Red/ET-mediated recombination (also referred to as recombineering) is a powerful method for DNA engineering. It allows precise cloning and modification of large DNA molecules at any chosen position. This method has initially been developed by the Steward Group at the European Molecular Biology Laboratories (EMBL) in Heidelberg (Germany)(26).
We have adopted and exploited this technique for DNA manipulation of streptomycetes DNA, e.g. generation of gene deletions. The strategy is to replace a chromosomal sequence within a BAC, fosmid or cosmid clone by a selectable resistance marker that has been generated by amplification of a disruption cassette using primers with only ~40 nt homology extensions. The lambda-RED or the corresponding RecE/RecT functions have the unique advantage in that they can catalyze efficient recombination with very short regions of sequence homology. Inclusion of an oriT in the disruption cassette allows conjugation to be used to introduce the modified DNA into a wide range of actinomycetes. Targets for site-specific recombinases, such as loxP or FRT sites are added on the flanks of the disruption cassettes allowing the subsequent removal of the cassettes by activating expression of site-specific recombinases such as Cre or Flp. The potent methyl-specific restriction system of some streptomycetes is circumvented by passaging DNA through a methylation-deficient E. coli host.
Recombineering was used to generate more than 200 gene disruptions so far, gene fusions, promoter replacements, gene cluster assemblies and expression of biosynthetic gene clusters in heterologous hosts(6-8). Recombineering is now well established as standard procedure for engineering streptomycetes DNA and has been adopted by many research groups worldwide.
