Applied Microbiology and Biotechnology

, Volume 101, Issue 15, pp 6039–6048 | Cite as

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome

  • Kai Huang
  • Tao Zhang
  • Bo JiangEmail author
  • Xin Yan
  • Wanmeng Mu
  • Ming Miao
Biotechnologically relevant enzymes and proteins


A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg R.pyc cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg R.pyc cassette sandwiched between “front” and “back” homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5 U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze l-arginine with a 97.2% molar yield, showing potential application in the food industry.


l-ornithine Bacillus subtilis Rummeliibacillus pycnus arginase Integration Multi-copy Marker-less 



This work was financially supported by the 863 Project of China (No. 2013AA102102) and the Fundamental Research Funds for the Central Universities (SKLF-ZZA-201509).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2017_8355_MOESM1_ESM.pdf (542 kb)
ESM 1 (PDF 541 kb).


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Kai Huang
    • 1
  • Tao Zhang
    • 1
  • Bo Jiang
    • 1
    Email author
  • Xin Yan
    • 2
  • Wanmeng Mu
    • 1
  • Ming Miao
    • 1
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of AgricultureNanjing Agricultural UniversityNanjingChina

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