Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can
result in improved processability and an increase in wine quality by improving extraction
of essential compounds from the grapes during the maceration stage. Pectin is the only
important cell wall polysaccharide that can be degraded by wild-type Saccharomyces
cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the
PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and
PG activity is thus strain specific. The lack of activity in certain strains has been
attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the
PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present
but not transcribed. The lack in transcription has been shown to be due to the gene
having a dysfunctional promoter or to regulatory differences between strains. Results
published in the literature are contradictory. The primary aim of this investigation was to
clarify the regulation of PG activity in S. cerevisiae and to determine why there are
differences in PG activity between different strains. Regulation of PG activity between
several wine and laboratory strains with varying PG activities was compared by looking
at the sequence of the PGU1 gene and its promoter as well as transcription levels of
this gene and its main transcription factors, TEC1 and STE12. In order to identify
regulatory factors influencing PG activity, the S. cerevisiae genome was screened for
activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG
activity were identified during this screen. Real-time PCR analysis showed that the PG
activity is regulated by transcription of the PGU1 gene. A linear relationship was
demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some
of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing
by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its
subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the
PGU1 gene with its native regulatory machinery to a different position away from its
telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating
the epigenetic influence on PGU1 regulation. Results from this study suggested that the
strain related difference in PGU1 expression occurs at an epigenetic level, with steric
hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting
transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more
effective strategies to improve PG degradation by S. cerevisiae. The genetic model
describing regulation of PGU1 transcription was extended by this study and a novel
mechanism of regulation of PG activity was identified.
The secondary aim of this study written as an addendum to this thesis, focussed on
degradation of another grape cell wall polysaccharide xylan by recombinant strains of S.
cerevisiae. These strains were enabled to degrade this polysaccharide through
heterologous expression of novel xylanase encoding genes from various origins.
Xylanase activity of the recombinant strains generated was compared. Overexpressing
the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or
the functional domain xynAc within optimal conditions for these enzymes all conferred
very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase
activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity
under optimal conditions activity in wine making conditions would be negligible. The
genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively
yielded higher levels of activity. According to these results, only the expression of XYN2
and XYN4 could have a potential effect on wine
An effective strategy for improving pectin degradation can in future potentially be
combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in
order to engineer a wine yeast strain with improved polysaccharase abilities. / AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in
wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van
belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van
die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan
word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W)
geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem
aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf
as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele
alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al
bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele
promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige
studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die
hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom
daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG
aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van
PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die
DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om
reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae
ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time
PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen
en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee
hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat
geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te
wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit
dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van
die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van
chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander
posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie
van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1
geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die
resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die
PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie
van die geen sodoende in sommige rasse voorkom.
Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander
komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n
addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in
hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste
wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens,
asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk.
Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die
rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van
die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en
Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase
aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene
ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van
wyngiste te verbeter.
Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë
potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie
het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme
van regulering van toepassing op PGU1 te identifiseer.
As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer
word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om
sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4005 |
| Date | 03 1900 |
| Creators | Louw, Campbell Trout |
| Contributors | Van Rensburg, P., Divol, B., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Detected Language | Unknown |
| Format | 131 p. : ill. |
| Rights | University of Stellenbosch |
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