An alternative genome-integrated method for undomesticated <i>Bacillus subtilis</i> and related species
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- Mahipant Gumpanat
- Biological Sciences Program, Faculty of Science, Chulalongkorn University Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University
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- Kato Junichi
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University
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- Kataoka Naoya
- Division of Agricultural Sciences, Graduate School of Sciences and Technology for Innovation, Yamaguchi University Research Center for Thermotolerant Microbial Resources, Yamaguchi University
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- Vangnai Alisa S.
- Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University Center of Excellence on Hazardous Substance Management (HSM), Chulalongkorn University
抄録
<p>Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.</p>
収録刊行物
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- The Journal of General and Applied Microbiology
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The Journal of General and Applied Microbiology 65 (2), 96-105, 2019
公益財団法人 応用微生物学・分子細胞生物学研究奨励会