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Red recombinase assisted gene replacement in Klebsiella pneumoniae

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The Red recombinase system, the most convenient genetic tool applied in Escherichia coli and other bacteria, was introduced for gene replacement in Klebsiella pneumoniae. The novel K. pneumoniae gene replacement system comprised the Red and FLP recombinases expression vector pDK6-red and pDK6-flp, and linear DNA fragments which encompassed a selective marker gene with target gene flanking extensions; the latter were PCR amplified using a plasmid DNA template obtained by in vivo recombination in E. coli. In this study, dhak1 gene, encoding a subunit of dihydroxyacetone kinase II, was deleted markerlessly at a transformation ratio of 260 CFU/μg DNA, i.e., 1,000-fold higher than that achieved in the native way. Our studies provide an efficient method with detailed protocol to perform gene replacement in K. pneumoniae and has great potential to be developed as a routine genetic approach for this important industrial microorganism.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Grant No. 20906076).

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Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People’s Republic of China

    Dong Wei & Min Wang

  2. Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai, 201210, People’s Republic of China

    Dong Wei, Jiping Shi & Jian Hao

Authors
  1. Dong Wei
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  2. Min Wang
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  3. Jiping Shi
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  4. Jian Hao
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Correspondence to Min Wang or Jian Hao.

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Wei, D., Wang, M., Shi, J. et al. Red recombinase assisted gene replacement in Klebsiella pneumoniae . J Ind Microbiol Biotechnol 39, 1219–1226 (2012). https://doi.org/10.1007/s10295-012-1117-x

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  • DOI: https://doi.org/10.1007/s10295-012-1117-x

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