Multiple large segment deletion method for Corynebacterium glutamicum.
Appl. Microbiol. Biotechnol. 69: 151-161. 2005.
N. Suzuki, H. Nonaka, Y. Tsuge, S. Okayama, M. Inui and H. Yukawa.

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.