A high-throughput screening method to reengineer DNA polymerases for random mutagenesis

Area of Science:

• Molecular Biology
• Biotechnology
• Enzyme Engineering

Background:

• Directed evolution is crucial for tailoring enzyme properties.
• DNA polymerases with specific functionalities are needed for synthetic biology and genetic engineering.
• Existing methods for polymerase screening can be limiting, especially for enzymes with low processivity.

Purpose of the Study:

• To develop and validate a novel screening system for directed evolution of DNA polymerases.
• To improve the elongation efficiency of consecutive mismatches in a distributive polymerase.
• To create engineered polymerases suitable for diverse applications in genetic engineering.

Main Methods:

• Development of a screening system utilizing a fluorescent Scorpion probe as a reporter.
• Application of the screening system to directed evolution of the distributive DNA polymerase Dpo4 from Sulfolobus solfataricus.
• Validation through sequence saturation mutagenesis experiments to assess improvements in mutation frequency.

Main Results:

• The developed Scorpion probe screening system was successfully validated.
• Engineered Dpo4 variants showed improved elongation efficiency, particularly for consecutive mismatches.
• One improved variant demonstrated a 2.5-fold increase in desirable consecutive transversion mutations compared to wild-type Dpo4.

Conclusions:

• The Scorpion probe screening system is effective for reengineering DNA polymerases with limitations in processivity and fidelity.
• This system facilitates the generation of diverse DNA polymerases for applications in directed protein evolution.
• The improved Dpo4 variants expand the toolkit for genetic engineering and synthetic biology.