Strategies for scarless chromosomal gene deletion, promoter replacement and RBS fine-tuning. (A) Genome editing cassettes are constructed by three rounds of PCR and recombinants after the first round of recombineering were selected by Tetr. (B) In the second step, the TetA marker were released by simultaneous induction of I-SceI and Red recombinase expression. (C) The genetic construct for promoter replacement after the first round of recombination. DR for duplicate region; I-sceI, I-SceI endonuclease recognition site.