Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker

作者：Ma Wenwen; Wang Xiaoyue; Mao Yufeng; Wang Zhiwen^*; Chen Tao; Zhao Xueming

来源：Biotechnology Letters, 2015, 37(3): 609-617.

DOI：10.1007/s10529-014-1718-8

• Corynebacterium glutamicum
• Double-strand break
• Markerless gene replacement
• Recombination
• I-SceI
• upp

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.

• 出版日期2015-3
• 单位天津大学