Student Theses and Dissertations

Date of Award

2012

Document Type

Thesis

RU Laboratory

Brady Laboratory

Abstract

The isolation of small molecule natural products from bacteria has led to the identification of many of the antibiotics currently in use today. Within the last 25 years, in fact, natural products (and their derivatives) account for almost three quarters of all antibiotic discoveries (Newman and Cragg). The discovery of novel natural product antibiotics, however, has witnessed a significant drop in numbers over this time period, suggesting that new sources of small molecules may be necessary to keep up with the need for new antibacterial compounds (Newman and Cragg). Due to the emergence of bacteria resistant to commonly used antibiotics, this need seems to be ever increasing, even as the pools of new antimicrobials are dwindling. One potential source of new small molecule diversity is the large quantity of uncultured bacteria found in any given environment. It is estimated that, in soil, greater than 99% of the bacteria present are recalcitrant to culture. This poses a challenge for the field of natural product discovery, considering the traditional route to small molecule discovery from soil bacteria is to organically extract pure cultures of an individual bacterium. New techniques emerging from the field of metagenomics take a culture-independent approach to examining the biosynthetic capabilities of soil bacteria by extracting the environmental DNA (eDNA) from the soil and cloning it into a host capable of maintaining and possibly heterologously expressing genes, which encode the production of small molecule secondary metabolites. These techniques have been employed in this thesis to expand not only a family of therapeutically relevant small molecule antibiotics (glycopeptides), but also to develop, in conjunction with new strategies, improvements on existing metagenomics approaches. My work towards the discovery of novel glycopeptide antibiotics has led to the identification of six novel glycopeptide biosynthetic pathways, via the use of homology-based metagenomics techniques, and the production of 15 novel congeners of this extremely important family of compounds. Additional work in collaboration with members of the Darst Laboratory at the Rockefeller University led to the structural and biochemical characterization of two sulfotransferases, enzymes responsible for the sulfonation of a glycopeptide substrate. This type of chemical modification is rarely seen in glycopeptide biosynthesis, and the collection of three sulfotransferases identified using metagenomics techniques therefore presented a unique opportunity to gain a better understanding of the reactivity and substrate restrictions of these enzymes. In addition to the work using existing, homology-based metagenomics techniques, an additional strategy was developed, utilizing the complementation of a well-studied biosynthetic pathway, responsible for the production of an iron-scavenging siderophore in E. coli, to select for eDNA clones likely to be rich in secondary metabolite biosynthetic genes. This strategy selects for clones containing 4’-phosphopantetheinyltransferases (PPtases), which are commonly proximally linked to the non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). PPtases are responsible for the post-translational modification of NRPSs and PKSs, which are genes commonly seen in secondary metabolite biosynthesis. This strategy was used to enrich an eDNA-derived metagenomic library for PKS and NRPS genes by over an order of magnitude compared to an un-enriched library.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/512

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