{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/122200"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/122200","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Bioinformatics-guided discovery and characterization of graspetides","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2025-12-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2025-12-01","abstract_has_math":false,"creators":["Ramesh, Sangeetha"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Microbiology","degree_department":null,"school":null,"contributors":["Mitchell, Douglas Alan","Kehl-Fie, Thomas Everett","Metcalf, William W","Nair, Satish K"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12","date_published":"2023-12","updated_at":"2026-07-22T22:25:00Z","subjects":["Natural Products","Genome Mining","Ripps","Atp-grasp Ligases","Graspetides","Rodeo"],"languages":["en","eng"],"rights":["Copyright 2023 Sangeetha Ramesh"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/122200","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Mitchell, Douglas Alan","Kehl-Fie, Thomas Everett","Metcalf, William W","Nair, Satish K"]},{"key":"dc:creator","label":"Author","values":["Ramesh, Sangeetha"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-12","2023-09-14"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"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","Genome Mining","Ripps","Atp-grasp Ligases","Graspetides","Rodeo"]}]},{"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 2023 Sangeetha Ramesh"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/122200"]}]},{"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 2025-12-01","The student, Sangeetha Ramesh, accepted the attached license on 2023-09-10 at 15:46.","The student, Sangeetha Ramesh, submitted this Dissertation for approval on 2023-09-10 at 15:59.","This Dissertation was approved for publication on 2023-09-14 at 16:32.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19825 on 2024-03-01 at 13:47:47","Natural products are molecules of biotic origin predominantly comprising secondary metabolites that make organisms unique. They frequently confer a selective advantage to the producer over other organisms under challenging life conditions. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse family of natural products featuring chemical modifications installed by enzymes on a peptide scaffold. Owing to increased access to genomic data and the availability of genome mining tools to parse this treasure trove of information, the RiPP research field has garnered a lot of traction these past few decades. Chapter-1 and Appendix A of this dissertation discusses new developments in the field of RiPP discovery, enzymology, engineering, and genome mining. Microviridins are serine protease inhibitory RiPPs comprising a cage-like tricyclic structure resulting from two macrolactones and a macrolactam installed by dedicated members of the ATP-grasp ligase enzyme family. Recent discovery of non-microviridin RiPPs, plesiocin and thuringinin, also featuring ATP-grasp ligase-mediated modifications suggested a much broader use of ATP-grasp ligases in RiPP biosynthesis beyond microviridins. This led us to reclassify microviridins, plesiocin, and thuringinin as members of the graspetide RiPP class after the enzymes that install the class-defining modifications (Chapter-1). Chapter-1 summarizes the current state of knowledge about graspetides and the class-defining ATP-grasp ligases. While several recent genome mining studies have expanded the sequence space of the graspetide class, their true biosynthetic diversity remained hidden owing to the lack of a genome mining tool to systematically parse the available genomic information. To address this issue, chapter-2 reports an update to Raid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides. RODEO-mediated genome mining identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups, from 12 to 24, defined on core consensus sequence and putative secondary modification. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Chapter-3 describes the characterization of a group-21 graspetide from Micromonospora rosaria that was newly identified in chapter-2. Group-21 graspetides display a novel tricylic structure as demonstrated through a combination of MS/MS, methanolysis, mutational and NMR spectroscopic analyses. Intriguingly, they also represent the first example of a graspetide that requires both a graspetide synthetase and a putative peptidyl prolyl isomerase (PPIase)/chaperone to observe the macrolactone modifications. Chapter-3 also delves into the initial characterization of the role of the PPIase/chaperone in graspetide biosynthesis. Overall, this dissertation provides a comprehensive overview of the graspetide biosynthetic landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries as exemplified in Appendix B by enabling open-access analysis of existing and emerging genomes."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Bioinformatics-guided discovery and characterization of graspetides"]}]}],"canonical_facts":{"dc:contributor":["Mitchell, Douglas Alan","Kehl-Fie, Thomas Everett","Metcalf, William W","Nair, Satish K"],"dc:creator":["Ramesh, Sangeetha"],"dc:date":["2023-12","2023-09-14"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2025-12-01","The student, Sangeetha Ramesh, accepted the attached license on 2023-09-10 at 15:46.","The student, Sangeetha Ramesh, submitted this Dissertation for approval on 2023-09-10 at 15:59.","This Dissertation was approved for publication on 2023-09-14 at 16:32.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19825 on 2024-03-01 at 13:47:47","Natural products are molecules of biotic origin predominantly comprising secondary metabolites that make organisms unique. They frequently confer a selective advantage to the producer over other organisms under challenging life conditions. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse family of natural products featuring chemical modifications installed by enzymes on a peptide scaffold. Owing to increased access to genomic data and the availability of genome mining tools to parse this treasure trove of information, the RiPP research field has garnered a lot of traction these past few decades. Chapter-1 and Appendix A of this dissertation discusses new developments in the field of RiPP discovery, enzymology, engineering, and genome mining. Microviridins are serine protease inhibitory RiPPs comprising a cage-like tricyclic structure resulting from two macrolactones and a macrolactam installed by dedicated members of the ATP-grasp ligase enzyme family. Recent discovery of non-microviridin RiPPs, plesiocin and thuringinin, also featuring ATP-grasp ligase-mediated modifications suggested a much broader use of ATP-grasp ligases in RiPP biosynthesis beyond microviridins. This led us to reclassify microviridins, plesiocin, and thuringinin as members of the graspetide RiPP class after the enzymes that install the class-defining modifications (Chapter-1). Chapter-1 summarizes the current state of knowledge about graspetides and the class-defining ATP-grasp ligases. While several recent genome mining studies have expanded the sequence space of the graspetide class, their true biosynthetic diversity remained hidden owing to the lack of a genome mining tool to systematically parse the available genomic information. To address this issue, chapter-2 reports an update to Raid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides. RODEO-mediated genome mining identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups, from 12 to 24, defined on core consensus sequence and putative secondary modification. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Chapter-3 describes the characterization of a group-21 graspetide from Micromonospora rosaria that was newly identified in chapter-2. Group-21 graspetides display a novel tricylic structure as demonstrated through a combination of MS/MS, methanolysis, mutational and NMR spectroscopic analyses. Intriguingly, they also represent the first example of a graspetide that requires both a graspetide synthetase and a putative peptidyl prolyl isomerase (PPIase)/chaperone to observe the macrolactone modifications. Chapter-3 also delves into the initial characterization of the role of the PPIase/chaperone in graspetide biosynthesis. Overall, this dissertation provides a comprehensive overview of the graspetide biosynthetic landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries as exemplified in Appendix B by enabling open-access analysis of existing and emerging genomes."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/122200"],"dc:language":["en","eng"],"dc:rights":["Copyright 2023 Sangeetha Ramesh"],"dc:subject":["Natural Products","Genome Mining","Ripps","Atp-grasp Ligases","Graspetides","Rodeo"],"dc:title":["Bioinformatics-guided discovery and characterization of graspetides"],"dc:type":["text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:00Z"}