Strategies for thiazole/oxazole-modified microcin discovery

Natural products continue to be an important source of therapeutically-relevant compounds. With the advent of inexpensive genome sequencing it has become apparent that bacteria produce a larger array of natural products than was previously believed. This new wealth in sequence data has potential to be helpful for the discovery of novel compounds by using genome mining. Although strategies of genome mining have become more efficient and capable of identifying novel biosynthetic gene clusters, it remains difficult to correlate gene clusters with natural products. In this dissertation I discuss limitations with the current methods of genome mining and correlating individual natural products with gene clusters. Furthermore, I characterize a rapidly growing family of natural products, the thiazole/oxazole-modified microcins (TOMMs), and discuss novel methods used to correlate the gene clusters to natural products from this family of metabolites. In chapter 2, I establish the sequence diversity and structural capability of bacteria and archaea to produce TOMM natural products. This genome mining characterization was used to identify nine novel classes of TOMMs, including one class from archaeal producers. In chapter 3, I discuss the utilization of genetic techniques to identify and isolate the TOMM natural product from the archaeal species Sulfolobus acidocaldarius. I demonstrate that although genetic manipulation has been previously used for the identification of natural products, comparative metabolomics is difficult to use for routine identification of low-abundance natural products such as the TOMM from S. acidocaldarius. Very few methods have been created to identify natural products from particular gene clusters. Therefore, in chapter 4, I discuss the creation of a novel method for the rapid identification of natural products following bioinformatics prioritization of antibiotic producing strains. This method utilizes the combination of genome mining and the chemical reactivity of natural products to discover new compounds. Dehydrated amino acids are modified residues commonly found in natural products such as TOMMs. I utilize the mild electrophilic chemical reactivity of dehydrated amino acids to label these natural products using a soft nucleophilic probe. These labeled natural products were easily detected using comparative mass spectrometry. Bacterial strains were prioritized by the genome mining established in chapter 2 to reduce the screening time to find a novel natural product. This dissertation presents the addition of novel genome mining and natural product discovery techniques to increase the discovery and production of therapeutically-relevant compounds