The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4-104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3-100%. Additionally, the recombination efficiencies were 2.5-45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.