title: Generation of an actagardine A variant library through saturation mutagenesis.
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.
authors.affiliation: Novacta Biosystems Ltd., BioPark Hertfordshire, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK. steve.boakes@cantabbio.com
authors.forename: Steven
authors.initials: S
authors.lastname: Boakes
authors.forename: Tania
authors.initials: T
authors.lastname: Ayala
authors.forename: Mark
authors.initials: M
authors.lastname: Herman
authors.forename: Antony N
authors.initials: AN
authors.lastname: Appleyard
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dawson
authors.forename: Jesús
authors.initials: J
authors.lastname: Cortés
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001430:Bacteriocins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015723:Gene Library; D014644:Genetic Variation; D006094:Gram-Positive Bacteria / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008848:Micromonosporaceae / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; C012993:gardimycin