Construction of Single-stranded Oligonucleotide-mediated Homologous Recombination in Bacillus amyloliquefaciens
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Abstract:
Bacillus amyloliquefaciens is an important industrial microorganism that has been widely used in agriculture, pharmaceutical, and food industries. In order to develop highly efficient genetic manipulation techniques in B. amyloliquefaciens, a single-stranded oligonucleotide (ssDNA)-mediated recombination method was introduced. First, the mutS gene involved in mismatch repair was knocked out, followed by introduction of the expression plasmid for the bet gene encoding the beta protein (a ssDNA-binding protein) into B. amyloliquefaciens, to construct the B. amyloliquefaciens XH7 host (mutS-，bet+), where the mutS gene was inactivated and the beta protein was expressed. Second, homologous recombination targeting the ropB gene was successfully achieved by designing an 88-bp ssDNA oligonucleotide and transforming it into the B. amyloliquefaciens XH7 host (mutS-，bet+) by electroporation, followed by antibiotic selection. The parameters for ssDNA-mediated recombination were optimized as follows: 75 μg ssDNA oligonucleotide and 6 h to 12 h of cell recovery from electroporation. Here, oligonucleotide-mediated recombination was successfully applied in B. amyloliquefaciens for the first time, providing a new approach for developing an effective genetic manipulation technique in B. amyloliquefaciens and other Bacillus spp. that are not naturally transformable.
