Directed evolution of λ integrase activity and specificity by genetic derepression

Area of Science:

• Molecular Biology
• Enzyme Engineering
• Genomics

Background:

• Genome engineering relies on novel enzymes with tailored specificity and activity.
• Existing methods for enzyme evolution require improvement for efficiency and precision.

Purpose of the Study:

• To develop a novel selection method for engineering recombinase enzymes.
• To generate improved lambda integrase variants for enhanced in vitro recombination.
• To establish a facile method for transgene integration in E. coli.

Main Methods:

• A novel selection strategy was devised coupling recombinase activity to enzyme function via a selectable phenotype.
• Beta-lactamase and its inhibitor were used to link recombinase activity to ampicillin resistance in E. coli.
• Lambda integrase variants were generated and screened for improved recombination of non-cognate substrates.

Main Results:

• The selection method successfully generated lambda integrase variants with improved in vitro recombination activity.
• One variant demonstrated enhanced catalytic activity and generalist properties.
• This variant enabled a single-step transformation to introduce large transgenes (up to 8.5 kb) into E. coli.

Conclusions:

• The developed selection method is effective for engineering recombinases with improved activity.
• The novel lambda integrase variants offer enhanced capabilities for genome engineering applications.
• A streamlined method for transgene insertion in E. coli was established using engineered integrase.