Bioinformatics-guided discovery and characterization of graspetides

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Ramesh, Sangeetha

Contributors dc:contributor

• Mitchell, Douglas Alan
• Kehl-Fie, Thomas Everett
• Metcalf, William W
• Nair, Satish K

• Natural Products
• Genome Mining
• Ripps
• Atp-grasp Ligases
• Graspetides
• Rodeo

Statement dc:rights

• Copyright 2023 Sangeetha Ramesh

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122200

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122200