Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 101, Issue 15, Pages 6039-6048Publisher
SPRINGER
DOI: 10.1007/s00253-017-8355-9
Keywords
L-ornithine; Bacillus subtilis; Rummeliibacillus pycnus arginase; Integration; Multi-copy; Marker-less
Categories
Funding
- 863 Project of China [2013AA102102]
- Fundamental Research Funds for the Central Universities [SKLF-ZZA-201509]
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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.
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