Abstract
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.
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Acknowledgments
We would like to thank Professor Tingyi Wen from Institute of Microbiology of Chinese Academy of Sciences for the kind donation of plasmid pWYE1088. This study was supported by National Program on Key Basic Research Project (2011CBA00804, 2012CB725203), National Natural Science Foundation of China (NSFC-21206112, NSFC-21176182, NSFC-21390201), National High-tech R&D Program of China (2012AA022103, 2012AA02A702).
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Supplementary Table 1—Primers used in this study.
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Ma, W., Wang, X., Mao, Y. et al. Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker. Biotechnol Lett 37, 609–617 (2015). https://doi.org/10.1007/s10529-014-1718-8
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DOI: https://doi.org/10.1007/s10529-014-1718-8