University of Illinois Urbana-Champaign

Exploration of the biosynthesis of lanthipeptides

Shi, Yanxiang

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Yanxiang_Shi.pdf

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https://hdl.handle.net/2142/46855

Description

Title
Exploration of the biosynthesis of lanthipeptides
Author(s)
Shi, Yanxiang
Issue Date
2014-01-16T18:18:47Z
Director of Research (if dissertation) or Advisor (if thesis)
van der Donk, Wilfred A.
Doctoral Committee Chair(s)
van der Donk, Wilfred A.
Committee Member(s)
  • Sweedler, Jonathan V.
  • Mitchell, Douglas A.
  • Kranz, David M.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2014-01-16T18:18:47Z
Keyword(s)
  • Lanthipeptides
  • Biosynthesis
  • Co-expression
Abstract
Natural products play important roles in the survival of organisms, and provide a large pool of candidates for drug discovery and pharmaceutical investigations. A major class of peptide-based natural products are ribosomally synthesized and post-translationally modified (RiPPs). Lanthipeptides, which possess (methyl)lanthionine structures, are a class of intensively studied RiPPs. Based on the biosynthesis enzymes that introduce the thioether motifs, lanthipeptides are classified into four classes. Class I lanthipeptides utilize two distinct enzymes, a dehydratase and a cyclase, while class II lanthipeptides adopt a bifunctional enzyme. Trifunctional enzymes with independent evolution pathways are employed by class III and class IV lanthipeptides. A major subclass of lanthipeptides possess antimicrobial activity, known as lantibiotics with known modes of action including lipid II binding, pore formation, inhibition of spore outgrowth, and phosphatidylethanolamine (PE) binding. Several lanthipeptides display no antimicrobial activities but have other functions important for bacterial life cycles, such as facilitating aerial hyphae formation during sporulation. In the past, most lanthipeptides were discovered based on their activity mainly through antimicrobial screening campaigns. With the advances in genome sequencing and bioinformatics, a variety of lanthipeptides have been identified by genome mining, presumably with more unexplored functions and molecular targets. Prochlorosins are an unusual group of lanthipeptides, which were discovered through genome mining and have no known antibiotic activities. Further biological function investigations of prochlorosins require pure products in adequate amounts, which was not achievable through natural product isolation or in vitro enzymatic assays. In this thesis, Chapter 2 describes the development of a novel co-expression system to produce prochlorosins by in vivo enzymatic modification in E. coli, providing prochlorosins with high purity and sufficient quantity for subsequent characterizations. Chapter 3 presents an example of utilizing the co-expression methodology to produce antimicrobial lanthipeptides, with additional modifications besides dehydration and cyclization. The precursor peptide of actagardine, one of the only two lantibiotics that have been evaluated in clinical trials thus far, was fully modified in E. coli as presented in Chapter 3. A luciferase-like monooxygenase located in the biosynthetic cluster of actagardine was also reconstituted, both in vitro and in vivo, to introduce a sulfoxide group onto actagardine and its derivatives. Based on the success of in vivo co-expression of lanthipeptides with identified or unidentified activities, Chapter 4 describes efforts towards the incorporation of non-canonical amino acids into lanthipeptides, in order to expand the structural diversity of these natural products, tune their activities, and introduce probes for subsequent functional investigations. The work presented in this thesis provides methods to effectively produce the peptide-based natural products lanthipeptides, in adequate quantities for subsequent investigations including study of substrate peptides and their modification enzymes, peptide structure elucidation, as well as sequence and structure diversification.
Graduation Semester
2013-12
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http://hdl.handle.net/2142/46855
Copyright and License Information
2013 Yanxiang (Nancy) Shi

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