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Title:Genome-guided, reactivity-based discovery and characterization of natural products
Author(s):Guo, XiaoRui
Director of Research:Mitchell, Douglas A
Doctoral Committee Chair(s):Mitchell, Douglas A
Doctoral Committee Member(s):van der Donk, Wilfred A; Metcalf, William W; Chan, Jefferson
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Polyene
macrolide
RiPP
lassopeptide
glycosyltransferase
graspetide
kineosporicin
cinnasporicin
chlorolassin
Abstract:Natural products, especially those derived from bioassay-guided isolation efforts, have revolutionized the fields of modern medicine and agriculture. The structural diversity and complexity that drive the bioactivity of these non-ribosomal peptide, polyketide, and ribosomally synthesized and post-translationally modified peptide (RiPP) metabolites is to be marveled. However, despite the impact that well known classes of antimicrobials such as beta-lactams, macrolides or tetracyclines have had, the threat of antibiotic resistance beckons the further discovery of new bioactives with novel mechanisms of action. However, traditional discovery efforts have been hounded by time consuming compound dereplication, which all too often leads to rediscovery. This situation is also true for the polyene macrolide class of antimycotics. This class of antifungals, while potent, have severe associated side effects and discovery efforts has largely stagnated. Chapter 1 provides an overview of the glycosylated polyene macrolide (GPM) class of natural products and ways to enhance discovery efforts. Chapters 2, and 3 both focus on the GPM class of polyketide natural products. While polyene macrolides like amphotericin B have been in use for almost 70 years, their precise mechanism of action is still not well understood and highly debated. The prevailing theory of channel formation being the primary mode of fungicidal action has been recently challenged by a sterol sponge model of sterol sequestration. Our work (Chapter 2) seeks to determine the unifying mechanism of action for all GPMs. Using a highly conserved polyene-hemiketal region of GPMs that we hypothesized to represent a conserved ergosterol binding domain, we bioinformatically mapped the entirety of the GPM sequence-function space and expanded the number of GPM biosynthetic gene clusters (BGCs) by 10-fold. We further leveraged bioinformatic predictions and tetrazine-based reactivity screening targeting the electron-rich polyene region of GPMs to discover a first-in-class methyltetraene-containing GPM, kineosporicin, that also uniquely displays vicinal diepoxidation and additional tetrahydropyran hydroxylation and to assign BGCs to new producers of previously reported members. Leveraging a range of structurally diverse known and newly discovered GPMs, we found that the sterol sponge mechanism of fungicidal action is conserved. Further and ongoing discovery efforts are described (Chapter 3) along with glycoengineering toward the chemoenzymatic production of a nontoxic but potent antifungal, C2’-epi-amphotericin. Chapter 4 deviates from polyketide natural product discovery and looks instead at the lasso peptide class of RiPPs. The lasso peptides are defined by an isopeptide linkage between the N-terminal amine and an internal Asp or Glu residue with the C-terminal sequence being threaded through the macrocycle. This lariat topology imparts the lasso peptides with heat and proteolytic stability. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of Arg deimination to yield citrulline (Cit). We describe the use of reactivity-based screening to detect Cit-bearing lasso peptides in bacterial extracts and report 13 new citrulassin variants. Partial phylogenetic profiling then enabled the identification of a distally encoded peptidyl arginine deiminase (PAD) gene potentially responsible for conversion of lasso peptide Arg to Cit. We show with heterologous expression of the PAD gene in a des-citrulassin producer that the PAD is unambiguously responsible for the deminiation of Arg to Cit. Lastly, Chapter 5 focuses on the graspetides, another class of RiPP natural product. This class of RiPPs contain potent serine protease inhibitors as evidenced by the microvidirins and plesiocins. Graspetides are characterized by the ATP-grasp ligase-dependent formation of macrolactone/macrolactam rings, which arise from serine, threonine or lysine donor residues linked to aspartate or glutamate acceptor residues. We report the expansion of this class of RiPP to over 3,200 members via a newly developed graspetide detection module for Rapid ORF Description and Evaluation Online (RODEO). We then leverage our dataset to heterologously produce and characterize a new graspetide, thatisin, from Lysobacter antibioticus. Using tandem mass spectrometry coupled with methanolysis, thatisin was found to contain two macrolactone linkages with an interlocking ring topology hitherto unseen among characterized graspetides. A substrate-scope study of the thatisin maturase revealed that the enzyme is tolerant of changes to residues not involved in macrolactone formation. This work provides a comprehensive overview of the graspetide genomic landscape and underscores the vast number of yet uncharacterized RiPP natural products.
Issue Date:2020-12-03
Type:Thesis
URI:http://hdl.handle.net/2142/109622
Rights Information:Copyright 2020 XiaoRui Guo
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12


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