Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of
mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the
major hemicellulose component in plant cell walls, is the most abundant polysaccharide
after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second
most abundant renewable monosaccharide in nature. Very few hemicelluloses are either
homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their
hydrolysis is more complex than the enzyme group responsible for the hydrolysis of
cellulose. Although the ability to degrade xylan is common among bacteria and
filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the
yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one
of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of
fermenting polymeric xylan directly to ethanol. However, it was shown that the natural
xylanolytic ability of this yeast is very weak.
In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast
to produce heterologous proteins, and to determine the enhancement of xylan utilisation
by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase
gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase
(PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to
express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was
shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL
promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not
function in P. stipitis .
When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene
(xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase
activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity
declined over time due to the presence of extracellular proteases, secreted by P. stipitis.
Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger
l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02)
with the S. cerevisiae mating factor secretion signal (MFal) under control of the
P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was
observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase
activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and
XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124
nkatlml, respectively.
The expression of the fungal xylanolytic genes in P. stipitis also led to an increased
biomass yield when the recombinant strains were cultured on birchwood xylan as sole
carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger
l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass
level than the control strain. Biomass levels of the recombinant strains were further
improved on average by 85% by growing them in a fermentor under conditions of high
oxygenation. The strains were also tested for direct conversion of xylan to ethanol and
the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding
genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield
over the reference strain. These strains represent a step towards the efficient degradation
and utilisation of hemicellulosic materials by ethanol-producing yeasts. / AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse
van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in
plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof
suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare
monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of
heeltemal linieêr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses
meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en
filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei
voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia
stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die
beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom
etanol vanafpolimeriese xilaan te produseer.
In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk
om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die
effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste
koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die
transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van
Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om
die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het
bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl
die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie
funksioneel in P. stipitis nie.
Ekstrasellulêre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon
waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii
~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie
aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulêre
proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulêre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak
tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen
(xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van
S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon
ekstrasellulêre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word.
Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik.
Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik.
Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe
biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood
xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees
suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die
biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon
gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n
fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om
xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6-
voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie
rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en
benutting van hemisellulose deur etanolproduserende giste.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53409 |
| Date | 12 1900 |
| Creators | Den Haan, Riaan |
| Contributors | Van Zyl, W. H., 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 | [xiv], 215 p. : ill. |
| Rights | Stellenbosch University |
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