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Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion
Thesis (PhD)--Stellenbosch University, 2019.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2019
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| _version_ | 1867613991197474816 |
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| access_status_str | Open Access |
| author | Davison, Steffi Angela |
| author2 | Van Zyl, Willem Heber |
| author_browse | Davison, Steffi Angela Van Zyl, Willem Heber |
| author_facet | Van Zyl, Willem Heber Davison, Steffi Angela |
| author_sort | Davison, Steffi Angela |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/106915 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:55.985Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/106915 Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion Davison, Steffi Angela Van Zyl, Willem Heber Den Haan, Riaan Stellenbosch University. Faculty of Science. Dept. of Microbiology. Saccharomyces cerevisiae -- Genetic engineering Yeast strains Saccharomyces cerevisiae -- Effect of of stress on Cellulase -- Secretion Phenotype Genetic diversity UCTD Thesis (PhD)--Stellenbosch University, 2019. ENGLISH SUMMARY: The yeast Saccharomyces cerevisiae is considered an important host for the consolidated bioprocessing (CBP) of plant biomass to fuels and commodity products, but the production of high titres of recombinant cellulases is required for efficient hydrolysis of heterogonous lignocellulosic substrates to fermentable sugars. Recently, it was shown that S. cerevisiae strain diversity represents a treasure trove of genetic determinants for industrially relevant traits, including secretory capacity for recombinant cellulases. Since recombinant protein secretion profiles vary significantly among different strain backgrounds, careful selection of robust strains with optimal secretion profiles is crucial. This dissertation addresses numerous central challenges surrounding S. cerevisiae CBP namely, (1) improving the yeast’s low secretion capacity for recombinant cellulase through the construction and screening of hybrids of natural and industrial strains; (2) the evaluation of different cellulolytic yeast strain configurations to handle the heterogeneity of lignocellulosic substrates; and (3) the identification of genetic elements associated with the complex, polygenic trait of heterologous cellulase production and secretion through whole genome sequencing of selected yeast strains. We detail a novel approach, which combines cellulase secretion profiles and phenotypic responses of strains to stresses known to influence the secretion pathway, for the development of a phenotypic screen. The construction and screening of haploids derived from natural strain isolates YI13, FINI and YI59, consequently yielded several haploid strains with enhanced general cellulase secretion. A clear distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and S288c, respectively. Our results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of novel strains capable of improved cellulase secretion. In an effort to find a suitable genetic background for efficient cellulase secretion, genetically diverse strains were created to produce core sets of fungal cellulases, namely, β-glucosidase, endoglucanase and cellobiohydrolase, in various combinations. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants released up to 1.34-fold higher glucose concentrations (g/L) than a control mixture composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted cellulase activity ratio of 1:15 (unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments, and yielded 4.05 g/L ethanol in fermentations. Finally, by performing pooled-segregant whole genome sequence analysis with subsequent quantitative trait loci mapping of an industrial strain (Ethanol Red) and a natural strain (YI13), we identified a large list of potential causative gene candidates linked to the high secretion phenotype. Some of these gene candidates were previously demonstrated to be active at different phases of secretion, ranging from the initiation of transcription, translation, post- translational modification to protein folding. Furthermore, we have identified several targets for future yeast strain improvement strategies. The yeast strains developed in this study therefore represent a new step towards efficient cellulase secretion for CBP bioethanol production. AFRIKAANS OPSOMMING: Die gis Saccharomyces cerevisiae word as 'n belangrike gasheer vir die gekonsolideerde bioprosessering (“consolidated bioprocessing” CBP) van plantbiomassa na brandstof en kommoditeitsprodukte beskou. Die produksie van hoë titers rekombinante sellulase word egter vir doeltreffende hidrolise van heterogene lignosellulotiese substrate tot fermenteerbare suikers benodig. Onlangs is aangetoon dat S. cerevisiae-stamdiversiteit 'n skatkis van genetiese determinante vir industriële relevante eienskappe verteenwoordig, insluitend sekresiekapasiteit vir rekombinante sellulases. Aangesien rekombinante proteïensekresieprofiele tussen verskillende stamagtergronde aansienlik verskil, is noukeurige seleksie van robuuste stamme met optimale sekresieprofiele van kardinale belang. Hierdie proefskrif adresseer ʼn aantal sentrale uitdagings rondom S. cerevisiae CBP naamlik, (1) die verbetering van die gis se lae sekresiekapasiteit vir rekombinante sellulases deur die konstruksie en sifting van hibriede van natuurlike en industriële stamme; (2) die evaluering van verskillende sellulolitiese gisstamkonfigurasies om die heterogeniteit van lignosellulolitiese substrate aan te pak; en (3) die identifikasie van genetiese elemente wat verband hou met die komplekse, poligeniese eienskap van sellulaseproduksie en -sekresie deur middel van heelgenoomvolgordebepaling van geselekteerde gisvasse. Ons beskryf 'n unieke benadering, wat sellulasesekresieprofiele van stamme en fenotipiese reaksies op stres, wat vir hul invloed op die sekresieweg bekend is, te kombineer en sodoende 'n fenotipiese siftingproses te ontwikkel. Die konstruksie en sifting van haploïede variante vanaf natuurlike stam-isolate YI13, FINI en YI59, het gevolglik verskeie haploïede met verhoogde algemene sellulasesekresie gelewer. 'n Duidelike onderskeid tussen die YI13 haploïede variante en die industriële en laboratorium-stamme, Ethanol Red en S288c, is waargeneem. Ons resultate het getoon dat 'n nuwe siftingstegniek gekombineer met 'n geteikende paringsbenadering 'n poel van nuwe stamme met verbeterde sellulasesekresievermoëns kon oplewer. In 'n poging om 'n geskikte genetiese agtergrond vir doeltreffende sellulasesekresie te vind, is geneties diverse stamme geskep om kernstelle van swamsellulases, naamlik β-glukosidase, endoglukanase en sellobiohidrolase, in verskillende kombinasies te produseer. Hoër sekresietiters deur sellulolitiese stamme met die YI13 genetiese agtergrond is verkry en sellulolitiese transformante het tot 1.34-voudige hoër glukosekonsentrasies (g/L) vrygestel in vergelyking met 'n kontrolemengsel met gelyke hoeveelhede van elke ensiemtipe. Die transformant wat BGLI en EGII saam in ʼn aktiwiteitsverhouding van 1:15 (eenheid per gram droë massa) geproduseer het, het 56.5% van die sellulose in mieliestronke tot glukose in hidrolise-eksperimente omgeskakel, en 4.05 g / L etanol in fermentasies opgelewer. Ten slotte het ons 'n omvattende lys van moontlike veroorsakende geenkandidate geïdentifiseer wat met ’n hoë sekresiefenotipe verband hou, deur middel van 'n gesamentlike segregante heelgenoomvolgorde-analise en die daaropvolgende kartering van kwantitatiewe eienskaplokusse van 'n industriële stam (Ethanol Red) en ʼn natuurlike stamisolaat (YI13). Ander navorsers jet getoon dat sommige van hierdie geenkandidate aktief by verskillende fases van sekresie, vanaf transkripsie, translasie, na-translasie-modifisering tot proteïenvouing, betrokke is. Daarbenewens het ons verskeie teikens vir toekomstige stamverbeteringstrategieë geïdentifiseer.Die stamme wat in hierdie studie ontwikkel is, verteenwoordig dus 'n nuwe stap na doeltreffende sellulasekresie vir CBP bio-etanolproduksie. Doctoral 2019-10-09T08:30:45Z 2019-12-11T06:38:49Z 2022-01-01T03:00:10Z 2019-12 Thesis http://hdl.handle.net/10019.1/106915 en_ZA Stellenbosch University xv, 203 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Saccharomyces cerevisiae -- Genetic engineering Yeast strains Saccharomyces cerevisiae -- Effect of of stress on Cellulase -- Secretion Phenotype Genetic diversity UCTD Davison, Steffi Angela Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title | Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title_full | Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title_fullStr | Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title_full_unstemmed | Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title_short | Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| title_sort | exploring phenotypic and genetic diversity of natural saccharomyces cerevisiae strains for improved recombinant cellulase secretion |
| topic | Saccharomyces cerevisiae -- Genetic engineering Yeast strains Saccharomyces cerevisiae -- Effect of of stress on Cellulase -- Secretion Phenotype Genetic diversity UCTD |
| url | http://hdl.handle.net/10019.1/106915 |
| work_keys_str_mv | AT davisonsteffiangela exploringphenotypicandgeneticdiversityofnaturalsaccharomycescerevisiaestrainsforimprovedrecombinantcellulasesecretion |