Culture independent studies (eDNA, metagenomics):
It is now widely recognized that the vast majority of bacteria are not readily cultured in the laboratory. The inability to culture these bacteria renders them incompatible with the most heavily relied upon techniques for characterizing bioactive natural products. Although it is still not possible to easily culture most bacteria from the environment, it is possible to extract microbial DNA directly from environmental samples and clone this DNA into model cultured bacteria where, for the first time, it can be functionally characterized. Our group has pioneered the use of culture independent methods to guide the discovery of novel bioactive secondary metabolites from natural microbial communities.
Homology approaches: We are developing homology-based approaches for studying gene clusters predicted to encode novel metabolites from within soil metagenomes. With homology screening strategies, clones containing genes known to be involved in the biosynthesis of natural product substructures commonly seen in bioactive compounds are recovered from environmental DNA (eDNA) libraries, and then each new gene cluster is assessed for its ability to encode novel bioactive small molecules in model cultured hosts. These studies are designed to provide access to both previously inaccessible derivatives of pharmacologically important classes of natural products, as well as completely novel natural products that are only linked to known metabolites by a single, highly conserved biosynthetic step. Gene clusters and the molecules identified in these studies form the basis for subsequent biosynthetic and mode action studies in the lab.
Functional approaches: The second strategy we are using to identify small molecule producing biosynthetic systems in large eDNA libraries is phenotype (expression-dependent) screening. With this approach, large insert eDNA libraries are examined directly in simple high throughput assays designed to identify clones exhibiting phenotypes traditionally associated with the production of bioactive small molecules (e.g. antibiosis, antifungal activity, cytotoxicity, etc.). In essentially all reported cases, these studies have been carried out in Escherichia coli. We are working on expanding the phylogenetic diversity of the model bacterial systems available for hosting and screening large eDNA libraries.
Molecular diversity studies: The true diversity of biosynthetic genes present in the environment remains a mystery. We are using next generation sequencing to assess the diversity of natural product biosynthetic genes present in diverse environments in an effort to provide a more complete picture of the natural product biosynthesis genes found in these libraries.
Small molecules play important roles in establishing and propagating bacterial infections. As resistance to currently available antibiotics increases, there has been a renewed interest in the identification of novel strategies to control infectious diseases. Cryptic small molecule biosynthetic gene clusters, gene clusters that do not appear to encode the biosynthesis of any known metabolites, are routinely found in sequenced bacterial genomes. In bacterial pathogens, these cryptic pathways represent the pool from which additional small molecule virulence factors will be found. We are using transcriptional activation and knockout strategies to study the role of cryptic natural product biosynthesis in pathogenesis. The knowledge gained from these studies is being used to develop new strategies for controlling bacterial pathogenesis.
The development of robust DNA-based methods to functionally access previously inaccessible natural product biosynthetic pathways found in the genomes of both cultured and as yet uncultured bacterial should significantly increase the number and diversity of natural products that are available to test as probes of biological processes. This in turn, should shed light on how best to use this collection of previously inaccessible small molecules "for the benefit of humanity".