Date of Award
Natural products have been a significant source of new drugs over the past 25 years. In fact, the origins of nearly 70% of antibacterial, antifungal, antiparasitic and antiviral small molecule drugs are in natural products (Newman and Cragg 2007). A significant proportion of natural product drugs and leads have been isolated from microbes (Newman and Cragg 2007). However, the discovery of novel natural products from microbes faces the growing hurdle of rediscovering known metabolites. Streptomyces is a microbe that has been a particularly rich source of natural products; however, it has been predicted that the last easily accessible novel Streptomyces metabolite will be found within the next few years (Watve, Tickoo et al. 2001). Finding a new source rich in natural products would be very beneficial to the development of new drugs. One potential new source of natural products is the plethora of uncultured bacteria found in environmental soil samples. It is estimated that greater than 99% of bacteria from soil samples are recalcitrant to culturing in a laboratory setting. A culture-independent approach of extracting environmental DNA (eDNA) from soil and cloning it into a library was leveraged to take advantage of the genetic diversity of uncultured microbes. Uncultured microbes have been found to be rich in type II polyketide synthase (PKS) gene clusters (Kieser, Bibb et al. 2000). PKS gene clusters produce a structurally diverse collection of aromatic natural products. The nascent polyketide in these pathways is produced by a conserved minimal PKS, composed of an Î±- ketosynthase, Î²-ketosynthase and acyl carrier protein. In order to access the structural diversity encoded by type II PKS gene clusters from uncultured bacteria, environmental DNA (eDNA) libraries were constructed from desert soil. These libraries were found to be rich in PKS genes and cosmids containing minimal PKS genes were recovered from the eDNA library using Î²-ketosynthase sequences as probes. The recovered clones containing minimal PKS genes from the eDNA library were screened for the ability to confer the production of clone-specific molecules to cultured Streptomyces albus. One of the recombinant Streptomyces clones was found to produce erdacin, a 26- carbon polyketide with both a novel carbon backbone and a novel pentacyclic ring arrangement. The novel structure and pentacyclic ring arrangement of erdacin raised questions as to the biosynthetic origins of this metabolite encoded by the eDNA-derived V167 clone. A biosynthetic analysis of wild type and transposon mutant V167 cultures indicated that erdacin arises from the heterodimerization of two 13-carbon monomer subunits that are both derived from octaketide precursors. These two 13-carbon subunits arise from two different second cyclizations, but both have the same three carbons excised from their 16-carbon octaketide precursors.
King, Ryan W., "Novel Molecules from Environmental DNA-Derived Type II Polyketide Biosynthetic Gene Clusters" (2013). Student Theses and Dissertations. 176.