Manipulating cell wall biosynthesis in yeast and higher plants

Horstmann, Carl Ulrich (2010-12)

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.

Includes bibliography.

Title page: Dept. of Genetics, Faculty of Science.

Thesis

ENGLISH ABSTRACT: Undeniably, changes in the environment and dwindling traditional energy resources have resulted in the search for viable, renewable energy alternatives such as biofuels. Cellulose is one of the most abundant polymers on earth and can be converted to simple sugars and fermented to ethanol biofuel fairly easily. Cellulose rich biomass that can serve to supply ethanol biofuel production can be sourced from unexploited agricultural waste. The main drawback to using vegetative tissue as opposed to harvested food stocks from crops results from the structural properties of plant cell walls. Although cellulose is abundant, the contaminating hemicellulose and lignin fibres within the cell wall matrix have a negative impact on the digestibility of the cellulose present. Thus, an important step in creating an effective biofuel production system from agricultural excess is developing crops with improved cell wall polymer characteristics that can be converted to ethanol more efficiently. This project consisted of two parts. Firstly, the aim was to assess lignin production in transgenic sugarcane transformed with a construct aimed at down-regulating the 4- (hydroxyl) cinnamoyl CoA ligase (4CL) gene in the lignin biosynthesis pathway. The second part of the project revolved around discovering the mechanism of impared cell growth caused by expressing the gene encoding cellulose synthase from a marine invertebrate, Ciona savignyi, in the yeast Saccharomyces cerevisiae. Several sugarcane lines that had been previously transformed with a hairpin RNAi construct aimed at down-regulating the 4CL gene in the monolignol biosynthesis pathway were subjected to analysis to determine if lignification had been reduced. Although the presence of the hairpin construct in the genomic DNA had been confirmed for all of the transgenic lines, there was no significant decrease in the lignin levels in any of the transgenic lines. PCR analysis of the mRNA and enzyme assays also confirmed that the 4CL gene was still being expressed. Ongoing work will determine the cause of the unsuccessful down-regulation. Previously, it had been proven that the cellulose synthase gene from C. savignyi could be functionally expressed in S. cerevisiae. However, cellulose production resulted in extremely retarded growth of colonies and cultures, to the point of the apparent death of the cultures. The aim of this part of the project was to determine the mechanism (either metabolic or physical) that causes this effect. To generate enough cell mass to perform metabolic analysis, several strategies to impede cellulose production in transgenic yeast were explored. Attempts to stop cellulose production and induce better growth by introducing Isoxaben (a traditional weed killer that targets cellulose synthases) into the growth medium used for the transgenic yeast proved unsuccessful. To control the expression of the transgene, it was attempted to clone the cellulose synthase gene into an expression system containing an inducible promoter. The cloning exercise proved extremely difficult and multiple attempts with several strategies proved unsuccessful. This process is still ongoing as the growth retarding process induced by cellulose production in yeast remains to be identified.

AFRIKAAANSE OPSOMMING: Dit is onontkenbaar dat veranderinge in die omgewing en minderwordende tradisionele energiebronne veroorsaak dat lewensvatbare en hernubare energiebronne soos biobrandstof gevind moet word. Sellulose is een van die mees volop polimere op aarde en kan redelik maklik omgeskakel word na eenvoudige suikers en gefermenteer word tot etanol-biobrandstof. Sellulose-ryk biomassa wat etanol-biobrandstof kan verskaf, kan herwin word van tot op hede ongebruikte landbou-afval. Die komplekse struktuur van plantselwande is die hoofstruikelblok in die omskakeling van vegetatiewe weefsel tot biobrandstof. Hoewel sellulose volop is, het die kontaminerende hemisellulose- en lignienvesels binne die selwand-matriks ’n negatiewe impak op die verteerbaarheid van die sellulose teenwoordig in die selwand. Daarom is ’n belangrike stap in die ontwikkeling van effektiewe biobrandstof-produksiesisteme vanaf landbou-afval om gewasse te ontwikkel met verbeterde selwandpolimeer-eienskappe wat etanol-produksie kan vergemakilik. Hierdie projek het bestaan uit twee dele. Eerstens was die doel om vas te stel of die lignienproduksie geaffekteer is in transgeniese suikerriet getransformeer met ’n konstruk wat mik om die 4-(hidroksie)-cinnamoyl CoA ligase (4CL) geen te af-reguleer in die lignienbiosintese- padweg. Die tweede deel van die projek het daarop gefokus om die meganisme te ondek wat die belemmerde selgroei veroorsaak, as gevolg van die uitdrukking van die geen wat kodeer vir sellulose-sintase in ’n mariene ongewerwelde, Ciona savignyi, in Saccharomyces cerevisiae. Verskeie suikerriet-lyne, wat voorheen getransformeer is met ’n haarnaald-RNAi-konstruk om die 4CL-geen te af-reguleer in die monolignol-biosintese-padweg, is onderwerp aan analise om vas te stel of lignifikasie verminder is. Hoewel die teenwoordigheid van die haarnaald-konstruk in die genomiese DNA bevestig is vir al die transgeniese lyne, was daar geen beduidende vermindering in die lignienvlakke in die transgeniese lyne nie. PKRanalise van die mRNA en ensiem-aktiwiteitstoetse het ook bevestig dat die 4CL-geen steeds uitgedruk word. Verdere ondersoek sal kan vasstel wat die oorsaak van die onsuksesvolle af-regulering is. Voorheen is bewys dat die sellulose-sintase-geen van C. savignyi funksioneel uitgedruk kon word in Saccharomyces cerevisiae. Egter, selluloseproduksie het die gevolg gehad dat groei in die transgeniese kolonies en kulture erg gestrem is, tot die punt dat die kulture dood voorgekom het. Die doel van hierdie deel van die projek was om vas te stel wat die meganisme (òf metabolies òf fisies) is wat hierdie verskynsel veroorsaak het. Om genoeg selmassa te genereer om metaboliese analise uit te voer, is verskeie strategieë om selluloseproduksie in transgeniese gis te verhinder, ondersoek. Pogings om selluloseproduksie te stop en om groei te verbeter deur Isoxaben by te voeg in die groeimedium gebruik vir transgeniese gis, was onsuksesvol. Isoxaben is ’n tradisionele onkruiddoder wat sellulose-sintases teiken en inhibeer. Om die uitdrukking van die transgeen te beheer, is ’n poging aangewend om dié sellulose-sintase-geen in ’n uitdrukking-sisteem te kloon met ’n induseerbare promotor. Die kloneringsoefening was uiters moeilik en veelvoudige pogings met verskeie strategieë was onsuksesvol. Hierdie proses moet verder gevoer word aangesien die groeistremmingsmeganisme veroorsaak deur selluloseproduksie in gis nog geïdentifiseer moet word.

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