Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon

  • Hadeel Waleed Abdulmalek (Dr. Orhan Ocalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University) ;
  • Ayten Yazgan-Karatas (Dr. Orhan Ocalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University)
  • Received : 2022.09.22
  • Accepted : 2022.12.07
  • Published : 2023.03.28
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Abstract

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

Keywords

Acknowledgement

This work was supported by the Istanbul Technical University Scientific Research Foundation (Grant No. 42659). Bacteria shapes in the graphical abstract were picked from Starline-Freepick.com. We would like to acknowledge J. Altenbucher for providing the plasmid pJOE9958.1. We thank our PhD student Busra Ozturk for providing technical assistance in the RT-qPCR experiments. This article is dedicated to the memory of Prof. Dr. Fatma Nese Kok (1970-2022), our beloved colleague, Head of the Department of Molecular Biology and Genetics at Istanbul Technical University (ITU), and Director of Dr. Orhan Ocalgiray Molecular Biology and Biotechnology and Genetics Research Center (ITU-MOBGAM), who passed away while this paper was being peer-reviewed.

References

  1. Walker JE, Abraham EP. 1970. The structure of bacilysin and other products of Bacillus subtilis. Biochem. J. 118: 563-570. https://doi.org/10.1042/bj1180563
  2. Sakajoh M, Solomon NA, Demain AL.1987. Cell-free synthesis of the dipeptide antibiotic bacilysin. J. Indust. Microbiol. 2: 201-208. https://doi.org/10.1007/BF01569541
  3. Yazgan A, Ozcengiz G, Ozcengiz E, Kilinc K, Marahiel MA, Alaeddinoglu NG. 2001. Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis. Enzyme Microbiol. Technol. 29: 400-406. https://doi.org/10.1016/S0141-0229(01)00401-X
  4. Tabata K, Ikeda H, Hashimoto S. 2005. ywfE in Bacillus subtilis codes for a novel enzyme l-amino acid ligase. J. Bacteriol. 187: 5195-5202. https://doi.org/10.1128/JB.187.15.5195-5202.2005
  5. Foster JW, Woodruff HB. 1946. Bacillin, a new antibiotic substance from a soil isolate of Bacillus subtilis. J. Bacteriol. 51: 363-369. https://doi.org/10.1128/jb.51.3.363-369.1946
  6. Newton GG. 1949. Antibiotics from a strain of B. subtilis; bacilipin A and B and bacilysin. Br. J. Exp. Pathol. 30: 306-319.
  7. Chen XH, Scholz R, Borriss M, Junge H, Mogel G, Kunz S, et al. 2009. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease. J. Biotechnol. 140: 38-44. https://doi.org/10.1016/j.jbiotec.2008.10.015
  8. Nannan C, Vu HQ, Gillis A, Caulier S, Nguyen TTT, Mahillon J. 2021. Bacilysin within the Bacillus subtilis group: gene prevalence versus antagonistic activity against Gram-negative foodborne pathogens. J. Biotechnol. 327: 28-35. https://doi.org/10.1016/j.jbiotec.2020.12.017
  9. Kenig M, Vandamme E, Abraham EP. 1976. The mode of action of bacilysin and anticapsin and biochemical properties of bacilysin-resistant mutants. Microbiology 94: 46-54. https://doi.org/10.1099/00221287-94-1-46
  10. Wang T, Liu XH, Wu MB, Ge S. 2018. Molecular insights into the antifungal mechanism of bacilysin. J. Mol. Model. 24: 1-9. https://doi.org/10.1007/s00894-017-3528-0
  11. Wu L, Wu H, Chen L, Yu X, Borriss R, Gao X. 2015. Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens. Sci. Rep. 5: 12975.
  12. Wu L, Wu H, Chen L, Xie S, Zang H, Borriss R, Gao X. 2014. Bacilysin from Bacillus amyloliquefaciens FZB42 has specific bactericidal activity against harmful algal bloom species. Appl. Environ. Microbiol. 80: 7512-7520. https://doi.org/10.1128/AEM.02605-14
  13. Ozcengiz G, Alaeddinoglu NG. 1991. Bacilysin production by Bacillus subtilis: effects of bacilysin, pH and temperature. Folia Microbiol. 36: 522-526.
  14. Vellanoweth RL, Rabinowitz JC. 1992. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol. Microbiol. 6: 105-114. https://doi.org/10.1111/j.1365-2958.1992.tb01548.x
  15. Shine J, Dalgarno L. 1974. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71: 1342-1346 https://doi.org/10.1073/pnas.71.4.1342
  16. Ringquist S, Shinedling S, Barrick D, Green L, Binkley J, Stormo GD, et al. 1992. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol. Microbiol. 6: 1219-1229. https://doi.org/10.1111/j.1365-2958.1992.tb01561.x
  17. Ma J, Campbell A, Karlin S. 2002. Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures. J. Bacteriol. 184: 5733-5745. https://doi.org/10.1128/JB.184.20.5733-5745.2002
  18. Salis HM, Mirsky EA, Voigt CA. 2009. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27: 946-950. https://doi.org/10.1038/nbt.1568
  19. Saito K, Green R, Buskirk AR. 2020. Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing. Elife 9: e55002.
  20. Chen H, Bjerknes M, Kumar R, Jay E. 1994. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 22: 4953-4957. https://doi.org/10.1093/nar/22.23.4953
  21. Volkenborn K, Kuschmierz L, Benz N, Lenz P, Knapp A, Jaeger KE. 2020. The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis. Microb. Cell Fact. 19: 1-12. https://doi.org/10.1186/s12934-019-1269-8
  22. Inaoka T, Takahashi K, Ohnishi-Kameyama M, Yoshida M, Ochi K. 2003. Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillussubtilis. J. Biol. Chem. 278: 2169-2176. https://doi.org/10.1074/jbc.M208722200
  23. Steinborn G, Hajirezaei MR, Hofemeister J. 2005. bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains. Arch. Microbiol. 183: 71-79. https://doi.org/10.1007/s00203-004-0743-8
  24. Rajavel M, Mitra A, Gopal B. 2009. Role of Bacillus subtilis BacB in the synthesis of bacilysin. J. Biol. Chem. 284: 31882-31892. https://doi.org/10.1074/jbc.M109.014522
  25. Mahlstedt SA, Walsh CT. 2010. Investigation of anticapsin biosynthesis reveals a four-enzyme pathway to tetrahydrotyrosine in Bacillus subtilis. Biochem. 49: 912-923. https://doi.org/10.1021/bi9021186
  26. Parker JB, Walsh CT. 2012. Olefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis. Biochem. 51: 3241-3251. https://doi.org/10.1021/bi300254u
  27. 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
  28. Haldenwang WG. 1995. The sigma factors of Bacillus subtilis. Microbiol. Rev. 59: 1-30. https://doi.org/10.1128/mr.59.1.1-30.1995
  29. Altenbuchner J. 2016. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Appl. Environ. Microbiol. 82: 5421-5427. https://doi.org/10.1128/AEM.01453-16
  30. Perry D, Abraham EP. 1979. Transport and metabolism of bacilysin and other peptides by suspensions of Staphylococcus aureus. Microbiol. 115: 213-221. https://doi.org/10.1099/00221287-115-1-213
  31. Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL. 2015. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10: e0124633.
  32. Ozcengiz G, Alaeddinoglu NG, Demain AL. 1990. Regulation of biosynthesis of bacilysin by Bacillus subtilis. J. Ind. Microbiol. 6: 91-100. https://doi.org/10.1007/BF01576428
  33. Guiziou S, Sauveplane V, Chang HJ, Clerte C, Declerck N, Jules M, Bonnet J. 2016. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res. 44: 7495-7508. https://doi.org/10.1093/nar/gkw624
  34. Zhang K, Duan X, Wu J. 2016. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci. Rep. 6: 27943.
  35. So Y, Park SY, Park EH, Park SH, Kim EJ, Pan JG, Choi SK. 2017. A highly efficient CRISPR-Cas9-mediated large genomic deletion in Bacillus subtilis. Front. Microbiol. 8: 1167.
  36. Zheng X, Li S, Zha GP, Wang J. 2017. An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis. Biochem. Biophys. Res. Commun. 485: 768-774. https://doi.org/10.1016/j.bbrc.2017.02.129
  37. Kenig M, Vandamme E, Abraham EP. 1976. The mode of action of bacilysin and anticapsin and biochemical properties of bacilysin-resistant mutants. J. Gen. Microbiol. 94: 47-45.
  38. Koroglu TE, Ogulur I, Mutlu, Yazgan-Karatas, A, Ozcengiz G. 2011. Global regulatory systems operating in bacilysin biosynthesis in Bacillus subtilis. J. Mol. Microbiol. Biotechnol. 20: 144-155. https://doi.org/10.1159/000328639
  39. Studer SM, Joseph S. 2006. Unfolding of mRNA secondary structure by the bacterial translation initiation complex. Mol. Cell 22: 105-115. https://doi.org/10.1016/j.molcel.2006.02.014
  40. Eriksen M, Sneppen K, Pedersen S, Mitarai N. 2017. Occlusion of the ribosome binding site connects the translational initiation frequency, mRNA stability and premature transcription termination. Front. Microbiol. 8: 362.
  41. Ozcengiz G, Alaeddinoglu NG. 1991. Bacilysin production and sporulation in Bacillus subtilis. Curr. Microbiol. 23: 61-64. https://doi.org/10.1007/BF02092250
  42. Moeller R, Schuerger AC, Reitz G, Nicholson WL. 2011. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions. Icarus 215: 204-210. https://doi.org/10.1016/j.icarus.2011.06.035
  43. Toymentseva AA, Altenbuchner J. 2019. New CRISPR-Cas9 vectors for genetic modifications of Bacillus species. FEMS Microbiol. Lett. 366: fny284.
  44. Zhu MM, Lawman PD, Cameron DC. 2002. Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate. Biotechnol. Prog.18: 694-699. https://doi.org/10.1021/bp020281+
  45. Nowroozi FF, Baidoo EEK, Ermakov S, Redding-Johanson AM, Batth TS, Petzold CJ, et al. 2014. Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly. Appl. Microbiol. Biotechnol. 98: 1567-1581. https://doi.org/10.1007/s00253-013-5361-4
  46. Jin P, Kang Z, Yuan P, Du G, Chen J. 2016. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. Metab. Eng. 35: 21-30. https://doi.org/10.1016/j.ymben.2016.01.008
  47. Niu J,Yan R, Shen J, Zhu X, Meng F, Lu Z, Lu. F. 2022. Cis-Element engineering promotes the expression of Bacillus subtilis type I L-asparaginase and its application in food. Int. J. Mol. Sci. 23: 6588.
  48. Youngman P, Perkins JB, Losick R. 1984. Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid 12: 1-9. https://doi.org/10.1016/0147-619X(84)90061-1
  49. Berg L, Lale R, Bakke I, Burroughs N, Valla S. 2009. The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA. Microb. Biotechnol. 2: 379-389. https://doi.org/10.1111/j.1751-7915.2009.00107.x