Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates
Thesis (PhD)--Stellenbosch University, 2018.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2018
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613750363684864 |
|---|---|
| access_status_str | Open Access |
| author | De Witt, Riaan Neethling |
| author2 | Volschenk, Heinrich |
| author_browse | De Witt, Riaan Neethling Volschenk, Heinrich |
| author_facet | Volschenk, Heinrich De Witt, Riaan Neethling |
| author_sort | De Witt, Riaan Neethling |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2018. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/105150 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:06.301Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/105150 Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates De Witt, Riaan Neethling Volschenk, Heinrich Van Zyl, Willem Heber Stellenbosch University. Faculty of Science. Dept. of Microbiology. quantitative trait loci, lignocellulosic inhibitors, bulk segregant analysis, natural isolate, Saccharomyces cerevisiae, shotgun proteomics Saccharomyces cerevisiae -- Genetic engineering Lignocellulosic inhibitors Lignocellulosic biomass Genotype-environment interaction Phenotype UCTD Thesis (PhD)--Stellenbosch University, 2018. ENGLISH SUMMARY: The innate general robustness, ease of genetic manipulation and high ethanol tolerance of Saccharomyces cerevisiae has cemented its position as an industrial power. Furthermore, as one of the most well studied eukaryotic model systems it has been crucial to the elucidation of human diseases and genetic defects. The genetic architecture of S. cerevisiae is perhaps its most interesting aspect and reason for ubiquity in nature. However, with the availability of omics technologies reaching out to the broader scientific community, the genetic aspects and peculiarities of S. cerevisiae are being rethought and interpreted from perspectives previously obscured from view. Inhibitor tolerance has been the subject of intense scrutiny and investigation in attempts to underpin the mechanisms thereof because of the complexity, but also its importance to commer- cial environments. Tolerance to lignocellulosic inhibitors is governed by multiple genes and elicits a intricate response which is not well understood. Current strains capable of tolerat- ing lignocellulosic inhibitors during the conversion of lignocellulosic biomass to biofuels and biochemicals perform sub optimally and require costly hydrolysate detoxification steps. In an effort to understand tolerance mechanisms and to create strains tailored to specific industrial environments, large scale modelling, synthetic biology and strain engineering projects have been founded. Industrial and laboratory strains are far removed from their natural counterparts from a genetic perspective as they have been adapted to a very specific and synthetic environment. The consequence being a loss of genetic diversity in domesticated strains where natural isolates have maintained their diversity to survive in a range of habitats. Many of the environments inhabited by natural isolates are representative of industrial conditions. Several reports have presented findings of natural isolates performing similar or better than industrial strains when exposed to commercial environments as a result of cross tolerance. The genetic diversity and agility of natural S. cerevisiae isolates has been identified as the cornerstone of industrially tractable phenotypes and a valuable resource for the improvement of yeast strains for industrial processes. To this means, methods to screen and identify the genetic factors which influence and govern traits of commercial concern have advanced from the single gene, single trait format to a more holistic approach. We therefore aimed to elucidate the mechanism employed by lignocellulosic inhibitor tolerant natural S. cerevisiae isolates via bulk segregant analysis and subsequent QTL mapping and discovery based proteomics. Screening of 54 natural isolates on a synthetic inhibitor cocktail representative of lignocellu- losic hydrolysates produced several isolates with tolerance levels superior to current industrial strains. Additionally, 2 isolates exhibited extreme tolerance characteristics superior to all other isolates and industrial strains. One superior (YI38) and two intermediate (J11 and MF1) tolerant isolates were selected for further analysis. Pooled-segregant whole-genome sequence analysis and subsequent quantitative trait loci (QTL) mapping of two hybrid strains (YI38/J11 and YI38/MF1) revealed the presence of 12 candidate QTLs involved in lignocellulosic inhibitor tolerance. This findings is in agreement with the current view that inhibitor tolerance is a complex trait. Furthermore, none of the QTLs are shared between the two hybrid strains which confirm the background specific nature of the response as observed by others. Subsequent investigation of the most promising QTL by reciprocal hemizygosity and comple- mentation analysis revealed the uncharacterized ORF YGL176C and BUD13/XRN1 as the causative genes. However, involvement of BUD13/XRN1 could not be separated from each other as no knockouts were recovered for XRN1 and the proximity of the genes to each other could affect the RHA and complementation assays. Furthermore, the finding of a background specific response of alleles suggest epistatic interactions are involved in the response. This is the first study to implicate YGL176C in a phenotypic response. Comparative shotgun proteomic analysis of isolate J11 and YI38 showed a core proteomic response which was shared between both isolates; in agreement with current research. Redox homeostasis, energy management and detoxification of compounds were the primary processes regulated. However, a background specific response to the exposure to lignocellulosic inhibitors was also observed. Surprisingly, the background unique and shared core response regulated similar processes. Our observation is that the level of tolerance is defined by the ability to rapidly and accurately regulate and fine-tune processes as described above. This study has added to the knowledgebase of inhibitor tolerance and the genetic and proteomic units which govern it. Furthermore, we have identified several targets for future strain improve- ment strategies. AFRIKAANS OPSOMMING: Die gis, Saccharomyces cerevisiae, is ’n gunsteling organisme met betrekking tot industriële prosesses waartydens ongewenste toestande heers. Ingebore robuustheid, gemak van genetiese manipulasie en hoë etanol toleransie is die hoofsaaklike redes vir die gevestigde posisie van industriële mag wat dié gis beklee. S. cerevisiae is ook een van die bes bestudeerde eukariotiese model sisteme wat krities bydrae gelewer het tot die uitleg van menslike kwale en genetise defekte. Dit is juis die genetiese argitektuur van dié gis wat verantwoordelik is vir sy interes- sante aspekte en alomteenwoordigheid in die natuur. Maar, met die toeganklikheid van omika tegnologieë wat versprei is na die breër navorsings gemeenskap, word die genetiese aspekte en eienaardighede van S. cerevisiae herdink en oorweeg vanaf nuwe perspektiewe wat voorheen verduister was. As ’n gevolg van die kompleksiteit, asook kommersiële belange van inhibitor toleransie, is dit gereeld die onderwerp van wysigings en ondersoek met die doel om die meganismes wat dit onderlê, tot lig te bring. Die verstaan van die ingewikkelde beheer wat veelvuldige gene uitoefen oor die regeer van die meganismes betrokke by toleransie tot lignosellulotiese inhibitors is nog skraps en onvoldoende. Huidige lignosellulotiese inhibitor tolerante stamme ly aan sub- optimale opbrengste gedurende bio-omskakeling van plant biomassa tot bio-brandstof en bio- chemikalieë. Om opbrengste te verbeter vereis die toevoeging van addisionele stappe tot die bogenoemde prosses, soos detoksifikasie van hidrolisaat, wat addisionele kostes toevoeg. In ’n poging om die meganismes van toleransie beter te verstaan is verskeie grootskaalse modellerings, syntetiese biologie en stam ingenieurswese projekte gestig met die doel om stamme to ontwikkel, pasgemaak vir industriële kondisies. Industriële en laboratoriumstamme is van ’n genetiese perspektief, ver verwyder van hul natuur- like eweknieë aangesien hul aangepas is tot baie spesifieke en sintetiese toestande. Die gevolg hiervan is ’n verlies aan genetiese diversiteit van hierdie stamme waar natuurlike isolate hul diversiteit behou het om in ’n verskeidenheid omgewings te oorleef. Baie van die omgewings wat deur natuurlike isolate bewoon word, is verteenwoordigend van industriële toestande en verskeie verslae het bevind dat natuurlike isolate wat soortgelyk of beter werk verrig as industriële stamme wanneer blootgestel aan kommersiële omgewings. Hierdie bevinding word toegeken aan kruistoleransie. Die genetiese diversiteit en ratsheid van natuurlike S. cerevisiae isolate word beskou as die hoeksteen van industriële trakbare fenotipes en ’n waardevolle hulpmiddel vir die verbetering van gisstamme vir industriële prosesse. Die gevolg is dat metodes om die genetiese faktore, wat eienskappe van kommersiële belang beïnvloed te identifiseer, gevorder het vanaf die enkelgeen, enkel-eienskapformaat tot ’n meer holistiese benadering. Met die doel om die meganismes van toleransie tot inhibitore afkomstig van lignosellulose toleransie beter te verduidelik; het ons tolerante natuurlike isolate ondersoek deur middel van grootmaat segregante analise en daaropvolgende QTL kartering asook ontdekking gebaseerde proteomika. Sifting van 54 natuurlike isolate op ’n sintetiese inhibitor mengsel, verteenwoordigend van lignosellulosiese hidrolisaat, het verskeie isolate met toleransievlakke beter as huidige industriële stamme opgelewer. Daarbenewens het 2 isolate ekstreme tolerante eienskappe uitgestal. Een ekstreem (YI38) en twee intermediêre (J11 en MF1) tolerante isolate is geselekteer vir verdere analise. Verenigde-segregante heel genoom volgorder bepaling analise en die daaropvolgende kwanti- tatiewe karaktertrek loci (KKL) kartering van twee basterstamme (YI38 / J11 en YI38 / MF1) het die teenwoordigheid van 12 kandidaat KKL wat betrokke was by lignosellulosiese inhibitor toleransie, geopenbaar. Hierdie bevindings stem ooreen met die huidige siening dat inhibitor toleransie ’n komplekse eienskap is. Verder word geen van die KKL gedeel tussen die twee basterstamme nie. Hierdie bevinding bevestig, so ook deur ander waargeneem, die genetise agtergrondspesifieke aard van die response. Daaropvolgende ondersoek van die mees belowende KKL deur middel van wederkerige hemisigotiese- (WHA), asook komplementasie-analise, het die ongekarakteriseerde ORF YGL176C en BUD13/XRN1 as die veroorsakende gene geopenbaar. Die betrokkenheid van BUD13/XRN1 kon egter nie van mekaar geskei word nie, aangesien geen uitslaan rasse van XRN1 geskep kon word nie. Verder, die nabyheid van die gene aan mekaar kan die WHA en komplementasie-analise se resultate beïnvloed. Verder, stel die bevinding van ’n agtergrondspesifieke respons van die allele voor dat epistatiese interaksies by die respons betrokke is. Hierdie is die eerste studie wat YGL176C in ’n fenotipie impliseer. Vergelykende haelgeweer proteomiese analise van isolaat J11 en YI38 het ’n kern proteomiese respons vertoon wat tussen beide isolate gedeel word; in ooreenstemming met huidige navorsing. Redoks homeostase, energiebestuur en detoksifisering van verbindings was die primêre prosesse wat gereguleer word in die kern-respons. ’N Agtergrondspesifieke reaksie op die blootstelling aan lignosellulosiese inhibeerders is egter ook waargeneem. Verrassend was dat die agtergrond- spesifieke en kern-respons soortgelyke prosesse gereguleer. Ons waarneming is dat die vlak van toleransie bepaal word deur die gis se vermoë om prosesse vinnig en akkuraat te reguleer, maar meer belangrik is die verfyning daarvan. Hierdie studie het bygevoeg aan die kennisbasis van inhibitor toleransie en die genetiese en proteomiese eenhede wat dit reguleer. Verder het ons verskeie teikens geïdentifiseer vir toekom- stige strategieë vir die verbetering van gis rasse. Doctoral 2018-10-03T13:04:39Z 2018-12-10T06:34:44Z 2018-10-03T13:04:39Z 2018-12-10T06:34:44Z 2018-12 Thesis http://hdl.handle.net/10019.1/105150 en_ZA Stellenbosch University xxi, 199 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | quantitative trait loci, lignocellulosic inhibitors, bulk segregant analysis, natural isolate, Saccharomyces cerevisiae, shotgun proteomics Saccharomyces cerevisiae -- Genetic engineering Lignocellulosic inhibitors Lignocellulosic biomass Genotype-environment interaction Phenotype UCTD De Witt, Riaan Neethling Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title | Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title_full | Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title_fullStr | Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title_full_unstemmed | Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title_short | Proteogenomic analysis of the inhibitor tolerance phenotype in natural Saccharomyces cerevisiae isolates |
| title_sort | proteogenomic analysis of the inhibitor tolerance phenotype in natural saccharomyces cerevisiae isolates |
| topic | quantitative trait loci, lignocellulosic inhibitors, bulk segregant analysis, natural isolate, Saccharomyces cerevisiae, shotgun proteomics Saccharomyces cerevisiae -- Genetic engineering Lignocellulosic inhibitors Lignocellulosic biomass Genotype-environment interaction Phenotype UCTD |
| url | http://hdl.handle.net/10019.1/105150 |
| work_keys_str_mv | AT dewittriaanneethling proteogenomicanalysisoftheinhibitortolerancephenotypeinnaturalsaccharomycescerevisiaeisolates |