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A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium
Abstract:
A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.
Insights
A novel plasmid vector, pHY481, demonstrates stable maintenance and enhanced gene expression in Bacillus brevis 47. Amplified copy number mutants significantly increase protein production, aiding genetic engineering.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Bacillus brevis 47 is a key protein-producing bacterium.
- Development of stable plasmid vectors is crucial for genetic manipulation in B. brevis.
- Existing vectors may lack stability or efficient gene expression.
Purpose of the Study:
- To construct and characterize a novel low-copy-number plasmid vector (pHY481) for B. brevis 47.
- To assess the stability and gene expression capabilities of pHY481.
- To develop B. brevis 47 mutants with amplified plasmid copy numbers for enhanced protein production.
Main Methods:
- Construction of pHY481 by combining Staphylococcus aureus and Bacillus brevis cryptic plasmids.
- Introduction of pHY481 into B. brevis 47 and assessment of plasmid stability without antibiotics.
- Subcloning of a Bacillus megaterium alpha-amylase gene into pHY481 and comparison with an S. aureus plasmid.
- Isolation of B. brevis 47 mutants with amplified pHY481 copy numbers.
Main Results:
- pHY481 exhibited high stability in B. brevis 47 without selective pressure.
- The alpha-amylase gene was retained more stably on pHY481 than on an S. aureus plasmid.
- B. brevis 47 mutants with approximately 10-fold amplification of pHY481 copy number were generated.
- Enzyme production increased severalfold in these amplified copy number mutants.
Conclusions:
- The constructed plasmid vector pHY481 is stable and effective for gene cloning in B. brevis 47.
- Copy-number amplified mutants of B. brevis 47 enhance foreign gene expression.
- This system provides a valuable tool for genetic engineering and protein production in B. brevis.