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New plasmid expression vectors for Bacillus subtilis.

G Grandi, M Del Bue, E Palla

    Plasmid
    |July 1, 1986
    PubMed
    Summary
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    New cloning vectors for Bacillus subtilis were created by combining pE194 and pUB110 DNA. These vectors facilitate the efficient insertion and expression of heterologous genes in B. subtilis, as demonstrated with E. coli enzymes.

    Area of Science:

    • Molecular Biology
    • Microbial Genetics
    • Biotechnology

    Background:

    • Bacillus subtilis is a key industrial microorganism, but efficient gene expression systems are crucial for its biotechnological applications.
    • Existing cloning vectors may have limitations in accommodating diverse heterologous gene insertions and ensuring high-level expression.
    • Development of versatile cloning vectors is essential for expanding the genetic engineering capabilities of B. subtilis.

    Purpose of the Study:

    • To construct and characterize novel cloning vectors for Bacillus subtilis.
    • To enable the efficient cloning and expression of heterologous genes within B. subtilis.
    • To provide a platform for the production of foreign proteins in B. subtilis.

    Main Methods:

    • In vitro joining of plasmid DNA fragments from pE194 and pUB110.

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  • Construction of vectors with a cloning site downstream of the erythromycin resistance gene's promoter and ribosome binding site.
  • Insertion and expression of Escherichia coli beta-lactamase and EcoRI methylase genes.
  • Verification of enzyme synthesis and activity in B. subtilis.

    • Main Results:

    • Novel cloning vectors for B. subtilis were successfully constructed.
    • The vectors allow for the insertion and expression of both sticky and blunt-ended DNA fragments.
    • Efficient synthesis of functional Escherichia coli beta-lactamase and EcoRI methylase was achieved in B. subtilis.
    • The engineered vectors demonstrate high expression levels for heterologous genes.

    Conclusions:

    • The developed cloning vectors offer a robust and versatile tool for genetic manipulation of Bacillus subtilis.
    • These vectors significantly enhance the potential for using B. subtilis as a host for recombinant protein production.
    • The efficient expression of tested heterologous genes highlights the broad applicability of these new vectors.