Analysis of the Tetronomycin Gene Cluster: Insights into the Biosynthesis of a Polyether Tetronate Antibiotic

110

A diverse collection of 60 marine-sediment-derived Actinobacteria representing 52 operational taxonomic units was screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were employed to specifically target adenylation domains associated with nonribosomal peptide synthetases (NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne polyketide synthases (PKSs). In total, two-thirds of the strains yielded a sequence-verified PCR product for at least one of these biosynthetic types. Genes associated with enediyne biosynthesis were detected in only two genera, while 88% of the ketosynthase sequences shared greatest homology with modular PKSs. Positive strains included representatives of families not traditionally associated with secondary-metabolite production, including the Corynebacteriaceae, Gordoniaceae, Intrasporangiaceae, and Micrococcaceae. In four of five cases where phylogenetic analyses of KS sequences revealed close evolutionary relationships to genes associated with experimentally characterized biosynthetic pathways, secondary-metabolite production was accurately predicted. Sequence clustering patterns were used to provide an estimate of PKS pathway diversity and to assess the biosynthetic richness of individual strains. The detection of highly similar KS sequences in distantly related strains provided evidence of horizontal gene transfer, while control experiments designed to amplify KS sequences from Salinispora arenicola strain CNS-205, for which a genome sequence is available, led to the detection of 70% of the targeted PKS pathways. The results provide a bioinformatic assessment of secondarymetabolite biosynthetic potential that can be applied in the absence of fully assembled pathways or genome sequences. The rapid identification of strains that possess the greatest potential to produce new secondary metabolites along with those that produce known compounds can be used to improve the process of naturalproduct discovery by providing a method to prioritize strains for fermentation studies and chemical analysis.Microbial natural products represent the primary resource from which new medicines are derived, accounting for approximately half of the antibiotics discovered as of 2002 (6). Over the past several decades, however, drug discovery efforts have moved away from microbial products (29), in part due to a reduction in the ratio of new chemical entities discovered relative to the isolation of known metabolites (3) and the challenges associated with developing effective "dereplication" methods to improve the efficiency of the discovery process. More recently, the rise in drug-resistant pathogens has left many current antibiotics obsolete, while the limited success of alternative discovery strategies, such as combinatorial chemistry, have created a void in the pipeline of new drug leads (39). The response to this need includes renewed interest in a group of actinobacteria commonly called actinomycetes (defined here as ba...

Section: Distribution Of Biosynthetic Genesmentioning

confidence: 99%

Sequence-Based Analysis of Secondary-Metabolite Biosynthesis in Marine Actinobacteria

Gontang

Gaudêncio

Fenical

et al. 2010

110

“…So far, five polyether biosynthesis gene clusters have been identified and characterized for the biosynthesis of monensin (27), nanchangmycin (37), lasalocid (35), nigericin (15), and tetronomycin (9). Based on in silico and experimental analysis, the polyene backbones, assembled by a type I polyketide synthase (PKS), could undergo oxidative cyclization catalyzed by an epoxidase and one or more epoxide hydrolases to generate the characteristic pattern of ether bonds.…”

mentioning

confidence: 99%

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Jiang

Wang

Kang

et al. 2012

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.

“…However, five complete polyether ionophore gene clusters now have been cloned and fully sequenced: monensin (31), nanchangmycin (43), nigericin (20), tetronomycin (12), and lasalocid (34,41). The complete polyether ionophore gene clusters contain a conserved tailoring enzyme gene encoding a flavin-linked epoxidase, which is responsible for the epoxidation of unsaturated polyether intermediates.…”

mentioning

confidence: 99%

Genetic Screening Strategy for Rapid Access to Polyether Ionophore Producers and Products in Actinomycetes

Wang

Ning

et al. 2011

Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of Actinomycetales. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including Micromonospora, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.Programs aimed at the discovery of new secondary metabolites with interesting biological activities from microbial sources have found an impressive number of compounds during the past 50 years. Actinomycetes represent one of the most prolific microbial sources for the discovery of bioactive metabolites (5, 6), many of which are produced by polyketide synthase (PKS) systems (6, 42). Polyether ionophores (Fig.