Scarless Genomic Point Mutation to Construct a Bacillus subtilis Strain Displaying Increased Antibiotic Plipastatin Production
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Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.
Abstract
Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.
참고문헌 (24)
- Aftab MN, "Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6", Braz. J. Microbiol., 43, pp.78~88 (2012)[DOI]
- Leonard CA, "Random mutagenesis of the Aspergillus oryzae genome results in fungal antibacterial activity", Int. J. Microbiol., 2013, pp.901697~ (2013)
- Medema MH, "The future of industrial antibiotic production: from random mutagenesis to synthetic biology", Bioeng. Bugs, 2, pp.230~233 (2011)[DOI]
- Tsuge K, "The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production", Antimicrob. Agents Chemother., 43, pp.2183~2192 (1999)
- Inaoka T, "ScoC regulates bacilysin production at the transcription level in Bacillus subtilis", J. Bacteriol., 191, pp.7367~7371 (2009)[DOI]
- Vargas-Bautista C, "Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis", J. Bacteriol., 196, pp.717~728 (2014)[DOI]
- Hamoen LW, "The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis", Proc. Natl. Acad. Sci. USA, 97, pp.9246~9251 (2000)[DOI]
- Mader U, "Bacillus subtilis functional genomics: genomewide analysis of the DegS-DegU regulon by transcriptomics and proteomics", Mol. Genet. Genomics, 268, pp.455~467 (2002)[DOI]
- Koumoutsi A, "DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42", Appl. Environ. Microbiol., 73, pp.6953~6964 (2007)[DOI]
- Msadek T, "Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU", J. Bacteriol., 172, pp.824~834 (1990)[DOI]
- Dahl MK, "The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis", J. Biol. Chem., 267, pp.14509~14514 (1992)
- Jeong DE, "Genome engineering using a synthetic gene circuit in Bacillus subtilis", Nucleic Acids Res, 43, pp.e42~ (2015)[DOI]
- Hill JE, "Yeast", E. coli shuttle vectors with multiple unique restriction sites, 1986, pp.2, 163~167
- Bae JH, "An efficient genome-wide fusion partner screening system for secretion of recombinant proteins in yeast", Sci. Rep., 5, pp.12229~ (2015)[DOI]
- Lee SJ, "Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis", J. Biotechnol., 149, pp.16~20 (2010)[DOI]
- So Y, "A highly efficient CRISPR-Cas9-mediated large genomic deletion in Bacillus subtilis", Front. Microbiol., 8, pp.1167~ (2017)[DOI]
- Kim JH, "Enhancement of commercial antifungal agents by kojic acid", Int. J. Mol. Sci., 13, pp.13867~13880 (2012)[DOI]
- el-Bendary MA, "Bacillus thuringiensis and Bacillus sphaericus biopesticides production", J. Basic Microbiol., 46, pp.158~170 (2006)[DOI]
- Chen X, "Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot", Postharvest Biol. Technol., 115, pp.113~121 (2016)[DOI]
- Gao L, "Translocation of the thioesterase domain for the redesign of plipastatin synthetase", Sci. Rep., 6, pp.38467~ (2016)[DOI]
- Saida F, "Expression of highly toxic genes in E. coli: special strategies and genetic tools", Curr. Protein Pept. Sci., 7, pp.47~56 (2006)[DOI]
- Gibson DG, "One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome", Proc. Natl. Acad. Sci. USA, 105, pp.20404~20409 (2008)[DOI]
- Stein T, "Bacillus subtilis antibiotics: structures, syntheses and specific functions", Mol. Microbiol., 56, pp.845~857 (2005)[DOI]
- Murray EJ, "A pivotal role for the response regulator DegU in controlling multicellular behaviour", Microbiology, 155, pp.1~8 (2009)[DOI]