Student Theses and Dissertations

Jeffrey Kim

Date of Award

2011

Document Type

Thesis

RU Laboratory

Brady Laboratory

Keywords

uncultured bacteria, soil derived DNA fragments, natural product biosynthesis, eDNA libraries

Abstract

A single gram of soil can contain thousands of unique bacterial species, only a small fraction of which is regularly cultured in the laboratory.Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has begun to provide access to the chemical diversity encoded in the genomes of previously uncultured bacteria. The systematic exploration of natural product biosynthesis in uncultured bacteria, however, still faces several challenges that we sought to experimentally address. First, many natural product gene clusters cannot be detected in functional screens due to cloning and expression limitations of metagenomic library host strains. Second, the lack of robust and scalable gene cluster assembly methods precludes the functional characterization of a large number of natural product biosynthetic gene clusters from cosmid-based eDNA libraries. Third, the large-scale analysis of metagenomic natural product chemical diversity and the phylogenetic context it is found in were previously unaddressed due to the complexities of microbial communities. To address these questions, we have: 1) Demonstrated that sequence-based screens can be used to systematically discover a diverse range of natural product gene clusters by screening two of the largest recombinant eDNA libraries reported to date. This approach circumvents many of the challenges of using functional screens to discover novel biosynthetic gene clusters. (Chapter 2) 2) Shown that transformation associated recombination in S. cerevisiae can be used to functionally reassemble large natural product gene clusters that exceed conventional eDNA cloning limits. This approach overcomes a significant barrier which prevented the functional characterization of many natural product gene clusters from eDNA libraries. (Chapter 3) 3) Developed a high throughput sequencing analysis framework to characterize environmental biosynthetic capacity. These results suggest that the continued construction and screening of soil-based eDNA libraries should provide access to additional novel pools of biosynthetic enzyme diversity. (Chapter 4)

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/10209/392

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