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 restructuring 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 promoters: (i) an arabinose-inducible promoter that drives expression of lambda-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, 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 performed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacterial 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.