Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum

Area of Science:

• Microbiology
• Synthetic Biology
• Genetic Engineering

Background:

• High spontaneous mutation rates are essential for discovering new phenotypes and understanding gene-phenotype relationships.
• Increasing mutation frequency is a key challenge in microbial strain development and genetic exploration.

Purpose of the Study:

• To develop a practical and controllable evolutionary tool (oMut-Cgts) for Corynebacterium glutamicum.
• To significantly increase mutation rates through dual genetic level modification.

Main Methods:

• Engineered RNA polymerase α subunit and DNA helicase Cgl0854 as anchors for cytidine deaminase (pmCDA1).
• Localized pmCDA1 to transient single-stranded DNA (ssDNA) regions during transcription and replication.
• Combined and optimized dual genetic level modifications.

Main Results:

• Achieved a 1.02 × 10^4-fold increase in mutation rate.
• Demonstrated uniform and efficient C:G→T:A transitions genome-wide.
• Successfully evolved Corynebacterium glutamicum for enhanced stress tolerance (acid, oxidative, ethanol).

Conclusions:

• The oMut-Cgts tool enables rapid genome evolution and phenotype enhancement in Corynebacterium glutamicum.
• This method is effective for multi-dimensional biological engineering, including gene function mining and protein evolution.
• The strategy is potentially applicable to all prokaryotic cells for rapid genome evolution.