Dissertation (DPhil)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is
the only potential renewable and sustainable source of energy available to mankind at
present, especially in the production of transportation fuels. Transportation fuels such as
gasoline can be blended with or completely replaced by ethanol produced exclusively
from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce
carbon emissions and also the dependence on foreign oil (mostly from the Middle East
and Africa) for many countries.
Bio-ethanol can be produced from both starch and cellulose present in plants,
even though cellulosic ethanol has been suggested to be the more feasible option.
Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic
action of acids or enzymes, which can, in turn, be broken down to monosaccharides
such as hexoses and pentoses. These simple sugars can then be fermented to ethanol
by microorganisms. Among the innumerable microorganisms present in nature, the
yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial
scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the
‘workhorse’ of the alcohol industry.
Although S. cerevisiae has arguably a relatively wide substrate utilisation range,
it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose
constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield
from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of
this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have
been made. Through molecular cloning methods, xylose pathway genes from the
natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have
been cloned and expressed separately in various S. cerevisiae strains. However,
recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose
reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and
fermented this pentose sugar to xylitol.
The main focus of this study was to improve xylose utilisation by a recombinant
S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic
fermentation conditions. This has been approached at three different levels: (i) by
creating constitutive carbon catabolite repression mutants in the recombinant
S. cerevisiae background so that a glucose-like environment is mimicked for the yeast
cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase
gene from the natural xylose-degrading fungus Neurospora crassa through functional
complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant
XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting
mutant that showed an altered product profile after anaerobic xylose fermentation. From
the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed,
although ethanol formation appears to be a highly regulated process in the cell.
In summary, this work exposits three different methods of improving xylose
utilisation under anaerobic conditions through manipulations at the molecular level and
metabolic level. The novel S. cerevisiae strains developed and described in this study
show improved xylose utilisation. These strains, in turn, could be developed further to
encompass other polysaccharide degradation properties to be used in the so-called
consolidated bioprocess. / AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is.
Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat
tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe.
Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van
plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang
word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande
ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en
Afrika) te verminder.
Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word,
maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan
deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek
word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses
afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol
gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die
gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy
unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die
alkoholbedryf gemaak.
Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit
nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten
minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die
etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie
benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae
te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg
uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam,
Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante
ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk,
het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis.
Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n
rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder
anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader:
(i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante
S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting
vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike
xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele
komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van
die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n
gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige
benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting
van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk
te wees.
Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om
xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die
molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie
ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle
beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit
wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/18705 |
| Date | 12 1900 |
| Creators | Thanvanthri Gururajan, Vasudevan |
| Contributors | Cordero Otero, Ricardo R., Pretorius, Isak S., Van Rensburg, Pierre, Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | ii, 183 leaves : ill. |
| Rights | Stellenbosch University |
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