Browsing by Subject "natural product biosynthesis"

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    Borosins: The biosynthesis of ribosomal alpha-N-methylated peptides from fungi and bacteria
    (2020-05) Miller, Fredarla
    Natural products are bioactive small molecules synthesized by living organisms. They are often used in medicine and industry as pharmaceuticals, flavors, pigments, and more. α-N-Methylation of the peptide backbone, as seen in the immunosuppressant natural product cyclosporin A, confers desirable pharmacokinetic characteristics such as resistance against proteolytic degradation, enhanced rigidity/target specificity, membrane permeability, and drug oral bioavailability. Until recently, it was thought that this backbone modification was only naturally accessible through non-ribosomal peptide synthesis. Our discovery of the borosin family of peptide natural products challenged that assumption by identifying nematocidal metabolites as α-N-methylated, ribosomally synthesized and posttranslationally modified peptides (RiPPs, a quickly growing class of natural products known for their modular and potentially engineerable biosynthesis). The first borosins we discovered, found primarily in basidiomycete fungi, are signified by a unique autocatalytic mechanism for incorporating α-N-methylations, where the natural product sequence is tethered in a single protein to an iteratively acting autocatalytic methyltransferase. In-depth bioinformatics analysis has since revealed an even more diverse subfamily of putative α-N-methylated peptides that have natural product precursors encoded in trans to the methyltransferase. These so-called “split borosins” are found primarily in bacteria. This thesis describes how borosins fit into the larger field of natural product and RiPP biosynthesis. It includes the discovery of additional domain architectures for the fused and split systems as well as a structural and biochemical analysis of a putative split borosin system in the bacterium Shewanella oneidensis MR-1. We have also begun to investigate the native role the split borosin peptide natural product may play in S. oneidensis MR-1 through the creation of genetic knockouts and in vivo phenotyping experiments.
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    Microbial biosynthesis of β-lactone natural products: from mechanisms to machine learning
    (2020-06) Robinson, Serina
    Natural products with β-lactone (2-oxetanone) rings often have potent antibiotic, antifungal and antitumor properties. These reactive pharmacophores are known to covalently inhibit enzymes from over 20 different families including lipases, proteases, and fatty acid synthases. Since the discovery of the first β-lactone natural product, anisatin, in 1952, over 30 compounds with β-lactone moieties have been isolated from bacteria, fungi, plants and insects. Now in the post-genomic era, the field of natural product drug discovery is in the midst of a transformation from traditional ‘grind and find’ methods to targeted genome mining approaches. However, genomics-guided discovery of new β-lactone natural products was hampered by a lack of understanding of the enzymes that catalyze β-lactone ring formation. In 2017, our lab reported the first standalone β-lactone synthetase enzyme, OleC, in a bacterial long-chain hydrocarbon biosynthesis pathway from Xanthomonas campestris. This thesis builds on this initial breakthrough through biochemical characterization of the substrate specificity, kinetics, and mechanism of X. campestris OleC. Using these biochemical data, I trained machine learning classifiers to predict the substrate specificity of β-lactone synthetases and related adenylate-forming enzymes. I developed this into a web-based predictive tool and mapped the biochemical diversity of adenylate-forming enzymes in >50,000 candidate biosynthetic gene clusters across bacterial, plant, and fungal genomes. This global genomic analysis led to my discovery and characterization of the biosynthetic gene cluster for an orphan β-lactone natural product, nocardiolactone. To more broadly investigate enzymatic production of β-lactone compounds, a library of 1,095 distinct enzyme-substrate combinations for OleA family of enzymes upstream in the β-lactone biosynthesis pathway were screened. Overall, this body of work advanced progress towards the discovery of new β-lactone natural products and combinatorial biosynthesis of β-lactone compound libraries.