Aureusimine B

Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.

Overexpression of a putative type III polyketide synthase (PKSIII) from the marine myxobacterium Enhygromyxa salina SWB007 in Streptoinyces coelicolor MI 146 led to the accumulation of a novel monoketopiperazine consisting of phenylalanine and isoleucine. This compound was named phileucin and shows high structural similarity to phevalin (aureusimine B). The protease inhibiting activity was tested against human cathepsin L, human leukocyte elastase; bovine trypsin and bovine chymotrypsin. In contrast to phevalin, no protease inhibition was observed.

Drug resistant infections are an increasing problem world-wide, responsible for an estimated 700,000 annual mortalities. The use of antibiotics to treat such infections has resulted in the development of resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). One potential alternative strategy for treating drug resistant bacterial infections is to inhibit the production of toxins, thereby making the bacteria less harmful to the host, a so called "anti-virulence" approach. In MRSA, the agr quorum sensing system is one of the major regulators of toxin production, and quorum sensing inhibitors that target this system are a promising anti-virulence strategy. With this study, we developed a method that enables the activity of quorum sensing inhibitors to be measured using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). This method is an improvement over existing methods because it can be employed to distinguish antimicrobial activity from quorum sensing inhibition activity based on the UPLC-MS data. This is possible by simultaneously tracking production of metabolites regulated by the agr quorum sensing system (AIP-I and formylated δ-toxin) and a metabolite that appears not to be agr regulated under the conditions of this study (aureusimine B). The newly developed method provides more nuanced indication of how metabolite production changes over time and in response to quorum sensing or growth inhibition than is possible with commonly employed spectroscopic methods.