Discovery of novel microbial natural products using a chemical screening approach and identification of gene clusters responsible for biosynthesis

Many of the pharmaceutical agents that are used clinically were developed from natural products and their derivatives. Natural products are often observed to have potent and specific interactions with biological targets, and are produced by a variety of macro and microorganisms. Actinomycetes have been an especially prolific source of natural products with antimicrobial applications and are responsible for approximately 70% of all clinically used agents. This has resulted in extensive investigation of these bacteria for the discovery of novel secondary metabolites, causing reisolation of known compounds to occur frequently. Novel techniques are needed to identify the production of new natural products to continue investigating natural sources for the discovery of new pharmaceutical agents. To overcome the reisolation issue that hinders natural products discovery, a chemical based screening method was employed to prioritize strains for scale-up and further investigation. This method involved analyzing crude fermentation extracts using LC-HRMS, followed by principal component analysis to visualize the results. Utilization of this technique to analyze libraries of actinomycetes resulted in the identification of novel natural products more effectively than the tradition bioassay-guided method. This led to the selection of Streptomyces sp. RKKD-790 and Kitasatospora cystarginea NRRL B-16505 for further investigation. A novel lasso peptide natural product tentatively named RKKD790_1592, was isolated from the fermentation broth of Streptomyces sp. RKKD-790. This group of peptides are characterized by their unique threaded structures, which provide them with superior stability compared to linear peptides. Computation modelling, MS/MS and stability assays were used to confirm that this novel peptide contained the characteristic threaded structure. The strain K. cystarginea was found to produce three novel natural products, cystargamide, and cystargolides A and B. Unique structural features of cystargamide include the presence of the non-proteinogenic amino acids 4-hydroxyphenyl glycine and 5’-hydroxy typtophan and 2,3-epoxydecanoyl group. Cystargamide displayed the ability to activate production of pigmented metabolites by Streptomyces coelicolor in a disk diffusion assay. This interesting result indicated that cystargamide may be involved in bacterial communication, and may induce natural product biosynthesis in other bacteria. The gene cluster responsible for producing this novel lipopeptide was determined bioinformatically from the whole genome sequence of K. cystarginea. Cystargolides A and B are novel β-lactone containing peptides that were also produced by K. cystarginea. These peptides have very unique structures, and are the only natural products to have been described that contain a carboxyl and isopropyl substituted β-lactone group. Cystargolides A and B were found to be inhibitors of human 20S proteasome, showing that they are an excellent candidate for development as antiproliferating agents to treat cancer. Further investigations of these applications are ongoing by members of the Kerr group. The gene cluster involved in production of cystargolides A and B was predicted using bioinformatics and confirmed by generating a gene disruption mutant. The discovery of these novel bioactive natural products represent exciting opportunities for development as an inducer of bacterial natural products, and an anticancer agent. Members of the Kerr group are continuing to explore these interesting applications. Identification of their unique structures and biosynthesis also extend our knowledge of the natural product production capabilities of bacteria