Abstract
The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.
Highlights
Emerging multidrug resistant pathogens resistant to most known antibiotics [1], coupled with the ubiquitous spread of antibiotic resistance throughout the wider environment such as in rivers [2], waste water [3] and agriculture [4], has led to an urgent global need for new antibiotics [5]
To either keto-synthase (KS) or β-ketoacyl acyl carrier protein (ACP) synthases from PK-like gene clusters, and all non-ribosomal peptide synthetases (NRPS) clone and library hits matched adenylation (A) domains from non-ribosomal peptides (NRPs) gene clusters
Similar findings were reported for the NRPS assay, with recovered clones having similarity to sequences from a diverse range of bacterial classes belonging to the Actinobacteria and Proteobacteria phyla, such as the Delta Proteobacterium Myxococcus, a prolific antibiotic producer [41]
Summary
Emerging multidrug resistant pathogens resistant to most known antibiotics [1], coupled with the ubiquitous spread of antibiotic resistance throughout the wider environment such as in rivers [2], waste water [3] and agriculture [4], has led to an urgent global need for new antibiotics [5]. Functional metagenomics (whereby genes are captured in plasmid, fosmid or BAC libraries and expressed) has been successfully used to capture and express many functional genes such as those associated with antibiotic resistance [18, 19] This approach has not been widely adopted for evaluating the diversity of biosynthetic gene clusters. Much of the work to date performed on biosynthetic gene cluster diversity relies mostly on two PCR assays described over a decade ago [23, 26,27,28] that are based on higly degenerate primers which may not be beneficial for screening large metagenomic libraries. In the current study we aimed to design a new updated PCR assay for NRPS and PKS modules for use in screening metagenomes for biosynthetic pathways. These included samples from Mars Oasis in Antarctica, which has previously been shown to have a high prevalence of the prolific antibiotic-producing phylum Actinobacteria [30], a high biodiversity site in Cuba proven to be abundant in enzymatic activity [31], and a range of sites from across Europe representing both coastal, untreated hay meadow, and heavily polluted agricultural soil
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