eprintid: 10203744 rev_number: 17 eprint_status: archive userid: 699 dir: disk0/10/20/37/44 datestamp: 2025-02-28 13:50:18 lastmod: 2025-02-28 13:50:18 status_changed: 2025-02-28 13:50:18 type: thesis metadata_visibility: show sword_depositor: 699 creators_name: Vasciaveo, Sara title: Genome mining for novel sactipeptides ispublished: unpub divisions: UCL divisions: B02 divisions: C08 divisions: D09 note: Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. abstract: The continuing rise of antimicrobial resistance poses a challenge for medicine. The emerging class of natural products ribosomally synthesised and post-translationally modified peptides (RiPPs) are a diverse group of biologically active antibacterial molecules and therefore an encouraging addition to antibiotics. Increasing numbers of RiPP natural products have been identified in recent decades thanks to the advances in genome sequencing. The biosynthetic gene clusters (BGCs) of known RiPP classes such as lanthipeptides, ranthipeptides, and sactipeptides, are identified by the presence of conserved genes, and the product’s structure can be predicted depending on the type of enzymes encoded in the cluster. Previous research on genome mining has shown that the use of bioinformatics tools such as BLAST and RODEO can be used in mapping of RiPP classes such as sactipeptides via the radical S-adenosylmethionine (rSAM) enzymes, uncovering hundreds of new BGCs. In this study, the promiscuity of the rSAM enzyme AlbA was investigated to explore its potential to create a sactipeptide library. Enzymatic studies proved challenging and thus genome mining was employed to identify putative sactipeptides and their BGCs. This led to the discovery of a novel sactipeptide named SVP1 in the bacteriocin-producing bacterium Bacillus smithii DSM 4216. SVP1 was extracted and purified, and its structure was characterised through high-resolution mass spectrometry, tandem mass spectrometry, and circular dichroism spectroscopy. Its antimicrobial activity was also tested against a panel of Gram-positive bacteria. Furthermore, the svpA gene was heterologously expressed in subtilosin producer Bacillus subtilis 168, successfully leading to the production of the peptide SVP1. This exciting result indicates the promiscuity of the post-translational modifying enzymes in the subtilosin biosynthetic pathway, including the thioether forming radical SAM enzyme, AlbA. The promiscuity of these enzymes is contrary to that reported in recent literature and opens new avenues raising important questions regarding sactipeptide biosynthetic pathways. date: 2025-01-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_open thesis_award: Ph.D language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 2354295 lyricists_name: Vasciaveo, Sara lyricists_id: SVASC98 actors_name: Vasciaveo, Sara actors_id: SVASC98 actors_role: owner full_text_status: public pages: 198 institution: UCL (University College London) department: UCL School of Pharmacy thesis_type: Doctoral editors_name: Dickman, Rachael editors_name: Barry, Sarah citation: Vasciaveo, Sara; (2025) Genome mining for novel sactipeptides. Doctoral thesis (Ph.D), UCL (University College London). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10203744/9/Vasciaveo_10203744_Thesis_Corrected%20version.pdf document_url: https://discovery.ucl.ac.uk/id/eprint/10203744/8/Sara%20Vasciaveo%20-%2014053725%20Supplementary%20Data.zip