{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/117552"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/117552","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Discovery, enzymology, and engineering of bacterial natural product pathways","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2024-12-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2024-12-01","abstract_has_math":false,"creators":["Simon, Max Andrew"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Bioengineering","degree_department":null,"school":null,"contributors":["van der Donk, Wilfred A","Metcalf, William W","Zhao, Huimin","Perez-Pinera, Pablo"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-12","date_published":"2022-12","updated_at":"2026-07-22T22:24:56Z","subjects":["Natural Products","Phosphonate","Enzymology","Lanthipeptide","Genome Mining","Biosynthesis"],"languages":["en","eng"],"rights":["Copyright 2022 Max Andrew Simon"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/117552","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["van der Donk, Wilfred A","Metcalf, William W","Zhao, Huimin","Perez-Pinera, Pablo"]},{"key":"dc:creator","label":"Author","values":["Simon, Max Andrew"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2022-12","2022-11-28"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Bioengineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Natural Products","Phosphonate","Enzymology","Lanthipeptide","Genome Mining","Biosynthesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2022 Max Andrew Simon"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/117552"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2024-12-01","The student, Max Simon, accepted the attached license on 2022-11-14 at 20:39.","The student, Max Simon, submitted this Dissertation for approval on 2022-11-18 at 16:18.","This Dissertation was approved for publication on 2022-11-28 at 16:02.","DSpace SAF Submission Ingestion Package generated from Vireo submission #18577 on 2023-04-12 at 11:34:55","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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Discovery, enzymology, and engineering of bacterial natural product pathways"]}]}],"canonical_facts":{"dc:contributor":["van der Donk, Wilfred A","Metcalf, William W","Zhao, Huimin","Perez-Pinera, Pablo"],"dc:creator":["Simon, Max Andrew"],"dc:date":["2022-12","2022-11-28"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2024-12-01","The student, Max Simon, accepted the attached license on 2022-11-14 at 20:39.","The student, Max Simon, submitted this Dissertation for approval on 2022-11-18 at 16:18.","This Dissertation was approved for publication on 2022-11-28 at 16:02.","DSpace SAF Submission Ingestion Package generated from Vireo submission #18577 on 2023-04-12 at 11:34:55","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."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/117552"],"dc:language":["en","eng"],"dc:rights":["Copyright 2022 Max Andrew Simon"],"dc:subject":["Natural Products","Phosphonate","Enzymology","Lanthipeptide","Genome Mining","Biosynthesis"],"dc:title":["Discovery, enzymology, and engineering of bacterial natural product pathways"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Bioengineering"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:56Z"}