Enduracidin A

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr(8), is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.

A new multi-residue method for the determination of seven polypeptides, namely, polymixin B1, polymixin B2, polymixin E1 (colistin A), polymixin E2 (colistin B), enduracidin A (enramycin A), enduracidin B (enramycin B), and bacitracin A, in food of animal origin was developed and validated for chicken muscle tissue. Chicken muscle tissue was extracted with acidified methanol (1 % TFA). After homogenization, shaking, and centrifugation, the acidified methanol extract was decanted. A second extraction was performed with methanol (1 % TFA) and formic acid (1 %) 25:75, v/v. The pooled extract was cleaned up and concentrated on a solid-phase extraction cartridge. The retained analytes were eluted with methanol/acetonitrile. The extract was evaporated to dryness, reconstituted in mobile phase, filtered, and quantified by LC-MS/MS under ESI conditions. The method has a LOQ of 50.0 μg/kg for polymixin E2 (colistin B), 39.0 μg/kg for polymixin E1 (colistin A), 74.0 μg/kg for polymixin B1, 71.0 μg/kg for polymixin B2, 66.0 μg/kg for enduracidin A, 50.0 μg/kg for enduracidin B, and 30.0 μg/kg for bacitracin A in chicken muscle tissues. This is the first sensitive, suitable, multi-residue method reported for the seven polypeptide drug residues in chicken muscle tissue.