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Generation of an actagardine A variant library through saturation mutagenesis

  • Applied genetics and molecular biotechnology
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Abstract

The lantibiotic actagardine A is nineteen amino acids in length and comprises three intertwined C-terminal methyllanthionine-bridged rings and an N-terminal lanthionine-bridged ring. Produced by the actinomycete Actinoplanes garbadinensis ATCC 31049, actagardine A demonstrates antibacterial activity against important Gram-positive pathogens. This activity combined with its ribosomal synthesis makes it an attractive target for the generation of lantibiotic variants with improved biological activity. A variant generation system designed to allow the specific substitution of amino acids at targeted sites throughout the actagardine A peptide has been used to generate a comprehensive library by site-directed mutagenesis. With the exception of residues involved in bridge formation, each amino acid in the actagardine A peptide as well as the alanine (ala(0)) at position −1 relative to the mature peptide, has been systematically substituted with all remaining 19 amino acids. A total of 228 mutants have been engineered with 44 produced in good yield. The mutant V15F in particular demonstrates improved activity against a range of notable Gram-positive pathogens including Clostridium difficile, when evaluated alongside actagardine A. The scope of variants generated provides an insight into the flexibility of the actagardine A processing machinery and will undoubtedly assist in future mutational studies.

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Notes

  1. Actagardine A was previously designated actagardine; the suffix was introduced in Boakes et al. (2010).

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Acknowledgments

We thank Prof. Ian Chopra of the University of Leeds for the provision of strains, and we gratefully acknowledge the funding awarded to Novacta Biosystems Ltd by the East of England Development Agency.

Declaration of a conflict of interests

A conflict of interest is declared in that the authors SB, MD and JC are shareholders of Novacta Biosystems Limited, while MD is on the board of directors for Novacta Biosystems Limited.

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Authors and Affiliations

  1. Novacta Biosystems Ltd., BioPark Hertfordshire, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX, UK

    Steven Boakes, Tania Ayala, Mark Herman, Antony N. Appleyard, Michael J. Dawson & Jesús Cortés

  2. Cantab Biopharmaceuticals Ltd., BioPark Hertfordshire, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX, UK

    Steven Boakes, Antony N. Appleyard & Michael J. Dawson

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  1. Steven Boakes
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  2. Tania Ayala
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  3. Mark Herman
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  4. Antony N. Appleyard
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  5. Michael J. Dawson
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  6. Jesús Cortés
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Correspondence to Steven Boakes.

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Boakes, S., Ayala, T., Herman, M. et al. Generation of an actagardine A variant library through saturation mutagenesis. Appl Microbiol Biotechnol 95, 1509–1517 (2012). https://doi.org/10.1007/s00253-012-4041-0

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  • DOI: https://doi.org/10.1007/s00253-012-4041-0

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