Continuous Evolution of Protein through T7 RNA Polymerase-Guided Base Editing in Corynebacterium glutamicum

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Directed evolution is crucial for protein engineering.
• Targeted mutagenesis systems are essential for efficient protein modification.
• Developing robust systems for continuous evolution in bacteria like Corynebacterium glutamicum is needed.

Purpose of the Study:

• To develop an efficient and targeted mutagenesis system for continuous gene evolution in Corynebacterium glutamicum.
• To optimize the system for high mutant frequency and low off-target effects.
• To demonstrate the system's utility in improving protein function.

Main Methods:

• Sequential fusion of cytosine deaminase and uracil-DNA glycosylase inhibitor to T7 RNA polymerase to create the CgMutaT7 system.
• Optimization of the CgMutaT7 system (resulting in CgMutaT7^4).
• High-throughput sequencing to analyze mutation profiles and frequencies.

Main Results:

• The optimized CgMutaT7^4 system achieved a 1.12 × 10^4-fold increase in target gene mutant frequency.
• Low off-target mutation rates were observed.
• Efficient and uniform C → T transitions were confirmed across a 1.8 kb DNA region.
• Continuous evolution of xylose isomerase to enhance xylose utilization was successfully demonstrated.

Conclusions:

• The CgMutaT7 system provides an efficient tool for targeted, continuous mutagenesis in Corynebacterium glutamicum.
• The system demonstrates significant potential for protein function and expression component evolution.
• This technology can accelerate the development of improved enzymes and biological components.