The accurate replacement of long genome region more than several hundreds kilobases in Bacillus subtilis

Competent cell transformation with DNA obtained by the gentle lysis of protoplasts (LP transformation) was used to replace a large genomic region in this study. Discontinuity was detected in the replacement of the donor region tested, probably due to multiple crossover events involving a single donor genome fragment. To overcome discontinuous replacement, we inverted the genomic region to be replaced in the donor used for LP transformation. The replaced region in the transformant was identified to have a continuous genomic region originating from the donor genome. Furthermore, the genome region to be replaced was inverted in the recipient, and the same region and the flanking 10 kb region of both ends was inverted in the donor genome. LP transformation was conducted with the two inversion mutants and it is possible to restrict homologous recombination to the 10 kb flanking regions. Using this method, the 99 kb yxjG-yxbA region, the 249 kb pbpG-yxbA region and the 602 kb yvfT-yxbA region were suggested to be replaced continuously and accurately.