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The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system
Thesis (MSc)--Stellenbosch University, 2022.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613783002710016 |
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| access_status_str | Open Access |
| author | Faure, Lindsay Marie |
| author2 | Dicks, Leon Milner Theodore |
| author_browse | Dicks, Leon Milner Theodore Faure, Lindsay Marie |
| author_facet | Dicks, Leon Milner Theodore Faure, Lindsay Marie |
| author_sort | Faure, Lindsay Marie |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126017 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:37.777Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126017 The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system Faure, Lindsay Marie Dicks, Leon Milner Theodore Van Staden, Anton du Preez Stellenbosch University. Faculty of Science. Dept. of Microbiology. Class I lanthipeptides Green fluorescent protein (GFP) Escherichia coli -- Genetics Peptides -- Biotechnology Antimicrobial peptides UCTD Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Antimicrobial resistance is currently one of the greatest threats to public health. Bacteria have the natural ability to acquire resistance however, the misuse and over prescription of various antimicrobials has accelerated this crisis. Several alternatives to antimicrobials are being explored, one of which are lanthipeptides. Lanthipeptides are ribosomally synthesized peptides characterized by the presence of lanthionine and methyl-lanthionine residues, which are introduced by post-translational modification enzymes. The interest in lanthipeptides has increased not only due to their antimicrobial activity against several clinically relevant multi-drug resistant bacteria but also various other bioactivities including antiviral, anticancer and pain relief. The main stumbling block hindering the applications of lanthipeptides is their production. Their production is complex, due to the difficulty in cultivation and purification from native sources, as well as the enzymes required for post-translational modification, which ultimately leads to low yields. In this study we fused the green fluorescent protein (GFP) to the core peptides (already fused to the nisin leader peptide) of the known class I lanthipeptides, nisin, Pep5, Epilancin 115x and epidermin and co-expressed the fusion with the post-translational modification enzymes NisB, NisC and EpiD using Escherichia coli (E. coli) as the host. The results showed that the post- translational enzymes were not obstructed by the GFP fusion and were able to modify the core peptides. Furthermore, antimicrobial activity was detected for nisin, Pep5 and epidermin following in-vitro leader peptide cleavage using nisin’s native protease, NisP. Preliminary yield estimates detected 2 mg/L of nisin following cleavage and purification. Following the favourable outcome of the GFP-fusion expression system, 16 putative class I lanthipeptides were mined from the genomes of numerous bacteria. The peptides were produced using the system and antimicrobial activity was observed for six of the peptides. The outcomes of the two-part study showed that E. coli is a suitable host to produce lanthipeptides, and an optimized GFP-fusion system can yield functional class I lanthipeptides. Furthermore, the system can be used to produce putative lanthipeptides discovered via genome mining techniques. These outcomes can prove valuable in the production of lanthipeptides for their use in the fight against multi-drug resistant bacteria as well as various other potential bioactivities. AFRIKAANSE OPSOMMING: Antimikrobiese weerstandigheid is tans een van die grootste bedreigings vir openbare gesondheid. Bakterieë het egter die natuurlike vermoë om weerstand te bied. Die misbruik en die voorskrif van verskillende antimikrobiese middels het hierdie krisis versnel. Verskeie alternatiewe vir antimikrobiese middels word ondersoek, waarvan een lantipeptiede is. Lantipeptiede is ribosomaal gesintetiseerde peptiede wat gekenmerk word deur die teenwoordigheid van lantionien- en metielantionienreste, wat deur post-translasionele modifikasie-ensieme ingestel word. Die belangstelling in lantipeptiede het toegeneem nie net as gevolg van hul antimikrobiese aktiwiteite teen verskeie klinies relevante multi-medikamentbestande bakterieë nie, maar ook verskeie ander bioaktiwiteite, waaronder antivirale, antikanker en pynverligting. Die belangrikste struikelblok in die toepassings van lantipeptiede is hulle produksie. Produksie is ingewikkeld as gevolg van die probleme met die sintese en suiwering vanuit natuurlike bronne, sowel as die ensieme wat benodig word vir post- translasionele modifikasie, wat uiteindelik tot lae opbrengste lei. In hierdie studie het ons groen fluoresserende proteïen (GFP) aan die kernpeptiede (versmelt tot die nisin-leier peptied) van die bekende klas I lantipeptiede, nisin, Pep5, epilancin 115x en epidermin en het die samesmelting saam met die post-translasionele modifikasie-ensieme NisB, NisC en EpiD uitgedruk, met behulp van Escherichia coli (E. coli) as gasheer. Die resultate het getoon dat die post-translasionele ensieme nie deur die GFP-fusie belemmer is nie en die kernpeptiede kon gemodifiseer word. Verder is antimikrobiese aktiwiteit vir nisin, Pep5 en epidermin opgespoor na die peptied-splitsing van die leier peptied in vitro met behulp van nisin se natuurlike protease, NisP. Voorlopige opbrengskramings het 2 mg/L van nisien opgespoor na splitsing en suiwering. Na die gunstige uitkoms van die GFP-fusie- uitdrukkingstelsel, is 16 putatiewe klas I-lantipeptiede uit die genome van talle bakterieë ontgin. Die peptiede is met behulp van die stelsel vervaardig en antimikrobiese aktiwiteit is vir 6 van die peptiede waargeneem. Die uitkomste van die tweedelige studie het getoon dat E. coli 'n geskikte gasheer is om lantipeptiede te produseer, en 'n geoptimaliseerde GFP-fusiestelsel kan funksionele klas I lantipeptiede oplewer. Verder kan die stelsel gebruik word om ledige lantipeptiede te vervaardig wat via genoommyntegnieke ontdek word. Hierdie uitkomste kan waardevol wees in die produksie van lantipeptiede vir die gebruik daarvan in die stryd teen multi-medikamentbestande bakterieë, sowel as verskillende moontlikhede. Masters 2022-11-18T08:12:16Z 2023-01-16T12:45:53Z 2022-11-18T08:12:16Z 2023-01-16T12:45:53Z 2022-12 Thesis http://hdl.handle.net/10019.1/126017 en_ZA Stellenbosch University vii, 181 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Class I lanthipeptides Green fluorescent protein (GFP) Escherichia coli -- Genetics Peptides -- Biotechnology Antimicrobial peptides UCTD Faure, Lindsay Marie The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title | The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title_full | The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title_fullStr | The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title_full_unstemmed | The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title_short | The production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| title_sort | production of class i lanthipeptides in escherichia coli using a green fluorescent protein fusion system |
| topic | Class I lanthipeptides Green fluorescent protein (GFP) Escherichia coli -- Genetics Peptides -- Biotechnology Antimicrobial peptides UCTD |
| url | http://hdl.handle.net/10019.1/126017 |
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