.
• The system holds promise for applications in protein engineering and drug discovery.