Assessment of the antibiotic production potential of marinederived actinomycetes via bioactivity screening and targeted genetic analysis

Drug discovery from marine organisms is gaining momentum and research focusing on marine actinomycete diversity has yielded numerous novel secondary metabolites with unique chemical structures. This study was based on targeted analyses of marine actinobacteria selected from a library of ca. 10.000 isolates cultivated from sediment- and sponge-samples in the Trondheim fjord, Norway. The two small sub-collections used in this study consisted of 35 shallow-water sediment-derived actinomycetes (“sub-library I”) and 27 deep-water sediment- and sponge-derived actinobacterial isolates (“sub-library II”). The isolates in these collections were analyzed for: (i) the influence of seawater on growth; (ii) their phylogenetic diversity based on molecular taxonomy, and (iii) their antibiotic production potential. The presence of seawater had different effects on the growth of the studied isolates. A preference for seawater or an obligatory requirement indicated the presence of indigenous marine actinomycetes in sub-library I and was used as a criterion for selection of isolates in sub-library II. The preliminary classification of isolates was performed by 16S rRNA gene sequence analysis which revealed significant biodiversity. Representatives of 11 different actinomycete genera were identified in sublibrary I and members of 8 genera confirmed in sub-library II. The results of a PCR screening for poly ketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes demonstrated the genetic potential to produce secondary metabolites with poly ketide and/or non-ribosomal peptide backbones for the majority of isolates in both sub-libraries. Whole-cell based antimicrobial assays involving microtitre cultivations and multiple fermentation media were performed using isolates of sub-library II with the objective to find producers of new antibacterial or antifungal compounds. Extracts with antibacterial or antifungal activity were identified for more than 50% of the isolates subjected to this screening. Nine isolates produced compounds with activity against multi-resistant bacterial and/or fungal strains. Fermentation extracts of isolate Nocardiopsis sp. TFS65-07 from sub-library II displayed high activity against vancomycin-resistant Enterococcus faecium. The active antibacterial compound was purified from fermentation extracts of TFS65-07 and structure elucidation identified it as a new member of the thiopeptide antibiotic family. The gene cluster for biosynthesis of the new thiopeptide, TP-1161, was identified by mining a draft genome sequence obtained for the producing organism. The proposed identity of the cluster was confirmed by gene inactivation experiments. Bioinformatics analyses of genes constituting the cluster and their products allowed proposition of the biosynthetic pathway for TP-1161. A cosmid containing the TP-1161 cluster (tpa) was isolated from a genomic cosmid library constructed for TFS65-07 and used for further studies of tpa gene functions. For heterologous expression, the cosmid was modified inserting the ΦC31 integration function and a selective marker, and successfully introduced into the genome of Streptomyces coelicolor M512. Heterologous production of TP-1161, however, could not be detected in M512. Further studies involving e.g. alternative hosts could pave the way for a systematic functional analysis of all genes involved in the TP-1161 biosynthesis