Student Theses and Dissertations

Hala Iqbal

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Brady Laboratory

Abstract

Bacteria are a prolific source of therapeutically and industrially relevant natural products. Traditional routes of natural product discovery focus on cultured bacteria, a strategy that eludes 99% of the microbiome. Metagenomics offers a route to study uncultured bacteria by extracting DNA present in environmental samples and cloning this eDNA into a vector to make metagenomic libraries. Libraries can then be screened in various ways to find natural products with desired characteristics and activities. Functional screening uses phenotypically identifiable traits such as changes in color or antibiotic activity to discover clones of interest in a metagenomic library. This mode of screening is contingent upon the bacterial host being able to heterologously express genes present in library clones, and therefore the choice of host is critical in determining the natural products found. In this thesis, I explored the use of two heterologous hosts for functional screening: Ralstonia metallidurans and Streptomyces albus, and found that both are powerful hosts for finding a wide range of natural products. Functional screening of a 700,000 cosmid clone library in R. metallidurans for antibiotic activity led the discovery of a diverse set of antibacterially active enzymes. Clones displaying zones of inhibition of growth of an assay strain in top agar bacterial overlays were predicted through bioinformatic analysis to encode peptidases, lipases and cell-wall lytic enzymes conferring antibiotic activity, and confirmed using transposon mutagenesis and subcloning. These antibacterial activities could not be replicated when clones were transformed into Escherichia coli, indicating the powerful ability of R. metallidurans to find diverse antibacterially active enzymes in metagenomic screens. Streptomyces is an excellent candidate for finding small molecules from functional metagenomic screens because it natively and heterologously produces a diverse range of small molecules. Functional screening in Streptomyces has not been successful to date: small libraries have been built in Streptomyces lividans and screening has not resulted in many small molecules being found. A collection of 39 Streptomyces strains was analyzed for transformation efficiency and heterologous expression. S. albus was identified as a strain with high heterologous expression capabilities. To transform a large metagenomic library into Streptomyces albus, a high quality library was built in E. coli using an E. coli – Streptomyces shuttle vector and gel-purified to remove any empty vector. Transformation of this library into S. albus was optimized by varying mating conditions. A 1.5 million clone library was built in S. albus, making this the first metagenomic library reported in S. albus, and is approximately 100x larger than any other library reported in Streptomyces to date. Preliminary screens of this library for colonies displaying changes in color were used to identify 12 clones, of which 8 contained carotenoid pathways, 1 contained a type III polyketide pathway and 3 contained pathways that could not be easily identified. Clone specific small molecules from these hits will be further analyzed for novel secondary metabolites. This thesis represents efforts at solving one of the problems limiting the success of functional screening, namely choice of heterologous host. Screens in two hosts, R. metallidurans and S. albus, are described that lead to the identification of different types of natural products: antibacterially active enzymes and small molecule biosynthetic pathways. This reiterates the importance of heterologous host choice in functional screening of metagenomic libraries for natural products with desired characteristics and activities.

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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