Dissertation
Natural product discovery of thiopeptide producers and functional characterization of adenylation domains in thiostrepton and siomycin biosynthesis
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2021
DOI:
https://doi.org/10.17918/00000571
Abstract
The antibiotic resistance crisis has led to the need for new antimicrobials. Natural product producers, including bacteria, are an abundant source for new antibiotics. Streptomycetes are avid natural product producers. These bacteria have large genomes approximately 8-12 Mb that contain many secondary metabolite biosynthetic gene clusters (smBGCs). We completed de novo whole genome sequencing of Streptomyces actuosus ISP-5337, Streptomyces sioyaensis B-5408, and Actinospica acidiphila B-2296 using PacBio RSII technology. Bioinformatic mining of each genome identified 75 smBGCs and 9 "unknown" clusters predicted to encode for bacteriocin, lanthipeptide, and nutrient-scavenging bioactive compounds. Fractionation of the crude extracts and subsequent antibacterial challenge against Staphylococcus aureus showed potent antibiotic activity. In parallel, fractions were analyzed by liquid chromatography mass spectrometry and detailed MS/MS analysis of the fractions showed production of nosiheptide and siomycin. Next, we used our sequenced genomes to analyze the BGCs of nosiheptide, thiostrepton and siomycin. Recent identification of non-ribosomal machinery, including an adenylation domain and carrier protein domain, encoded in the ribosomally-encoded BGC of nosiheptide led us to functionally characterize these domains in the BGCs of thiostrepton and siomycin. We determined the substrate specificities of heterologously expressed and purified adenylation domains TsrJ and SioQ using a coupled pyrophosphate release assay. In vitro characterization demonstrated both adenylation domains have substrate specificities for quinaldic acid derivative 4,8-dihydroxyquinoline-2-carboxylic acid. Next, we used our genome sequences to clone a 36 kb thiopeptide BGC from Streptomyces azureus into Escherichia coli, for heterologous production and engineering purposes. These results will facilitate targeted bioengineering of thiopeptides to produce novel antibiotics with improved chemical properties and address the need for new antibiotics.
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Details
- Title
- Natural product discovery of thiopeptide producers and functional characterization of adenylation domains in thiostrepton and siomycin biosynthesis
- Creators
- Haley Morgan Majer
- Contributors
- Joris Beld (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 150 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991014972849504721