Production of a New Thiopeptide Antibiotic,TP-1161, by a Marine Nocardiopsis Species

Twenty-seven marine sediment- and sponge-derived actinomycetes with a preference for or dependence on seawater for growth were classified at the genus level using molecular taxonomy. Their potential to produce bioactive secondary metabolites was analyzed by PCR screening for genes involved in polyketide and nonribosomal peptide antibiotic synthesis. Using microwell cultures, conditions for the production of antibacterial and antifungal compounds were identified for 15 of the 27 isolates subjected to this screening. Nine of the 15 active extracts were also active against multiresistant Gram-positive bacterial and/or fungal indicator organisms, including vancomycin-resistant Enterococcus faecium and multidrug-resistant Candida albicans. Activity-guided fractionation of fermentation extracts of isolate TFS65-07, showing strong antibacterial activity and classified as a Nocardiopsis species, allowed the identification and purification of the active compound. Structure elucidation revealed this compound to be a new thiopeptide antibiotic with a rare aminoacetone moiety. The in vitro antibacterial activity of this thiopeptide, designated TP-1161, against a panel of bacterial strains was determined.Natural products remain the most prolific source of new antimicrobials, and the chemical diversity of natural compounds is still unmatched by combinatorial chemistry approaches (9, 31). While the latter has been successfully applied for lead optimization, it basically failed to deliver genuinely new pharmacophores, especially in the field of antimicrobials (31), mainly due to limitations in the structural variety of compounds represented in combinatorial libraries.Most of the antibiotics in clinical use today have been developed from compounds isolated from bacteria and fungi, with members of the actinobacteria being the dominant source (34). Traditionally, most of these antimicrobials have been isolated from soil-derived actinomycetes of the genus Streptomyces. However, isolation strategies in recent years have been directed to unexploited environments like marine sources (40). Bioprospecting efforts focusing on the isolation and screening of actinobacteria from ocean habitats (25, 27) have added new biodiversity to the order Actinomycetales and revealed a range of novel natural products of pharmacological value. The existence of marine actinobacterial species physiologically and phylogenetically distinct from their terrestrial relatives is now widely accepted, and new taxonomic groups of marine actinomycetes have been described for at least six different families within the order Actinomycetales (12). Apart from being phylogenetically distinct from their terrestrial relatives, marine isolates have been shown to possess specific physiological adaptations (e.g., to high salinity/osmolarity and pressure) to their maritime surroundings and many were found to produce novel and chemically diverse secondary metabolites (10, 13, 35).Most streptomycetes and other filamentous actinomycetes possess numerous gene clusters for the biosynthesis of secondary metabolites (2, 32), and genome sequence studies have shown that large portions of their genomes are devoted to secondary metabolite biosynthesis. Twenty gene clusters coding for known or predicted secondary metabolites were identified in the 8.7-Mb genome of Streptomyces coelicolor A3(2) (2), and 6.4% of the 8.7-Mb genome of Streptomyces avermitilis is dedicated to gene clusters for secondary metabolite biosynthesis (32). The marine actinomycete Salinispora allocates nearly 10% of its 5.2-Mb genome to 17 diverse biosynthetic loci, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and several hybrid clusters (4, 43). Many medicinally important natural products, including antibacterials and antifungals, are synthesized by these multimodular assembly lines (14), and genome mining for secondary metabolite gene clusters has become a common tool to assess the genetic capability of bacteria to produce novel bioactive compounds. However, even for well-studied model antibiotic producers like S. coelicolor A3(2), discrepancies between the number of known metabolites on the one hand and the number of pathways identified from genomic data on the other hand are tremendous (2). These discrepancies can only be explained by the facts that most gene clusters for secondary metabolites are silenced under standard laboratory cultivation conditions and that an expression or upregulation of these pathways is only triggered in response to certain environmental signals. It has been shown that by cultivating bacteria under a range of conditions, it is possible to obtain products of many of these “orphan” biosynthetic pathways (4). Using the OSMAC (one strain-many compounds) approach, Bode et al. were able to isolate more than 100 compounds comprising 25 structural classes from only six microorganisms (4).In this study, marine sediment-derived actinomycete isolates were analyzed for the production of antimicrobial secondary metabolites by using microwell plate fermentations and a range of media and conditions. This approach led to the isolation of a new thiopeptide antibiotic, designated TP-1161, produced by a marine sediment-derived Nocardiopsis isolate. Here we report the isolation and structural and biological characterization of TP-1161.