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We have developed an improved and rapid genomic engineering procedure for the construction of custom-designed microorganisms. This method, which can be performed in 2 days, permits restruc- turing of the Escherichia coli genome via markerless deletion of selected genomic regions. The deletion process was mediated by a special plasmid, pREDI, which carries two independent inducible promot- ers: (i) an arabinose-inducible promoter that drives expression of j-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cas- sette, and (ii) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which stimulates deletion of the introduced marker by double-strand breakage-mediated intramolecular recombination. This genomic deletion was per- formed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacter- ial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette were nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

Rapid and efficient construction of markerless deletions in the Escherichia coli genome