Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Scarless Genomic Point Mutation to Construct a Bacillus subtilis Strain Displaying Increased Antibiotic Plipastatin Production

  • Jeong, Da-Eun (Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • So, Younju (Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lim, Hayeon (Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Park, Seung-Hwan (Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Choi, Soo-Keun (Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • Received : 2017.10.23
  • Accepted : 2018.04.06
  • Published : 2018.06.28
Download PDF
⟨ Previous Next ⟩

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.

Keywords

References

  1. Aftab MN, Haq I-U, Baig S. 2012. Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6. Braz. J. Microbiol. 43: 78-88. https://doi.org/10.1590/S1517-83822012000100009
  2. Leonard CA, Brown SD, Hayman JR. 2013. Random mutagenesis of the Aspergillus oryzae genome results in fungal antibacterial activity. Int. J. Microbiol. 2013: 901697.
  3. Medema MH, Alam MT, Breitling R, Takano E. 2011. The future of industrial antibiotic production: from random mutagenesis to synthetic biology. Bioeng. Bugs 2: 230-233. https://doi.org/10.4161/bbug.2.4.16114
  4. Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M. 1999. The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob. Agents Chemother. 43: 2183-2192.
  5. Inaoka T, Wang G, Ochi K. 2009. ScoC regulates bacilysin production at the transcription level in Bacillus subtilis. J. Bacteriol. 191: 7367-7371. https://doi.org/10.1128/JB.01081-09
  6. Vargas-Bautista C, Rahlwes K, Straight P. 2014. Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis. J. Bacteriol. 196: 717-728. https://doi.org/10.1128/JB.01022-13
  7. Hamoen LW, Van Werkhoven AF, Venema G, Dubnau D. 2000. The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 97: 9246-9251. https://doi.org/10.1073/pnas.160010597
  8. Mader U, Antelmann H, Buder T, Dahl MK, Hecker M, Homuth G. 2002. Bacillus subtilis functional genomics: genomewide analysis of the DegS-DegU regulon by transcriptomics and proteomics. Mol. Genet. Genomics 268: 455-467. https://doi.org/10.1007/s00438-002-0774-2
  9. Koumoutsi A, Chen XH, Vater J, Borriss R. 2007. DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. Appl. Environ. Microbiol. 73: 6953-6964. https://doi.org/10.1128/AEM.00565-07
  10. Msadek T, Kunst F, Henner D, Klier A, Rapoport G, Dedonder R. 1990. 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: 824-834. https://doi.org/10.1128/jb.172.2.824-834.1990
  11. Dahl MK, Msadek T, Kunst F, Rapoport G. 1992. 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: 14509-14514.
  12. Jeong DE, Park SH, Pan JG, Kim EJ, Choi SK. 2015. Genome engineering using a synthetic gene circuit in Bacillus subtilis. Nucleic Acids Res. 43: e42. https://doi.org/10.1093/nar/gku1380
  13. Hill JE, Myers AM, Koerner TJ, Tzagoloff A. 1986. Yeast/ E. coli shuttle vectors with multiple unique restriction sites. Yeast 2: 163-167. https://doi.org/10.1002/yea.320020304
  14. Bae JH, Sung BH, Kim HJ, Park SH, Lim KM, Kim MJ, et al. 2015. An efficient genome-wide fusion partner screening system for secretion of recombinant proteins in yeast. Sci. Rep. 5: 12229. https://doi.org/10.1038/srep12229
  15. Lee SJ, Pan JG, Park SH, Choi SK. 2010. Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis. J. Biotechnol. 149: 16-20. https://doi.org/10.1016/j.jbiotec.2010.06.021
  16. So Y, Park S-Y, Park E-H, Park S-H, Kim E-J, Pan J-G, et al. 2017. A highly efficient CRISPR-Cas9-mediated large genomic deletion in Bacillus subtilis. Front. Microbiol. 8: 1167. https://doi.org/10.3389/fmicb.2017.01167
  17. Kim JH, Chang PK, Chan KL, Faria NC, Mahoney N, Kim YK, et al. 2012. Enhancement of commercial antifungal agents by kojic acid. Int. J. Mol. Sci. 13: 13867-13880. https://doi.org/10.3390/ijms131113867
  18. el-Bendary MA. 2006. Bacillus thuringiensis and Bacillus sphaericus biopesticides production. J. Basic Microbiol. 46: 158-170. https://doi.org/10.1002/jobm.200510585
  19. Chen X, Zhang Y, Fu X, Li Y, Wang Q. 2016. Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot. Postharvest Biol. Technol. 115: 113-121. https://doi.org/10.1016/j.postharvbio.2015.12.021
  20. Gao L, Liu H, Ma Z, Han J, Lu Z, Dai C, et al. 2016. Translocation of the thioesterase domain for the redesign of plipastatin synthetase. Sci. Rep. 6: 38467. https://doi.org/10.1038/srep38467
  21. Saida F , Uzan M, Odaert B , Bontems F. 2006. Expression of highly toxic genes in E. coli: special strategies and genetic tools. Curr. Protein Pept. Sci. 7: 47-56. https://doi.org/10.2174/138920306775474095
  22. Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, et al. 2008. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl. Acad. Sci. USA 105: 20404-20409. https://doi.org/10.1073/pnas.0811011106
  23. Stein T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56: 845-857. https://doi.org/10.1111/j.1365-2958.2005.04587.x
  24. Murray EJ, Kiley TB, Stanley-Wall NR. 2009. A pivotal role for the response regulator DegU in controlling multicellular behaviour. Microbiology 155: 1-8. https://doi.org/10.1099/mic.0.023903-0

Cited by

  1. Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751 vol.68, pp.41, 2018, https://doi.org/10.1021/acs.jafc.0c03694
  2. Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.] vol.12, pp.None, 2021, https://doi.org/10.3389/fmicb.2021.789362