Antony Nicholas Appleyard

Dissecting Structural and Functional Diversity of the Lantibiotic Mersacidin

Summary Mersacidin is a tetracyclic lantibiotic with antibacterial activity against Gram-positive pathogens. To probe the specificity of the biosynthetic pathway of mersacidin and obtain analogs with improved antibacterial activity, an efficient system for generating variants of this lantibiotic was developed. A saturation mutagenesis library of the residues of mersacidin not involved in cycle formation was constructed and used to validate this system. Mersacidin analogs were obtained in good yield in approximately 35% of the cases, producing a collection of 82 new compounds. This system was also used for the production of deletion and insertion mutants of mersacidin. The outcome of these studies suggests that this system can be extended to produce mersacidin variants with multiple changes that will allow a full investigation of the potential use of modified mersacidins as therapeutic agents.

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

The biosynthetic pathway of the type B lantibiotic actagardine (formerly gardimycin), produced by Actinoplanes garbadinensis ATCC31049, has been cloned, sequenced and annotated. The gene cluster contains the gene garA that encodes the actagardine prepropeptide, a modification gene garM, involved in the dehydration and cyclization of the prepeptide, several putative transporter and regulatory genes as well as a novel luciferase‐like monooxygenase gene designated garO. Expression of these genes in Streptomyces lividans resulted in the production of ala(0)‐actagardine while deletion of the garA gene from A. garbadinensis generated a strain incapable of producing actagardine. Actagardine production was successfully restored however, by the delivery of the plasmid pAGvarX. This plasmid contains an engineered cassette of the actagardine encoding gene garA and offers an alternative route to generating extensive libraries of actagardine variants. Using this plasmid, an alanine scanning library has been constructed and the mutants analysed. Further modifications include the removal of the novel garO gene from A. garbadinensis. Deletion of this gene resulted in the production of deoxy variants of actagardine, demonstrating that the formation of the sulfoxide group is enzyme catalysed and not a spontaneous chemical modification as previously believed.

Organization of the biosynthetic genes encoding deoxyactagardine B (DAB), a new lantibiotic produced by Actinoplanes liguriae NCIMB41362

Deoxyactagardine B (DAB) is a hitherto unknown type B lantibiotic, produced by Actinoplanes liguriae NCIMB41362. The mature peptide is 19 amino acids in length and structurally analogous to actagardine, differing by two amino acids (V15L and I16V) and the absence of a sulfoxide bond between residues 14 and 19. The biosynthetic genes encoding DAB are clustered, and in addition to the structural gene ligA include genes believed to encode for the proteins responsible for the modification, transport and regulation of DAB synthesis. Surprisingly, despite the presence of a gene that shares significant homology to the monooxygenase garO from the actagardine biosynthetic gene cluster, the oxidized form of DAB has not been detected. A lanA gene encoding the DAB peptide has been introduced into the plasmid pAGvarX and delivered into a strain of Actinoplanes garbadinensis lacking the structural gene for actagardine, garA (A. garbadinensis ΔgarA). Expression of this gene in A. garbadinensis ΔgarA resulted in the production of actagardine B, an oxidized form of DAB.

Generation of an actagardine A variant library through saturation mutagenesis

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.

Synthesis of Peptides Containing Overlapping Lanthionine Bridges on the Solid Phase: An Analogue of Rings D and E of the Lantibiotic Nisin

A methodology for the solid-phase synthesis of the overlapping lanthionine bridges found in many lantibiotics has been developed. A novel Teoc/TMSE-protected lanthionine derivative has been synthesized, and this lanthionine, and an Aloc/allyl-protected lanthionine derivative, have been incorporated into a linear peptide using solid-phase peptide synthesis. Selective deprotection of the silyl protecting groups, followed by sequential cyclization, deprotection of the allyl protecting groups, and further cyclization, enabled the regioselective formation of an analogue of rings D and E of nisin.

Chapter 22 Whole‐Cell Generation of Lantibiotic Variants

The generation of modified lantibiotics in whole cells has proved to be of value for the investigation of the specificity of the lantibiotic-processing enzymes and their tolerance to mutations in the primary sequence of lantibiotics. The development of methods to produce new lantibiotic variants has also enabled the investigation of the structure-activity relationships of these compounds and hence an evaluation of this hitherto underexploited class of natural products as a source of potential therapeutic drug candidates. We report the methods and strategies that have been used to engineer new lantibiotic variants and practical methods to analyze libraries of new compounds with a view toward optimizing drug properties.