Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid
environment. This aggregation of cells, also referred to as "floc formation", will in most
cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast
strains, the floes can move to the surface of the liquid growth substrate to form a thin layer,
called a "velum", that has been compared to other microbial biofilms.
The factors that trigger flocculation can be divided into two groups, physical/chemical
(e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol
concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that
are either directly or indirectly involved in flocculation). In top-fermenting yeast strains,
several physical and chemical factors that trigger the process have been described,
including ethanol concentration, the presence of organic solvents, the absence of
molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors
appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors,
no specific genes have thus far been associated with flocculation in top fermenting yeast
strains.
In bottom-fermenting yeast strains, the physical and chemical factors that affect the
process are similar to the ones described for top-fermenting yeast strains, but include,
more specifically, the concentration of hexoses in the media (mannose or glucose), which
may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been
divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type
(inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various
genes have been associated with the flocculation of bottom-fermenting yeast strains.
Through genetic analysis, the genes have been categorised into dominant genes, semidominant
genes and recessive genes.
In order to better understand the role of some of the proteins responsible for
flocculation in S. cerevisiae, and to create strains whose flocculation properties would
correspond to those wanted in the wine and beer industries, three of the dominant
flocculation genes, FL01, FL05 and FL011, were placed under the control of the
promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved
by replacing the native promoters of the flocculation genes with the heterologous
promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent
due to the absence of the transcription factor that is required for flocculation,
F108p,was used as a model system.
Some of the transformed strains showed high flocculation, especially when the genes
were placed under control of the ADH2 promoter. In addition to this, the strains carrying a
modified FL011 gene showed increased adhesion to solid agar media and were able to
invade the growth substrate. These strains also showed an increased velum-forming ability
when grown in media containing only non-fermentable carbon sources. / AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n
vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie,
en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste,
beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie
te vorm. Hierdie verskynsel was ook al gevind in ander organismes.
Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore
kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie,
die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie,
pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek
betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie
beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore
waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die
teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en
die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of
hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot
dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie.
Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit
van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit
in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose),
wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se
flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur
die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur
slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die
effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie
gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en
ressessiewe flokkulerende gene.
Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste
effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om
spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë,
was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder
regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was
verkry deur die vervanging van die wilde tipe promotors van die drie gene met die
stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die
laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van
'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike
gene in flokkulasie, was gebruik as 'n wilde tipe ras.
Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer
onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar
en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om
'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare
koolstofbronbevattende media opgegroei word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/49788 |
| Date | 04 1900 |
| Creators | Domingo, Jody L. (Jody Lawren) |
| Contributors | Bauer, F. F., Pretorius, I. S., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology and Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 41 p. : ill. |
| Rights | Stellenbosch University |
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