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Purification of Peptide Antimicrobials and Thioether-Stabilized Molecules Produced In Vivo by Lantibiotic Modification Machineries

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Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

The presence of posttranslational modifications is a key feature for the biological activity and stability of many natural products, including lanthipeptides. Lanthipeptides are a group of posttranslationally modified peptides containing lanthionine residues in their structures. Among lanthipeptides, the subgroup of lantibiotics is receiving a renewed interest in the last years for their high antimicrobial activity. The development of production platforms for novel lantibiotics and peptides containing posttranslational modifications is taking place in parallel to the understanding of the mechanistic aspects of the enzymes responsible for these modifications. It is now possible to combine pieces (i.e., enzymes and regulatory elements) of different biosynthesis routes of natural products in order to modify peptides of interest in a predictable manner. In this chapter we detail the production of lanthipeptides and thioether-stabilized peptides using two well-established production platforms for Escherichia coli and Lactococcus lactis. These are based on the dehydration and cyclization reactions carried out by the enzymes NisB and NisC, respectively, on peptides fused to the nisin leader peptide. Furthermore, we provide appropriate protocols for the purification and characterization of the peptides produced and modified with these biosynthetic tools.

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References

  1. Arnison PG, Bibb MJ, Bierbaum G et al (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Knerr PJ, van der Donk WA (2012) Discovery, biosynthesis, and engineering of lantipeptides. Annu Rev Biochem 81:479–505

    Article  CAS  PubMed  Google Scholar 

  3. Dischinger J, Basi Chipalu S, Bierbaum G (2014) Lantibiotics: promising candidates for future applications in health care. Int J Med Microbiol 304:51–62

    Article  CAS  PubMed  Google Scholar 

  4. Mota-Meira M, LaPointe G, Lacroix C, Lavoie MC (2000) MICs of mutacin B-Ny266, nisin A, vancomycin, and oxacillin against bacterial pathogens. Antimicrob Agents Chemother 44:24–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cotter PD, Ross RP, Hill C (2013) Bacteriocins – a viable alternative to antibiotics? Nat Rev Microbiol 11:95–105

    Article  CAS  PubMed  Google Scholar 

  6. Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788

    Article  CAS  PubMed  Google Scholar 

  7. Boucher HW, Talbot GH, Bradley JS et al (2009) Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 48:1–12

    Article  PubMed  Google Scholar 

  8. Van Heel AJ, Montalban-Lopez M, Kuipers OP (2011) Evaluating the feasibility of lantibiotics as an alternative therapy against bacterial infections in humans. Expert Opin Drug Metab Toxicol 7:675–680

    Article  PubMed  Google Scholar 

  9. Oman TJ, van der Donk WA (2010) Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nat Chem Biol 6:9–18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Abts A, Montalban-Lopez M, Kuipers OP, Smits SH, Schmitt L (2013) NisC binds the FxLx motif of the nisin leader peptide. Biochemistry 52:5387–5395

    Article  CAS  PubMed  Google Scholar 

  11. Plat A, Kuipers A, Rink R, Moll GN (2013) Mechanistic aspects of lanthipeptide leaders. Curr Protein Pept Sci 14(2):85–96

    Article  CAS  PubMed  Google Scholar 

  12. Plat A, Kluskens LD, Kuipers A, Rink R, Moll GN (2011) Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage. Appl Environ Microbiol 77:604–611

    Article  CAS  PubMed  Google Scholar 

  13. Willey JM, van der Donk WA (2007) Lantibiotics: peptides of diverse structure and function. Annu Rev Microbiol 61:477–501

    Article  CAS  PubMed  Google Scholar 

  14. Montalbán-López M, Zhou L, Buivydas A, van Heel AJ, Kuipers OP (2012) Increasing the success rate of lantibiotic drug discovery by Synthetic Biology. Expert Opin Drug Discovery 7:695–709

    Article  Google Scholar 

  15. Chatterjee C, Patton GC, Cooper L, Paul M, van der Donk WA (2006) Engineering dehydro amino acids and thioethers into peptides using lacticin 481 synthetase. Chem Biol 13:1109–1117

    Article  CAS  PubMed  Google Scholar 

  16. Furgerson Ihnken LA, Chatterjee C, van der Donk WA (2008) In vitro reconstitution and substrate specificity of a lantibiotic protease. Biochemistry 47:7352–7363

    Article  CAS  PubMed  Google Scholar 

  17. Levengood MR, van der Donk WA (2008) Use of lantibiotic synthetases for the preparation of bioactive constrained peptides. Bioorg Med Chem Lett 18:3025–3028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Levengood MR, Knerr PJ, Oman TJ, van der Donk WA (2009) In vitro mutasynthesis of lantibiotic analogues containing nonproteinogenic amino acids. J Am Chem Soc 131:12024–12025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang X, van der Donk WA (2007) On the substrate specificity of dehydration by lacticin 481 synthetase. J Am Chem Soc 129:2212–2213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Caetano T, Barbosa J, Möesker E, Süssmuth RD, Mendo S (2014) Bioengineering of lanthipeptides in Escherichia coli: assessing the specificity of lichenicidin and haloduracin biosynthetic machinery. Res Microbiol 165:600–604

    Article  CAS  PubMed  Google Scholar 

  21. Breukink E, Wiedemann I, van Kraaij C, Kuipers OP, Sahl H, de Kruijff B (1999) Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286:2361–2364

    Article  CAS  PubMed  Google Scholar 

  22. Hasper HE, Kramer NE, Smith JL et al (2006) An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II. Science 313:1636–1637

    Article  CAS  PubMed  Google Scholar 

  23. Lubelski J, Rink R, Khusainov R, Moll GN, Kuipers OP (2008) Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell Mol Life Sci 65:455–476

    Article  CAS  PubMed  Google Scholar 

  24. Siegers K, Entian KD (1995) Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3. Appl Environ Microbiol 61:1082–1089

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kuipers OP, Beerthuyzen MM, Siezen RJ, De Vos WM (1993) Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem FEBS 216:281–291

    Article  CAS  Google Scholar 

  26. Kuipers OP, Beerthuyzen MM, de Ruyter PG, Luesink EJ, de Vos WM (1995) Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem 270:27299–27304

    Article  CAS  PubMed  Google Scholar 

  27. Van der Meer JR, Polman J, Beerthuyzen MM, Siezen RJ, Kuipers OP, De Vos WM (1993) Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis. J Bacteriol 175:2578–2588

    Google Scholar 

  28. Siezen RJ, Kuipers OP, de Vos WM (1996) Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek 69:171–184

    Article  CAS  PubMed  Google Scholar 

  29. De Ruyter PG, Kuipers OP, Beerthuyzen MM, van Alen-Boerrigter I, de Vos WM (1996) Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis. J Bacteriol 178:3434–3439

    PubMed  PubMed Central  Google Scholar 

  30. Eichenbaum Z, Federle MJ, Marra D et al (1998) Use of the lactococcal nisA promoter to regulate gene expression in Gram-positive bacteria: comparison of induction level and promoter strength. Appl Environ Microbiol 64:2763–2769

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kluskens LD, Kuipers A, Rink R et al (2005) Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin. Biochemistry 44:12827–12834

    Article  CAS  PubMed  Google Scholar 

  32. Kuipers A, de Boef E, Rink R et al (2004) NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. J Biol Chem 279:22176–22182

    Article  CAS  PubMed  Google Scholar 

  33. Rink R, Kluskens LD, Kuipers A, Driessen AJM, Kuipers OP, Moll GN (2007) NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides. Biochemistry 46:13179–13189

    Article  CAS  PubMed  Google Scholar 

  34. Bosma T, Kuipers A, Bulten E, de Vries L, Rink R, Moll GN (2011) Bacterial display and screening of posttranslationally thioether-stabilized peptides. Appl Environ Microbiol 77:6794–6801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Van Heel AJ, Mu D, Montalbán-López M, Hendriks D, Kuipers OP (2013) Designing and producing modified, new-to-nature peptides with antimicrobial activity by use of a combination of various lantibiotic modification enzymes. ACS Synth Biol 2:397–404

    Article  PubMed  Google Scholar 

  36. Majchrzykiewicz JA, Lubelski J, Moll GN et al (2010) Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence. Antimicrob Agents Chemother 54:1498–1505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kluskens LD, Nelemans SA, Rink R et al (2009) Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog. J Pharmacol Exp Ther 328:849–854

    Article  CAS  PubMed  Google Scholar 

  38. Rink R, Arkema-Meter A, Baudoin I et al (2010) To protect peptide pharmaceuticals against peptidases. J Pharmacol Toxicol Methods 61:210–218

    Article  CAS  PubMed  Google Scholar 

  39. Khusainov R, Heils R, Lubelski J, Moll GN, Kuipers OP (2011) Determining sites of interaction between prenisin and its modification enzymes NisB and NisC. Mol Microbiol 82:706–718

    Article  CAS  PubMed  Google Scholar 

  40. Khusainov R, Moll GN, Kuipers OP (2013) Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC. FEBS Open Bio 3:237–242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Van Heel AJ, de Jong A, Montalbán-López M, Kok J, Kuipers OP (2013) BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides. Nucleic Acids Res 41:W448–W453

    Article  PubMed  PubMed Central  Google Scholar 

  42. Mu D, Montalbán-López M, Masuda Y, Kuipers OP (2013) Zirex: a novel zinc-regulated expression system for Lactococcus lactis. Appl Environ Microbiol 79:4503–4508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rink R, Kuipers A, de Boef E et al (2005) Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes. Biochemistry 44:8873–8882

    Article  CAS  PubMed  Google Scholar 

  44. Kuipers OP, de Ruyter PG, Kleerebezem M, de Vos WM (1997) Controlled overproduction of proteins by lactic acid bacteria. Trends Biotechnol 15:135–140

    Article  CAS  PubMed  Google Scholar 

  45. Poolman B, Konings WN (1988) Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. J Bacteriol 170:700–707

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Sambrook J, Russell D, Sambrook J (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  47. Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119–3123

    CAS  PubMed  PubMed Central  Google Scholar 

  48. De Ruyter PG, Kuipers OP, De Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667

    PubMed  PubMed Central  Google Scholar 

  49. Shi Y, Yang X, Garg N, van der Donk WA (2011) Production of lantipeptides in Escherichia coli. J Am Chem Soc 133:2338–2341

    Article  CAS  PubMed  Google Scholar 

  50. Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22

    Article  PubMed  Google Scholar 

  51. Lin Y, Teng K, Huan L, Zhong J (2011) Dissection of the bridging pattern of bovicin HJ50, a lantibiotic containing a characteristic disulfide bridge. Microbiol Res 166:146–154

    Article  CAS  PubMed  Google Scholar 

  52. Caetano T, Krawczyk JM, Mösker E, Süssmuth RD, Mendo S (2011) Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli. Chem Biol 18:90–100

    Article  CAS  PubMed  Google Scholar 

  53. Shi Y, Bueno A, van der Donk WA (2012) Heterologous production of the lantibiotic Ala(0)actagardine in Escherichia coli. Chem Commun (Camb) 48:10966–10968

    Article  CAS  Google Scholar 

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Acknowledgments

Andrius Buivydas and Manuel Montalban-Lopez are supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) projects ALW 821.02.018 and 855.01.162, respectively. M. Montalban-Lopez is also part of the EU Project Synpeptide.

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Authors and Affiliations

  1. Department of Molecular Genetics, University of Groningen, Nijenborgh 7, 9747AG, Groningen, The Netherlands

    Manuel Montalban-Lopez, Andrius Buivydas & Oscar P. Kuipers

Authors
  1. Manuel Montalban-Lopez
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  2. Andrius Buivydas
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  3. Oscar P. Kuipers
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Corresponding author

Correspondence to Oscar P. Kuipers .

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Editors and Affiliations

  1. Dept. Biological Sciences, University of Essex, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Braunschweig, Germany

    Kenneth N. Timmis

  3. Dept. Biologia, Univ de les Illes Balears, Palma de Mallorca, Spain

    Balbina Nogales Fernández

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Montalban-Lopez, M., Buivydas, A., Kuipers, O.P. (2015). Purification of Peptide Antimicrobials and Thioether-Stabilized Molecules Produced In Vivo by Lantibiotic Modification Machineries. In: McGenity, T., Timmis, K., Nogales Fernández, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_122

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  • DOI: https://doi.org/10.1007/8623_2015_122

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  • Print ISBN: 978-3-662-49126-3

  • Online ISBN: 978-3-662-49127-0

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