Characterization of atypical class I lanthipeptides from the marine bacterium thalassomonas viridans XOM25ᵀ

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dc.contributor.advisorDicks, Leon Milner Theodore en_ZA
dc.contributor.advisorVan Staden, Anton du Preezen_ZA
dc.contributor.authorVermeulen, Rossen_ZA
dc.contributor.otherTrindade, Marlaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Microbiology.en_ZA
dc.date.accessioned2024-02-09T14:08:10Z
dc.date.accessioned2024-04-27T02:01:35Z
dc.date.available2024-02-09T14:08:10Z
dc.date.available2024-04-27T02:01:35Z
dc.date.issued2024-03
dc.descriptionThesis (PhD)--Stellenbosch University, 2024. en_ZA
dc.description.abstractENGLISH ABSTRACT: The natural products (NPs) that are produced by living organisms have historically served as a pivotal starting point for numerous highly effective therapeutic agents. These compounds often possess distinct chemical structures that enable valuable mechanisms and modes of biological activity. Natural products are thus a promising reservoir that potentially contains the key to the treatment of diseases like cancer, infections, resistance, neurological disorders, and even a range of agricultural and environmental challenges. The rate of novel drug discovery is exhibiting a gradual decline. However, recent advancements in biotechnology, such as next-generation sequencing (NGS) and heterologous gene expression, are empowering researchers to access unexplored realms of biological and chemical diversity. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of NPs that have experienced significant growth by combining NGS and heterologous expression. Within the RiPP group, lanthipeptides are characterized by the post-translational installation of lanthionine rings or thioether cross-linkages. The incorporation of lanthionine rings offers several advantages such as enhanced stability and expanded structural diversity at low genetic cost. However, the process of post-translational modification poses distinct challenges when attempting to heterologously produce lanthipeptides exhibiting previously unobserved and markedly distinct characteristics. To date, the majority of described lanthipeptides play an antimicrobial role which provides a competitive advantage to the native producer by inhibiting the growth of similar or closely related species. Recently, a significant number of cryptic lanthipeptides have been heterologously produced which do not exhibit antimicrobial activity against species related to the native host. Many of these seemingly inactive and atypical lanthipeptides originate from Gram-negative bacteria associated with the marine environment. In this work, a significantly dissimilar lanthipeptide operon was identified in the genome of the marine Gram-negative marine bacteria Thalassomonas viridans XOM25T. The operon was used to construct an Escherichia coli BL21 expression system capable of co-producing the peptide substrates and post-translational modification machinery. The target peptides were subsequently isolated, confirmed to contain post-translationally installed lanthionine rings, and designated as the viridisin operon. Finally, the antimicrobial, stereochemical and pharmaceutical potential of the viridisins were assessed. The viridisins have similar stereochemistries to other lanthipeptides from Gram- negative bacteria, and likewise, they do not have antimicrobial properties. However, this study proved that the viridisin were able to modulate zebrafish larvae behavior. While the results of the zebrafish behavioral screening do not provide definitive proof of the mechanisms at play, such findings allude to the remarkable diversity of biological roles that RiPPs fulfil – intended or otherwise.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Natuurlike produkte (NPs), wat deur lewende organismes geproduseer word, dien as ‘n basis vir die ontwikkeling van verskeie hoogs doeltreffende terapeutiese middels. Hierdie verbindings besit dikwels kenmerkende chemiese strukture wat waardevolle meganismes en wyses van biologiese aktiwiteit moontlik maak. Gevolglik is natuurlike produkte 'n belowende bron wat potensieël die sleutel bevat tot die behandeling van siektes soos kanker, infeksies, weerstandigheid, neurologiese afwykings, en selfs 'n reeks landbou- en omgewingsuitdagings. Alhoewel die tempo van die ontdekking van nuwe geneesmiddels geleidelik afgeneem het, bemagtig onlangse vorderinge in tegnologie, soos volgende-generasie-volgordebepaling (VGV) en heteroloë geenuitdrukking, navorsers om onontginde gebiede van biologiese en chemiese diversiteit te verken. Ribosomaal-gesintetiseerde en post-translasie gewysigde peptiede (RiPPs) is 'n klas NPs waarvan ons kennis beduidend toegeneem het as gevolg van ʼn kombinasie van VGV en heteroloë geenuitdrukking. Binne die RiPP-groep word lanthipeptiede gekenmerk deur die vorming van lanthionienringe of thio-eter-kruiskoppelings na translasie. Die insluiting van lanthionienringe bied verskeie voordele, insluitend verbeterde stabiliteit en uitgebreide strukturele diversiteit teen 'n lae genetiese koste. Die proses van post-translasie modifikasie het egter spesifieke uitdagings wanneer gepoog word om lanthipeptiede in ‘n heteroloë gasheer te produseer wat voorheen onwaargenome en opmerkend duidelike eienskappe vertoon. Tot op hede speel die meerderheid van die beskryfde lanthipeptiede 'n antimikrobiese rol wat 'n mededingende voordeel aan die inheemse produsent verleen deur die groei van soortgelyke of nouverwante spesies te inhibeer. Onlangs is 'n beduidende aantal kriptiese lanthipeptiede in ‘n heteroloë gasheer geproduseer, maar vertoon geen antimikrobiese aktiwiteit teen spesies wat verwant is aan die inheemse gasheer nie. Baie van hierdie skynbaar onaktiewe lanthipeptiede het hul oorsprong in Gram-negatiewe bakterieë wat geassosieer word met die mariene omgewing. In hierdie werk is 'n beduidend uiteenlopende lanthipeptied-operon geïdentifiseer in die genoom van die mariene Gram-negatiewe bakterie Thalassomonas viridans XOM25T. Die operon is gebruik om 'n Escherichia coli BL21-geenuitdrukkingsisteem op te bou wat in staat is om die peptiedsubstrate en post-translasie modifiseringsmasjinerie terselftertyd te produseer. Die teikenpeptiede is vervolgens geïsoleer, bevestig om post-translasie geïnstalleerde lanthionienringe te bevat, en die Viridisin-operon genoem. Ten slotte is die antimikrobiese, stereochemiese en farmaseutiese potensiaal van die viridisins bestudeer. Alhoewel die stereochemie van viridisins soortgelyk is aan ander lanthipeptiede van Gram-negatiewe bakterieë en ook nie antimikrobiese eienskappe bevat nie, is hulle in staat om die gedrag van sebravis-larwes te moduleer. Alhoewel die gedrag van die sebravisse nie ʼn besliste antwoord bied oor die wyse waarop hierdie peptiede funksioneer nie, dui hierdie studie op die uitermate diversiteit van biologiese rolle wat RiPPs vervul – opsetlik of nie.af_ZA
dc.description.versionDoctorateen_ZA
dc.format.extentvi, 179 pages : illustrations (some color)en_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130669
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshNatural products -- Therapeutic useen_ZA
dc.subject.lcshPost-translational modificationen_ZA
dc.subject.lcshGenetics -- Techniqueen_ZA
dc.subject.lcshMarine bacteria -- Environmental aspectsen_ZA
dc.subject.lcshDrugsen_ZA
dc.subject.lcshLanthipeptides -- Genetic engineeringen_ZA
dc.subject.lcshGram-negative bacteria -- Genetic aspectsen_ZA
dc.subject.lcshPseudomonadaceae -- Identificationen_ZA
dc.subject.lcshPeptides -- Biotechnologyen_ZA
dc.subject.nameUCTDen_ZA
dc.titleCharacterization of atypical class I lanthipeptides from the marine bacterium thalassomonas viridans XOM25ᵀen_ZA
dc.typeThesisen_ZA
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Doctoral Degrees (Microbiology)