Characterization of an Antimicrobial, Non-Ribosomal Lipopeptide from the Flavobacterium, Aquimarina muelleri

Abstract

The alarming prevalence of multi drug-resistant bacterial pathogens has made novel antimicrobial drug development a focal point for current molecular biology research. Specifically, researchers have targeted small molecule products of non- ribosomal peptide synthetase (NRPS) enzymes, which produce structurally diverse peptides and lipopeptides (LPs). Many current drugs in clinical use today originate from or are semi-synthetic forms of such small molecules. Thus, efforts to isolate new sources of these peptides have been focused on genome mining for secondary metabolite-producing biosynthetic gene clusters (BGCs) across bacterial genomes, in the hopes of identifying novel natural sources for antimicrobial small molecules. Though Actinobacteria have been studied extensively as prolific producers of such compounds, genetic analysis and bioassay-based research from the Donia Lab has shown species of the phylum Bacteroidetes are also rich in bioactive compounds. One such compound, a predicted lipopeptide, from the marine bacterium, Aquimarina muelleri, has shown remarkable broad-spectrum antibacterial activity in disc diffusion bioassays [1]. Here, we present results from ongoing research into the purification and characterization of the identified compound, in addition to preliminary genetic analyses into the predicted BGCs encoding for the peptide in the A. muelleri genome. Our research included large-scale cultivation of and extraction from A. muelleri, in addition to a series of chemical separation experiments utilizing both gravity flow columns and HPLC columns. We aimed to isolate as pure a concentrated sample of the target compound as possible (>95%) to be able to use high resolution nuclear magnetic resonance spectroscopy (NMR) to confirm the molecular structure of the lipopeptide. High resolution tandem mass spectrometry (MS/MS) analysis proved unprofitable, due to poor fragmentation. Currently, we have an approximately 80- 85% pure sample and are continuing purifications before NMR analysis.