While bioassay driven screening of natural product extracts from easily cultured bacteria has identified many highly effective antibiotics, continuing to screen this same small pool of biosynthetic pathways in search of novel biologically active small molecules has proved to be counter productive. Unfortunately, the vast majority of bacteria present in nature remain recalcitrant to culturing and, as result, these bacteria have not yet been explored for the production of novel antibacterial agents. Uncultured bacteria are likely the largest remaining pool of biosynthetic diversity not yet examined for the production of secondary metabolites. Exploiting this genetic diversity should prove to be a useful strategy for uncovering new antibiotics that can serve as novel therapeutics against antibiotic resistant and ESKAPE pathogens. Nonribosomal peptides (NRPs) comprise a large fraction of pharmacologically relevant microbial secondary metabolites, many of which function as antibiotics. To access antibacterially active nonribosomal peptides within the genomes of environmental bacteria, DNA extracted directly from geographically diverse soils will be used to construct a set of large environmental DNA libraries. These libraries will be enriched for nonribosomal peptide biosynthetic gene clusters and the enriched libraries will be sequenced. Peptides encoded by these gene clusters will be bioinformatically predicted and chemo enzymatically synthesized en mass using solid phase peptide synthesis. Libraries of synthetic non-ribosomal peptides (syn-NRPs), all of which are based on sequences that have been evolutionarily selected for bioactivity, will be tested for antibacterial activity against antibiotic resistant and ESKAPE (Enterococcus faecium. Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Enriched libraries hosted in Streptomyces coelicolor and Burkholderia cenocepacia will also be screened in functional metagenomic screens for clones that exhibit antibacterial activity against model antibiotic resistant Gram-positive, Gram-negative and ESKAPE pathogens.
The fusion of metagenomics, bioinformatics and solid phase peptide synthesis that is outlined in this proposal will provide access to novel natural products that can, for the first time, be assesed for activity against diverse antibiotic resistant pathogens.