Abstract
Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
Similar content being viewed by others
References
Augustin J, Rosenstein R, Wieland B, Schneider U, Schnell N, Engelke G, Entian K.-D. & Götz F. (1992) Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur. J. Biochem. 204: 1149–1154
Banerjee S & Hansen JN (1988) Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J. Biol. Chem. 263: 9508–9514
Bierbaum G, Reis M, Szekat C & Sahl H-G (1994a) Construction of an expression system for engineering of the lantibiotic pep5. Appl. Environm. Microbiol. 60: 4332–4338
Bierbaum G, Reis M, Szekat C & Sahl H-G (1994b) Construction of an expression system for site directed mutagenesis in the pep5 biosynthesis operon. 2nd International Workshop on Lactibiotics, November 20–24 1994, Arnhem, the Netherlands
DeVos WM, Mulders JWM, Siezen RJ, Hugenholtz J & Kuipers OP (1993) Properties of nisin Z and the distribution of its gene, nisZ, in Lactococcus lactis. Appl. Environ. Microbiol. 59: 213–218
Dodd HM, Horn N, Hao Z & Gasson MJ (1992) A lactococcal expression system for engineered nisins. Appl. Environ. Microbiol. 58: 3683–3693
Dodd HM, Horn N & Gasson MJ (1994) Characterisation of IS905, a new multicopy insertion sequence identified in Lactococci. J. Bacteriol. 176: 3393–3396
Dodd HM, Horn N & Gasson MJ (1995) A cassette vector for protein engineering the lantibiotic nisin. Gene, submitted
Freund S, Jung G, Gutbrod O, Folkers G & Gibbons WA (1991) The three-dimensional solution structure of gallidermin determined bu NMR-based molecular graphics, p. 91–102. In G.Jung and H.-G.Sahl (ed.), Nisin and novel lantibiotics, ESCOM Science Publishers, Leiden, Netherlands.
Horn N, Dodd HM & Gasson MJ (1994) Strategies for engineering nisin biosynthesis. 2nd International Workshop on Lantibiotics, November 20–24 1994, Arnhem, the Netherlands
Jung G (1991) Lantibiotics-ribosomally synthesized biologically active polypeptides containing sulfide bridges and α,β-didehydroamino acids. Angew. Chemie Inc. Ed. Engl. 30: 1051–1068
Kuipers OP, Yap WMGJ, Rollema HS, Beerthuyzen MM, Siezen RJ & deVos WM (1991a) Expression of wild-type and mutant nisin genes in Lactococcus lactis in Nisin and Novel Lantibiotics, Sahl & Jung (Editors), Escom Publ, Leiden, 250–259
Kuipers OP, Boot HJ & deVos WM (1991b) Improved site-directed mutagenesis method using PCR, Nucl. Acids. Res. 19: 4558
Kuipers OP, Rollema HS, Yap WMGJ, Boot HJ, Siezen RJ & deVos WM (1992) Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J. Biol. Chem. 267: 24340–24346
Kuipers OP, Beerthuyzen MM, Siezen RJ & deVos WM (1993a) Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis Requirement of expression of the nisA and nisl genes for development of immunity. Eur. J. Biochem. 216: 281–291
Kuipers OP, Rollema HS, deVos WM & Siezen RJ (1993b) Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis. FEBS Letters 330: 23–27
Kuipers OP, Rollema HS, Bongers R, van den Bogaard P, Kosters H, de Vos WM & Siezen RJ (1994) Structure-function relationships of nisin studied by protein engineering. 2nd International Workshop on Lantibiotics, November 20–24 1994, Arnhem, the Netherlands
Kuipers OP, Beerthuyzen MM, deRuyter PGGA, Leusink EJ & deVos WM (1995) Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J. Biol. Chem. 270: 27299–27304
Liu W & Hansen JN (1992) Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis. J. Biol. Chem. 267: 25078–25085
Ottenwälder B, Kupke T, Gnau V, Metzger J, Jung G & Götz F (1994) Isolation and characterization of genetically engineered gallidermin and epidermin analogues. 2nd International Workshop on Lantibiotics, November 20–24 1994, Arnhem, the Netherlands
Rollema HS, Kuipers OP, Both P, deVos WM & Siezen RJ (1995) Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering. Appl. Environ. Microbiol. 61: 2873–2878
Sahl H-G, Jack RW & Bierbaum G (1995) Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur. J. Biochem. 230: 827–853
Schnell N, Entian K-D, Schneider U, Götz F, Zähner H, Kellner R & Jung G (1988) Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 333: 276–278
Siezen RJ, Rollema HS, Kuipers OP & deVos WM (1995) Homology modeling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin. Protein Engineering 8: 117–125
Spee J, deVos WM & Kuipers OP (1993) Efficient random mutagenesis method with adjustable mutation frequency using PCR and dITP. Nucl. Acdis. Res. 21: 777–778
Van derMeer JR, Polman J, Beerthuyzen MM, Siezen RJ, Kuipers OP & deVos 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
Van derMeer JR, Rollema HS, Siezen RJ, Beerthuyzen MM, Kuipers OP & deVos WM (1994) Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis. J. Biol. Chem. 269: 3555–3562
Van deVen FJM, van denHooven HW, Konings RNH & Hilbers CW (1991) NMR studies of lantibiotics. Eur. J. Biochem. 202: 1181–1188
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kuipers, O.P., Bierbaum, G., Ottenwälder, B. et al. Protein engineering of lantibiotics. Antonie van Leeuwenhoek 69, 161–170 (1996). https://doi.org/10.1007/BF00399421
Issue Date:
DOI: https://doi.org/10.1007/BF00399421