Deaminase-driven random mutation enables efficient DNA mutagenesis for protein evolution

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein evolution is vital for creating proteins with new functions.
• DNA mutagenesis is a key step in protein evolution.
• Current methods like error-prone PCR (epPCR) have limitations in mutation efficiency and diversity.

Purpose of the Study:

• To develop a novel DNA mutagenesis strategy with higher efficiency and diversity than epPCR.
• To introduce a broad spectrum of mutations in a single round of mutagenesis.
• To facilitate the discovery of novel protein mutants.

Main Methods:

• Developed deaminase-driven random mutation (DRM) using engineered cytidine deaminase A3A-RL and adenosine deaminase ABE8e.
• Introduced C-to-T, G-to-A, A-to-G, and T-to-C mutations on both DNA strands.
• Compared DRM to epPCR in terms of mutation frequency and diversity.

Main Results:

• DRM demonstrated a 14.6-fold higher DNA mutation frequency compared to epPCR.
• DRM produced a 27.7-fold greater diversity of mutation types than epPCR.
• DRM enables a more comprehensive exploration of the genetic landscape for protein engineering.

Conclusions:

• DRM is a powerful tool for protein evolution, offering superior mutagenic capability.
• This strategy significantly enhances the discovery of novel and improved protein mutants.
• DRM provides high-quality DNA products for protein engineering applications.