Skip to main content
SpringerLink
Log in

Heterologous expression of the novel α-helical hybrid peptide PR-FO in Bacillus subtilis

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

PR-FO is a novel α-helical hybrid antimicrobial peptide (AMP) with strong antimicrobial activities and high stability, and the potential to develop into a new generation of antimicrobial agents. In this study, the encoded gene sequence of SMT3-PR-FO was designed and transformed into B. subtilis WB800N. Fusion proteins with concentrations of 16 mg L−1 (SPamyQ) and 23 mg L−1 (SPsacB) were obtained after purification by a Ni–NTA resin column. A total of 3 mg (SPamyQ) and 4 mg (SPsacB) of PR-FO with a purity of 90% was obtained from 1 L fermentation cultures. Recombinant PR-FO exhibited high inhibition activities against both gram-negative bacteria and gram-positive bacteria, and low haemolytic activity against human red blood cells. These results indicated that the rSMT3-PR-FO could be expressed under the guidance of SPamyQ and SPsacB, and the maltose-induced expression strategy might be a safe and efficient method for the soluble peptides production in B. subtilis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Bacanli M, Başaran N (2019) Importance of antibiotic residues in animal food. Food Chem Toxicol 125:462–466

    Article  CAS  PubMed  Google Scholar 

  2. Wang JJ, Dou XJ, Song J, Lvy YF, Zhu X, Xu L, Li WZ, Shan AS (2018) Antimicrobial peptides: promising alternatives in the postfeeding antibiotic era. Med Res Rev 39:1–29

    Google Scholar 

  3. Hancock REW, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551–1557

    Article  CAS  PubMed  Google Scholar 

  4. Lai ZH, Tan P, Zhu YJ, Shao CX, Shan AS, Li L (2019) Highly stabilized α-helical coiled coils kill gram-negative bacteria by multi-complementary mechanisms under acidic condition. ACS Appl Mater Interfaces 11:22113–22128

    Article  CAS  PubMed  Google Scholar 

  5. Tan TT, Wu D, Li WZ, Zhu X, Li WF, Shan AS (2017) High Specific selectivity and membrane-active mechanism of synthetic cationic hybrid antimicrobial peptides based on the peptide FV7. Int J Mol Sci 18:339

    Article  PubMed Central  Google Scholar 

  6. Ma Z, Wei D, Yan P, Zhu X, Shan A, Bi Z (2015) Characterization of cell selectivity, physiological stability and endotoxin neutralization capabilities of α-helix-based peptide amphiphiles. Biomaterials 52:517–530

    Article  CAS  PubMed  Google Scholar 

  7. Wibowo D, Zhao CX (2019) Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides. Appl Microbiol Biot 103:659–671

    Article  CAS  Google Scholar 

  8. Winkler DFH, Tian K (2015) Investigation of the automated solid-phase synthesis of a 38mer peptied with difficult sequence pattern under different synthesis strategies. Amino Acids 47:787–794

    Article  CAS  PubMed  Google Scholar 

  9. Li Y (2013) Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin-SUMO dual fusion system. Protein Expr Purif 87:72–78

    Article  CAS  PubMed  Google Scholar 

  10. Elumalai P, Park YJ, Cho M, Shea PJ, Oh BT (2018) Red yeast rice fermentation with Bacillus subtilis B2 under blue light-emitting diodes increases antioxidant secondary products. Bioproc Biosyst Eng. https://doi.org/10.1007/s00449-018-2056-3

    Article  Google Scholar 

  11. Yang M, Zhang W, Chen Y, Gong Y (2010) Development of a Bacillus subtilis expression system using the improved Pglv promoter. Microb Cell Fact 9:1–8

    Article  CAS  Google Scholar 

  12. Chen J, Fu G, Gai Y, Zheng P, Zhang D, Wen J (2015) Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression. Microbial Cell Fact. https://doi.org/10.1186/s12934-015-0282-9

    Article  Google Scholar 

  13. Li D, Fu G, Tu R, Jin Z, Zhang D (2019) High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components. Microb Cell Fact. https://doi.org/10.1186/s12934-019-1066-4

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chen J, Zhao L, Fu G, Zhou W, Sun Y, Zheng P, Zhang D (2016) A novel strategy for protein production using non-classical secretion pathway in Bacillus subtilis. Microb Cell Fact. https://doi.org/10.1186/s12934-016-0469-8

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chen J, Gai Y, Gang F, Zhou W, Zhang D, Wen J (2015) Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotei. Biotechnol Lett 37:899–906

    Article  CAS  PubMed  Google Scholar 

  16. Lu Y, Lin Q, Wang J, Wu Y, Bao W, Lv F, Lu Z (2010) Overexpression and characterization in Bacillus subtilis of a positionally nonspecific lipase from Proteus vulgaris. J Ind Microbiol Biot 37:919–925

    Article  CAS  Google Scholar 

  17. Zhang J, Kang Z, Ling Z, Cao W, Liu L, Wang M, Du G, Chen J (2013) High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements. Bioresource Technol 146:543–548

    Article  CAS  Google Scholar 

  18. Parachin NS, Mulder KC, Viana AA, Dias SC, Franco OL (2012) Expression systems for heterologous production of antimicrobial peptides. Peptides 38:446–456

    Article  CAS  PubMed  Google Scholar 

  19. Kim DS, Kim SW, Song JM, Kim SY, Kwon KC (2019) A new prokaryotic expression vector for the expression of antimicrobial peptide abaecin using SUMO fusion tag. BMC Biotechnol 19:13

    Article  PubMed  PubMed Central  Google Scholar 

  20. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genom 5:75–86

    Article  CAS  Google Scholar 

  21. Lee CD, Sun HC, Hu SM, Chiu CF, Homhuan A, Liang SM, Leng CH, Wang TF (2008) An improved SUMO fusion protein system for effecitve procuction of native proteins. Protein Sci 17:1241–1248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bommariu B, Jenssen H, Elliott M, Kindrachuk J, Pasupuleti M, Gieren H, Jaeger KE, Hancock REW, Kalman D (2010) Post-effective expression and purfication of antimicrobial and host defense peptides in Escherichia coli. Peptides 31:1957–1965

    Article  Google Scholar 

  23. Motejadded H, Altenbuchner J (2009) Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing. Biotechnol Lett 31:543–549

    Article  CAS  PubMed  Google Scholar 

  24. Yang M, Zhang W, Ji S, Cao P, Chen Y, Zhao X (2013) Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system. PLoS ONE 8:e56321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jorgensen S, Vorgias CE, Antranikian G (1997) Cloning, Sequencing, Characterization, and expression of an extracellular-amylase from the hyperthermophilic archaeon pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J Biol Chem 272:16335–16342

    Article  CAS  PubMed  Google Scholar 

  26. Caimi PG, Mccole LM, Klein TM, Kerr PS (1996) Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB gene. Plant Physiol 110:355–363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ilk N, Schum CT, Bohle B, Egelseer EM, Sleytr UB (2011) Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy. Microb Cell Fact 10:1–12

    Article  Google Scholar 

  28. Stark M, Liu LP, Deber CM (2002) Cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents Ch 46:3585–3590

    Article  CAS  Google Scholar 

  29. Shao C, Li W, Tan P, Shan A, Dou X, Ma D, Liu C (2019) Symmetrical modification of minimized dermaseptins to extend the spectrum of antimicrobials with endotoxin neutralization potency. Int J Mol Sci 20:1417

    Article  CAS  PubMed Central  Google Scholar 

  30. Zhang L, Li G, Zhan N, Sun T, Cheng B, Li Y, Shan A (2019) Expression of a Pseudomonas aeruginosa-targeted antimicrobial peptide T9W in Bacillus subtilis using a maltose-inducible vector. Process Biochem 81:22–27

    Article  CAS  Google Scholar 

  31. Zhu X, Dong N, Wang Z, Ma Z, Zhang L, Ma Q, Shan A (2014) Design of imperfectly amphipathic α-helical antimicrobial peptides with enhanced cell selectivity. Acta Biomater 10:244–257

    Article  CAS  PubMed  Google Scholar 

  32. Veldhuizen EJA, Brouwer EC, Schneider VAF, Fluit AC (2013) Chicken cathelicidins display antimicrobial activity against multiresistant bacteria without inducing strong resistance. PLoS ONE 8:e61964

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expres Purif 80:260–267

    Article  CAS  Google Scholar 

  34. Zhang L, Li X, Wei D, Wang J, Shan A, Li Z (2015) Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector. J Ind Microbiol Biotechnol 42:1369–1376

    Article  CAS  PubMed  Google Scholar 

  35. Gimenez GG, Costa H, de Lima Neto QA, Fernandez MA, Ferrarotti SA, Matioli G (2019) Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800. Bioproc Biosyst Eng. https://doi.org/10.1007/s00449-018-02068-4

    Article  Google Scholar 

  36. Nguyen TT, Quyen TD, Le HT (2013) Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microb Cell Fact 12:79

    Article  PubMed  PubMed Central  Google Scholar 

  37. Chityala S, Venkata DV, Ahmad J, Prakasham RS (2015) High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N. Bioproc Biosyst Eng 38(11):2271–2284

    Article  CAS  Google Scholar 

  38. Sun H, Bie X, Lu F, Lu Y, Wu Y, Lu Z (2009) Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter. Can J Microbiol 55(8):1003–1006

    Article  CAS  PubMed  Google Scholar 

  39. Chen J, Zhu Y, Fu G, Song Y, Jin Z, Sun Y, Zhang D (2016) High-level intra- and extra-cellular production of d-psicose 3-epimerase via a modified xylose-inducible expression system in Bacillus subtilis. J Ind Microbiol Biotechnol 43(11):1577–1591

    Article  CAS  PubMed  Google Scholar 

  40. Phan HTT, Nhi NNY, Tien LT, Phuong CTB, Ngan LTP, Trang PPT, Nguyen HD (2019) Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter. Nat Sci 22:239–246

    Google Scholar 

  41. Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ (2013) Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biot 98(8):3651–3658

    Article  Google Scholar 

  42. Wang Y, Liu Y, Wang Z, Lu F (2014) Influence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis. Biotechnol Lett 36:1783–1789

    Article  CAS  PubMed  Google Scholar 

  43. Fu G, Liu JL, Li JS, Zhu BW, Zhang DW (2018) Systematic Screening of optimal signal peptides for secretory production of heterologous proteins in Bacillus subtilis. J Agric Food Chem 66:13141–13151

    Article  CAS  PubMed  Google Scholar 

  44. Zhang W, Yang M, Yang Y, Zhan J, Zhou Y (2016) Zhao X (2016) Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening. Appl Microbiol Biotechnol 100:8745–8756

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the financial support from the National Natural Science Foundation of China [31672434, 31872368, 31472104], the Natural Science Foundation of Heilongjiang Province [TD2019C001], and the China Agriculture Research System [CARS-35].

Author information

Author notes

  1. Licong Zhang and Dandan Wei contributed equally to this work.

Authors and Affiliations

  1. Institute of Animal Nutrition, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin, 150030, People’s Republic of China

    Licong Zhang, Dandan Wei, Na Zhan, Taotao Sun, Bingdong Shan & Anshan Shan

Authors
  1. Licong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dandan Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Na Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taotao Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bingdong Shan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anshan Shan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anshan Shan.

Ethics declarations

Conflict of interest

The authors have declared no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, L., Wei, D., Zhan, N. et al. Heterologous expression of the novel α-helical hybrid peptide PR-FO in Bacillus subtilis. Bioprocess Biosyst Eng 43, 1619–1627 (2020). https://doi.org/10.1007/s00449-020-02353-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-020-02353-1

Keywords

Search

Navigation