Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Brady Laboratory

Abstract

Natural products have historically served as a major source for most of the therapeutically relevant compounds. A significant fraction of these natural product-based drugs and leads are derived from microorganisms. Although the traditional culture-based strategy has proven very successful over the course of more than a century of investigation, it excludes a significant portion of the molecules present in environmental samples because majority of the bacteria are not readily amenable to culture-based strategies. This uncultured majority is believed to represent 99% of bacterial species in the environment. Therefore, it has remained a significant challenge to access the chemical diversity present in these microorganisms using culture-based methods. Most modern natural product discovery efforts are faced with the hurdle of high rediscovery rates (>99%) of known metabolites produced by easily cultured bacteria. Metagenomics has evolved as an alternative approach to conventional microbial screening. By directly cloning environmental DNA (eDNA or metagenome) in a surrogate host, one can exhaustively investigate the metagenome, independent of the culturability of the source organisms. Metagenomic approaches can thus provide access to previously untapped pools of chemical diversity. These techniques have been employed in the current thesis to search for a family of therapeutically relevant, polycyclic aromatic natural products, synthesized by Type II polyketide synthases (PKSs) in soil-borne bacteria. Through the functional characterization of soil eDNA-derived Type II PKS containing clones, herein we attempt to identify novel PKS systems that encode structurally diverse metabolites and explore their biological activity (Chapter 1). My doctoral work towards the discovery of novel type II PKS derived natural products has led to the elucidation of the mode of action of eDNA encoded antibiotics Fasamycins A and B. To identify the target of the fasamycins, fasamycin A resistant mutants were generated. Next generation sequencing of these mutants, along with in vitro biochemical assays, showed that fasamycins inhibit the FabF gene of the type II fatty acid biosynthetic system. Subsequently, candidate gene overexpression studies consolidated the mode of action of fasamycins. (Chapter 2) Insights into the genomes of uncultured microorganisms indicate that they are rich in Type II PKS gene clusters. In our attempt to identify novel aromatic polyketide natural products, we investigated the eDNA of soil samples collected from Texas region for unique Type II PKSs using homology-based metagenomic screening techniques. The eDNA-derived clones containing unique type II polyketide synthase genes identified in this screening were examined for the ability to produce clone-specific metabolites in Streptomyces. This led to the identification of hitherto unknown biosynthetic gene cluster for Seitomycin, a Type II PKS derived antibiotic. (Chapter 3) In addition to the work using homology-based metagenomic techniques outlined above, three unique Type II PKS clones were identified based on bioinformatics analysis and comparison with previously characterized PKS gene clusters. These novel pathways were functionally reconstructed from multiple overlapping clones using Transformation Associated Recombination (TAR). Upon complete reassembly, all of the three PKS pathways were demonstrated to produce clone-specific metabolites during heterologous expression studies in Streptomyces host. In future studies, characterization of these metabolites is likely to lead to novel Type II PKS derived natural products. (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

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