Multiple large segment deletion method for Corynebacterium glutamicum.
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.
Suzuki
Nobuaki
N
Microbiology Research Group, Research Institute of Innovative Technology for the Earth (RITE), Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto, Japan.
Nonaka
Hiroshi
H
Tsuge
Yota
Y
Okayama
Satoshi
S
Inui
Masayuki
M
Yukawa
Hideaki
H
D002872:Chromosome Deletion; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D017384:Sequence Deletion / Q000235:genetics*; D013379:Substrate Specificity