Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an
industrialised context, conversion to liquid biofuel is necessary, which could provide the
motor vehicle market with energy. To enable fermentation of both hexose and pentose
sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose
utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose.
The aim of this study was to increase D-xylose utilisation and lower the xylitol production
found with the eukaryotic pathway, thus redirecting carbon to the increased production of
ethanol.
In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae
transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis
and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the
parental strain with respect to D-xylose utilisation, lowered xylitol production and improved
ethanol production was obtained.
Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae.
Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic
pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose
isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not
previously been transformed into yeast. When the native xylA was expressed in E. coli
and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of
activity was surmised to be due to an amino acid modification, or possibly due to a vastly
different codon bias in yeast compared to the Bacteroides strain. Northern analysis
revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene
(SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for
S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with
the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole
carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents
a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for
bioethanol. / AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige
industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien,
is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide
heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende
navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra
om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om
D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid
xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol
opbrengs te handhaaf.
Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n
rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH)
gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik
geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose
kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie.
Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese
xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese
paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase
(XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis
getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is
geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan
aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse
(“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het
bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase
geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die
S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI)
getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste
koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te
verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol
met geneties gemodifiseerde S. cerevisiae.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17346 |
| Date | 04 1900 |
| Creators | De Villiers, Gillian K. |
| Contributors | Van Zyl, W.H., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xi, 111 leaves : ill. |
| Rights | University of Stellenbosch |
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