Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker.
Corynebacterium glutamicum is well-established for industrial and biotechnological applications. However, its genetic manipulation has generally lagged behind traditional genetic models. In this study, a counter-selectable marker gene upp was firstly confirmed to be more efficient than traditional sacB. Furthermore, a markerless gene replacement system was developed by combining upp with double-strand break repair caused by the exogenous endonuclease I-SceI. Finally, genetic modification using a dsDNA PCR fragment was carried out with the expression of recombinase/exonuclease RecE/RecT. Our results show that the genetic modification system allows precise and markerless gene replacement without altering the chromosome, with a simplified screening procedure to generate its modification.
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Wenwen
W
Ma
Xiaoyue
X
Wang
Yufeng
Y
Mao
Zhiwen
Z
Wang
Tao
T
Chen
Xueming
X
Zhao
D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012641:Selection, Genetic
D045522:Recombinases; D005090:Exodeoxyribonucleases; D004262:DNA Restriction Enzymes