Abstract
We herein adapted a markerless gene replacement method by combining a temperature-sensitive plasmid pKSV7 with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the poly-γ-glutamic acid (γ-PGA)-producing strain Bacillus amyloliquefaciens LL3. Deletion of the upp gene conferred LL3 5-fluorouracil (5-FU) resistance. Sensitivity to 5-FU was restored when LL3 Δupp was transformed with pKSV7-based deletion plasmid which carries a functional allele of the upp gene of Bacillus subtilis 168. These observations allowed us to adapt a two-step plasmid integration and excision strategy to perform markerless deletion of genes of interest. Deletion plasmid harboring a mutant allele of the target gene was first integrated in the genome by culturing cells under nonpermissive conditions for pKSV7 replication. Single-crossover recombinants were then grown without antibiotics to aid the second recombinational event. 5-FU was used to select for double-crossover recombinants with plasmid evicted from the chromosome. The resulting recombinants either harbored the wild-type or mutated allele of the target gene and could be identified by PCR and DNA sequencing. Using this method, we successively removed the amyA gene and a 47-kb fragment of the bae cluster from the genome of LL3, with higher efficiency compared with previous reports. We also investigated the effects of a transcriptional regulator, RocR, on γ-PGA production and cell growth. Specific γ-PGA production of the rocR mutant was increased by 1.9-fold, which represents a new way to improve γ-PGA production.
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Acknowledgments
This work was supported by the National Key Basic Research Program of China (“973”-Program) 2012CB725204; the National High Technology Research and Development Program of China (“863”-Program) 2012AA021505; the Natural Science Foundation of China Grant Nos. 31170030, 31300032, and 51073081; and the Project of Tianjin, China (13JCZDJC27800, 13JCYBJC24900, and 13JCQNJC09700). This work was also supported by the Ph.D. Candidate Research Innovation Fund of Nankai University.
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Zhang, W., Gao, W., Feng, J. et al. A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production. Appl Microbiol Biotechnol 98, 8963–8973 (2014). https://doi.org/10.1007/s00253-014-5824-2
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DOI: https://doi.org/10.1007/s00253-014-5824-2