Genomics-Guided Discovery and Characterization of Ancillary RiPP Modifications

Harris, Lonnie Alexander

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https://hdl.handle.net/2142/115933 Copy

Description

Title

Genomics-Guided Discovery and Characterization of Ancillary RiPP Modifications

Author(s)

Harris, Lonnie Alexander

Issue Date

2022-07-15

Director of Research (if dissertation) or Advisor (if thesis)

Mitchell, Douglas A

Doctoral Committee Chair(s)

Mitchell, Douglas A

Committee Member(s)

• van der Donk, Wilfred A
• Nair, Satish
• Chan, Jefferson

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• RiPP
• lasso peptides
• post-translational modifications
• citrulline
• chlorination
• prenylation
• citrulassin
• wygwalassin
• chlorolassin

Abstract

Natural products have traditionally been a fruitful source of chemical matter that has been developed into novel therapeutics. Actinomycetes and several other bacterial taxa are especially gifted in biosynthesizing natural products. However, many decades of intense bioactivity-based screening led to a large rediscovery problem, rendering industrial natural product discovery pipelines uneconomical. Numerous methods for circumventing the rediscovery problem have been developed, among them various chemistry-focused strategies, including reactivity-based screening. Emerging from the field of chemical proteomics, reactivity-based screening relies on a reactive probe that chemoselectively modifies a functional group of interest in the context of a complex biological sample. Reactivity-based probes for several distinct functional groups have been deployed to discover new polyketide and peptidic natural products. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is processed by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminal sequence threaded through the macrocycle. This unique lariat topology, which typically provides considerable stability towards heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Chapter one reviews the current state of natural product discovery, as well as RiPPs and lasso peptide biosynthesis. Chapter two and three both focus on ancillary tailoring in lasso peptide biosynthesis. In chapter two, the discovery of a novel bacterial peptidyl arginine deiminase (PAD) that modifies lasso peptides is described. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of Arg deimination to yield citrulline (Cit). Although a Cit-containing lasso peptide (i.e. citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for Arg deimination had remained unknown. We described the use of reactivity-based screening to discriminate bacterial strains that produce Arg- versus Cit-bearing citrulassins, yielding 13 new lasso peptide variants. Partial phylogenetic profiling identified a distally encoded PAD gene ubiquitous to the Cit-containing variants. Absence of this gene correlated strongly with lasso peptide variants only containing Arg (i.e. des-citrulassin). Heterologous expression of the PAD gene in a non-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in Arg deimination. The PADs were then bioinformatically surveyed to provide a deeper understanding of their taxonomic distribution, genomic contexts, and to facilitate future studies that will evaluate any additional biochemical roles for the superfamily. In chapter three, the discovery and preliminary investigation of two tryptophan- modified lasso peptides is discussed. Using a previously compiled dataset of predicted lasso peptide biosynthetic gene clusters (BGCs), a cluster was found in Lentzea jiangxiensis that encoded a putative tryptophan halogenase with high similarity to MibH, a previously described RiPP chlorinase involved in the biosynthesis of NAI-107 (microbisporicin). Screening of L. jiangxiensis revealed a mass consistent with a dichlorinated lasso peptide, dubbed chlorolassin. High resolution tandem mass spectrometry localized the sites of chlorination to two of three tryptophans in the core peptide. Due to low native titers, chromosomal integration of the chlorolassin gene cluster under ermEp expression in an engineered strain of Streptomyces albus was attempted, yielding ~100-fold increase in chlorolassin titers. Preliminary NMR experiments and Marfey’s analysis suggest the two tryptophan residues are chlorinated at the 5-position of the indole, similar to the regioselectivity seen in MibH. Integration of the chlorolassin gene cluster without the halogenase resulted in the production of des-chlorolassin, which lacked the two chlorine substituents, confirming the chlorinase activity of the gene product and suggesting that chlorination is not necessary for lasso formation. In addition, a small family of lasso peptide BGCs was discovered that contained a tryptophan dimethylallyltransferase (Trp-DMAT), PhyH-like oxidase, and a Cox15-CtaA-like heme oxygenase. Screening of several producers led to the identification of wygwalassin in Streptomyces iakyrus, which contained a prenylation and cryptic loss of 2 Da. A similar heterologous strategy in S. albus was accomplished and will allow for further dissection of the biosynthesis of wygwalassin. Future work will focus on finishing the chemical characterization of wygwalassin, as well as dissecting the biosynthetic timing of ancillary tailoring.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Lonnie Harris

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