Hardening yeast for cellulosic ethanol production
| dc.contributor.advisor | Van Zyl, Willem Heber | en_ZA |
| dc.contributor.advisor | Gorgens, Johann F. | en_ZA |
| dc.contributor.author | Brandt, Bianca Anina | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Microbiology. | en_ZA |
| dc.date.accessioned | 2019-01-30T09:17:38Z | |
| dc.date.accessioned | 2019-04-17T08:04:09Z | |
| dc.date.available | 2022-03-03T03:00:14Z | |
| dc.date.issued | 2019-04 | |
| dc.description | TThesis (PhD)--Stellenbosch University, 2019. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: The physico-chemical pretreatment of lignocellulose biomass is required to provide substrate access to enzymes responsible for the release fermentable sugars, but also releases microbial inhibitors such as furan aldehydes, weak acids, and various lignin-derived phenolic compounds, that detrimentally effect the subsequent bioconversion by Saccharomyces cerevisiae. Sustainable and cost effective lignocellulose bioconversion therefore relies heavily on the generation and optimization of robust S. cerevisiae strains with resistance to multiple inhibitors. In this context, the study aimed to improve the lignocellulose-hydrolysate fermentation efficiency of S. cerevisiae strains previously engineered for xylose fermentation capacity (introduced xylose isomerase - XI), using a combination of rational engineering and evolutionary adaptation to generate “hardened” strains exhibiting multi-inhibitor resistance phenotypes. A xylose capable S. cerevisiae was used as parental and industrial control strain for the rational engineering of seven genes identified by literature to address furan aldehyde, weak acid and phenolic-induced stresses. These genes included the TAL1, FDH1, ARI1, ADH6, and PAD1 genes that have been characterized to contribute to in situ inhibitor detoxification phenotypes. Enzymatic in situ detoxification was combined with plasma membrane modification whereby the FPS1 gene (membrane pore) was partially deleted, and the ICT1 gene (membrane modifier) overexpressed. The overexpression genes were integrated in novel combinations into a partial FPS1 deletion background, to identify gene combinations that confer multi-inhibitor resistance phenotypes, yet limit the impact on xylose metabolism. The study yielded transformants with novel gene combinations that conferred multi-inhibitor resistance phenotypes based on the sequence of integration, and increased both biomass and ethanol yield. Xylose utilization was negatively impacted by the overexpression of the gene combinations, however, transformants with specific gene combinations (TAL1-FDH1 + ARI1- ADH6) showed a limited impact. The resultant transformants were adapted in concentrated un-detoxified spent sulphite liquor (SSL) to further strengthen the introduced multi-inhibitor resistance phenotype and to include inhibitors typical to SSL waste streams. The adaptation followed a serial transfer in shake flask methodology, with increasing concentrations of concentrated SSL as selective pressure. The adapted isolates exhibited stable increased resistance phenotypes and increased ethanol yields relative to parental control, in 60% and 80% v/v SSL fermentations, however, there appeared to be a trade-off between the expressed phenotype and ethanol productivity. The adapted isolates exhibited an increased robust phenotypes, however, a regression on ethanol productivity was observed as adaptation progressed. Interestingly, the xylose utilization of the adapted isolates was only similar to that of the parental strain under stressed conditions, versus nonselective growth. Taken together, this study undertook the development of multi-inhibitor resistance phenotypes in a xylose capable S. cerevisiae and elaborated on its implications under industrially relevant conditions. The study highlighted novel gene combinations TF, AA and PI that can be used to “harden” yeast to generate robust biocatalyst strains, with limited impact on xylose utilization. This contributes towards furthering the understanding of inhibitor phenotypes and possible links to carbon metabolism. In addition, this study illustrated the use of rational engineering in combination with evolutionary adaptation to manipulate and improve a complex phenotype in an industrial strain. This paves the way forward towards developing inhibitor resistant xylose capable industrial strains for efficient cellulosic ethanol production. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Die fisies-chemiese voorbehandeling van lignosellulose biomassa wat vereis word om ensieme verantwoordelik vir die vrystelling van fermenteerbare suikers, toegang tot substraat te bied, stel ook mikrobiese inhibeerders soos furanaldehiede, swak sure en verskeie lignien-verwante fenoliese molekules vry wat die gevolglike omskakeling deur Saccharomyces cerevisiae nadelig beïnvloed. Volhoubare en koste-effektiewe lignosellulose-omskakeling berus dus sterk op die ontwikkeling en optimisering van robuuste S. cerevisiae-rasse met weerstand teen verskeie inhibeerders. In hierdie konteks was die studie daarop gerig om die lignosellulose- hidrolisaat fermentasie-doeltreffendheid van S. cerevisiae-rasse, wat voorheen vir xilose- fermentasie vermoë (toevoeging van xilose isomerase - XI) aangepas was, te verbeter deur gebruik te maak van 'n kombinasie van rasionele manipulasie en evolusionêre aanpassing om "geharde" rasse met multi-inhibeerder-weerstand fenotipes te genereer. 'n Xilose-bekwame S. cerevisiae-ras was gebruik as ‘n ouer- en industriële kontroleras vir die rasionele manipulasie van sewe gene wat uit die literatuur geïdentifiseer is om furanaldehied, swak sure en fenoliese geïnduseerde stres aan te spreek. Hierdie gene het TAL1- , FDH1-, ARI1-, ADH6- en PAD1-gene ingesluit, wat gekenmerk word vir hul bydrae tot in situ-inhibeerder detoksifisering-fenotipes. Ensimatiese in-situ detoksifikasie was met plasmamembraan-modifikasie gekombineer waardeur die FPS1-geen (membraanporie) gedeeltelik verwyder is en die ICT1-geen (membraanmoduleerder) ooruitgedruk is. Die ooruitdrukkingsgene is in nuwe kombinasies in 'n gedeeltelike FPS1-delesie agtergrond geïntegreer om geenkombinasies te identifiseer wat multi-inhibibeerder-weerstand fenotipes verleen, maar die impak op xilose-metabolisme beperk. Die studie het transformante gelewer met nuwe geenkombinasies wat multi-inhibeerder weerstand fenotipes vertoon, gebaseer op die volgorde van geen-integrasie, en wat beide biomassa en etanolopbrengste meebring. Xilose-benutting was negatief beïnvloed deur die ooruitdrukking van die geenkombinasies, maar transformante met spesifieke geenkombinasies (TAL1-FDH1 + ARI1-ADH6) het 'n beperkte impak gehad. Die transformante was in gekonsentreerde, nie-ontgiftigde, gebruikte sulfietloog (“spent sulphite liquor” – SSL) aangepas om die multi-inhibeerder weerstand fenotipes verder te versterk en om inhibeerders wat tipies is vir SSL-afvalstrome in te sluit. Die aanpassingsmetodiek was op 'n reeks oordrag in skudflesse gebaseer, met toenemende konsentrasies van gekonsentreerde SSL om selektiewe druk te verseker. Die aangepaste isolate het stabiele verhoogde weerstandfenotipes en verhoogde etanolopbrengste in fermentasies met 60% en 80% v/v SSL vertoon, in vergelyking met die ouer kontroleras. Daar was egter 'n kompromie tussen die uitgedrukte fenotipe en etanolproduktiwiteit. Die aangepaste isolate het verhoogde robuuste fenotipes vertoon, maar 'n afname in etanolproduktiwiteit was waargeneem soos verhoogde robuustheid toegeneem het. Interessant genoeg was die xilose- benutting van die aangepaste isolate soortgelyk aan dié van die ouer kontroleras onder strestoestande, teenoor nie-selektiewe groei. Hierdie studie het die ontwikkeling van multi-inhibeerder weerstandfenotipes in 'n xilose-bekwame S. cerevisiae-ras onderneem en die implikasies daarvan onder industriële relevante toestande ondersoek. Die studie het nuwe geenkombinasies TF, AA en PI opgelewer wat gebruik kan word om "geharde" gis te ontwikkel om robuuste biokatalise rasse te genereer, met beperkte impak op xilose-benutting. Dit dra by tot ‘n beter begrip van inhibeerderfenotipes en moontlike skakels met koolstofmetabolisme. Hierdie studie illustreer ook die gebruik van rasionele modifisering in kombinasie met evolusionêre aanpassing om 'n komplekse fenotipe in 'n industriële stam te manipuleer en te verbeter. Dit baan die weg vorentoe vir die ontwikkeling van inhibeerder-weerstandige, xilose-bekwame industriële rasse vir doeltreffende sellulose-etanolproduksie. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.embargo.terms | 2022-02-28 | |
| dc.format.extent | xviii, 154 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/105597 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Lignocellulose biomass -- Genetic engineering | en_ZA |
| dc.subject | Saccharomyces cerevisiae -- Effect of stress on | en_ZA |
| dc.subject | Ethanol | en_ZA |
| dc.subject | Phenotype -- Genetics | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Hardening yeast for cellulosic ethanol production | en_ZA |
| dc.type | Thesis | en_ZA |