The widespread use of antibiotics is crucial for the treatment of illnesses, however the prolonged use and mis-management of these pharmaceuticals has likely contributed to the global issue of antibiotic resistance. Antibiotics are efficient at killing certain strains of bacteria, but over time through natural selection, resistant bacteria emerge as the dominant strain among the bacterial community. The genes of unculturable bacteria may have potential pharmaceutical relevance that can be utilized to alleviate this global epidemic. Sequence-based metagenomics is a method used to screen these genes, and can be used to discover molecules with pharmaceutical properties. Many of these molecules are products of nonribosomal peptide synthetase (NRPS) enzymes, which use a non-traditional, modular method to assemble molecules. Products of NRPS enzymes have shown antibiotic, antitumor and immunosuppressant properties. Isolation of NRPS enzymes was accomplished through PCR of metagenomic DNA pools followed by successive rounds of PCR to identify individual clones in metagenomic libraries. Restriction digestion and gel electrophoresis were used to verify the clone that gave rise to any positive PCR product. Thus far, one NRPS enzyme from a Minnesota municipal wastewater treatment plant metagenomic library has been confirmed through sequencing. Preliminary identification shows that the clone is 90% identical to <i>Pseudomonas entomophila</i> at the nucleotide level, with likely higher similarity at the protein level. Fifteen positive PCR products have been identified and isolated. The genes encoding these potential NRPS enzymes from a Wisconsin apple orchard and the Minnesota municipal wastewater treatment plant are currently being sequenced.

Identification of Nonribosomal Peptide Synthetase (NRPS) enzymes to find naturally occurring secondary metabolites

253rd National Meeting (2017)

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