Directed evolution of λ integrase activity and specificity by genetic derepression

Protein Eng Des Sel. 2015 Jul;28(7):211-20. doi: 10.1093/protein/gzv015. Epub 2015 Mar 18.


Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using β-lactamase and the β-lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin. Using this method, we generated λ integrase variants displaying improved in vitro recombination of a non-cognate substrate present in the human genome. One generalist integrase variant displaying enhanced catalytic activity was further used in a facile, single-step transformation method to introduce transgenes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.

Keywords: directed evolution; integrase; recombineering; synthetic biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line, Tumor
  • Directed Molecular Evolution / methods*
  • Escherichia coli / genetics
  • Humans
  • Integrases / genetics*
  • Integrases / metabolism*
  • Mutation
  • Recombination, Genetic
  • Substrate Specificity
  • Transformation, Genetic
  • beta-Lactamases / genetics


  • Integrases
  • beta-Lactamases