| Summary: | Thesis (MSc (Genetics))--University of Stellenbosch, 2010. === Includes bibliography. === Title page: Dept. of Genetics, Faculty of Science. === 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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