Discovery, enzymology, and engineering of bacterial natural product pathways

Simon, Max Andrew

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https://hdl.handle.net/2142/117552 Copy

Description

Title

Discovery, enzymology, and engineering of bacterial natural product pathways

Author(s)

Simon, Max Andrew

Issue Date

2022-11-28

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)

• Metcalf, William W
• Zhao, Huimin
• Perez-Pinera, Pablo

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Natural products
• phosphonate
• enzymology
• lanthipeptide
• genome mining
• biosynthesis

Abstract

Phosphonates, which are characterized by their hydrolytically stable carbon-phosphorus bond, are prevalent in everyday life, such as in commercially available herbicides and an antibiotic used in the clinic for more than 50 years. Lanthipeptides, a large class of ribosomally-synthesized and post-translationally modified peptides (RiPPs), are characterized by their proteolytically stable thioether rings are found in nearly every microorganism and have been used as a preservative in food for more than 50 years. Production of these compounds in a heterologous host has been thwarted by complex biosynthetic pathways and other challenges. This thesis will include my contributions to understanding phosphonate biosynthetic chemistry in more detail, resulting in the discovery of a new antibiotic with a novel target, and the engineering of an expression strain for enhanced RiPP production and characterization. Fosfomycin is an antibiotic used for the treatment of cystitis. Its activity against both Gram-positive and Gram-negative pathogens has received strong recent interest. Fosfomycin is biosynthesized through two convergent routes in Streptomyces and Pseudomonas species. The pathways only share the first and last steps, which are catalyzed by phosphoenolpyruvate mutase and 2-hydroxypropylphosphonate epoxidase (HppE), respectively. Chapter 2 will describe investigations of the biosynthesis of fosfomycin by Pseudomonas through heterologous co-expression of genes from the biosynthetic gene cluster in Escherichia coli. Systematic evaluation of these genes led to the identification of an enzyme responsible for completing the biosynthetic pathway in pseudomonads, PsfC. The missing transformation involves the oxidative decarboxylation of the intermediate 2-phosphonomethylmalate (2-Pmm) to a new intermediate, 3-oxo-4-phosphonobutanoate (3-OBPn). Active site mutants were analyzed for activity to probe how PsfC interacts with 2-Pmm and its Fe2+ cofactor. This newly characterized protein family expands the chemical space of catalysis by metallohydrolase superfamily enzymes. Investigations into the activity of PsfC in Chapter 2 revealed a new phosphonate biosynthesis pathway in Burkholderia. Reconstitution of this pathway in E. coli and in vitro resulted in the discovery of a phosphonate with antibacterial activity against E. coli. Chapter 3 will discuss the investigations to identify the product of this pathway. Manipulation of the indicator strain used for zone of growth inhibition assays resulted in the identification of the putative target enzyme for this compound. In vitro enzyme assays determined that the final enzyme in this biosynthetic pathway produces different products than other known members of its enzyme class. This compound is the first natural product discovered thus far to inhibit the vitamin B2 biosynthesis pathway in bacteria. The most intensely studied class of RiPPs are lanthipeptides, designated by the presence of (methyl)lanthionine rings connecting dehydrated former-threonine and serine residues with cysteine residues through a thioether bond. These peptides have found use as broad-spectrum antibacterials (nisin), as well as narrow-spectrum antibiotics, like the lipolanthines that demonstrate antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Chapter 4 will discuss attempts to reconstitute the production of these peptides in a heterologous host, E. coli, and the challenges associated therein. A homolog of a protease within E. coli was demonstrated to recognize many of these peptides as substrates, resulting in poor production of these peptides in E. coli. A knockout of this protease was established in an E. coli expression strain, resulting in enhanced production of these peptides. Characterization of the peptide products resulted in a diverse structural landscape, doubling the number of characterized class IV lanthipeptides. Another knockout in E. coli was generated that enhances purification and analysis of these peptides by mass spectrometry techniques.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Max Andrew Simon

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