Browsing by Subject "Natural Products"

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    Borosin foray: Expanding a family of fungal autocatalytic α-N-methylating RiPP natural products
    (2020-07) Quijano, Marissa
    Backbone N-methylations impart several favorable characteristics to peptides including increased proteolytic stability and membrane permeability. Nonetheless, amide bond N-methylations incorporated as post-translational modifications are scarce in nature and were first demonstrated in 2017 for a single set of fungal metabolites. Here we expand on our previous discovery of iterative, autocatalytic α-N-methylating precursor proteins in the borosin family of ribosomally encoded peptide natural products. We identify over 50 putative pathways in a variety of ascomycete and basidiomycete fungi and functionally validate nearly a dozen new self-α-N-methylating catalysts. Significant differences in precursor size, architecture, and core peptide properties subdivide this new peptide family into three discrete structural types. Lastly, using targeted genomics, we link the biosynthetic origins of the potent antineoplastic gymnopeptides to the borosin natural product family. This work highlights the metabolic potential of fungi for ribosomally synthesized peptide natural products.
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    Discovery, Dereplication, And Functional Assignment Of Natural Products In Streptomyces Coelicolor M145
    (2021-11) Marshall, Andrew
    Natural products are structurally complex molecules produced from numerous organisms such as fungi, plants, and bacteria. Sought after primarily for their therapeutic properties, especially as antibacterial agents, screening of natural product-producing organisms extracts from 1920-1960s resulted in a “golden age” of antibiotic discovery. However, this did not last as antibacterial resistance and rediscovery of known molecules became rampant. Re-invigoration of natural product screening efforts occurred as genome sequencing became commonplace and the biosynthetic potential of natural product producers was revealed to be much greater than previously thought. This has led to new efforts to exploit their biosynthetic potential. Key improvements in analytical tools, namely mass spectrometry, has accelerated efforts to sensitively examine natural product mixtures. Additionally, computational tools have enabled the analysis of the large datasets that are a result of these sensitive analytical tools, including quantitation and identification of many molecules, providing a more comprehensive view of natural product extracts than ever before. Application of these tools towards discovery and functional annotation of natural products enables greater insight into the production of these important molecules. This thesis is focused on developing and applying modern mass spectrometry-based approaches to investigate the natural products produced from Streptomyces coelicolor M145, the most well-studied streptomycete. First, ion mobility mass spectrometry is investigated and shown to be an effective approach to separate and distinguish isobaric cyclic-prodiginines that are produced by S. coelicolor. Second, discovery and characterization of a novel actinorhodin-related molecule, θ-actinorhodin, is accomplished using a combined metabolomics and proteomics approach. This represents the first known trimeric benzoisochromanequinone natural product and illustrates the effective use of metabolomics and proteomics on the same liquid chromatography-mass spectrometer. Third, a comprehensive analysis of the time-resolved relationship between gene expression and metabolite abundance of S. coelicolor is enabled by the use of mass spectrometry-based tools and correlation network analysis. This resulted in important conclusions about the concerted expression of biosynthetic gene clusters and the resulting production of the linked metabolite. Finally, several strategies are presented for future analysis of natural product extracts including the use of proteomics to identify putative Streptomyces using a collated list of natural product synthases and an approach combining modern dereplication tools and chemoselective probes for prioritization of natural products for purification.