Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Single Crossover-Mediated Markerless Genome Engineering in Clostridium acetobutylicum

  • Lee, Sang-Hyun (Department of Biotechnology, Graduate School, Korea University) ;
  • Kim, Hyun Ju (Biosystems and Bioengineering Program, University of Science and Technology and Microbiomics and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Shin, Yong-An (R&D Center, GS Caltex Corporation) ;
  • Kim, Kyoung Heon (Department of Biotechnology, Graduate School, Korea University) ;
  • Lee, Sang Jun (Biosystems and Bioengineering Program, University of Science and Technology and Microbiomics and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2015.12.07
  • Accepted : 2016.01.15
  • Published : 2016.04.28
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Abstract

A novel genome-engineering tool in Clostridium acetobutylicum was developed based on single-crossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.

Keywords

References

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