Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: There is a global demand for technologies to reduce ethanol levels in wine without
compromising wine quality. While several chemical and physical methods have been developed
to reduce ethanol in finished wine, the target of an industrially applicable biological solution has
thus far not been met. Most attempted biological strategies have focused on developing new
strains of the main fermentative organism, the yeast Saccharomyces cerevisiae. Gene
modification approaches have primarily focused on partially redirecting yeast carbon
metabolism away from ethanol production towards glycerol production. These techniques have
met with some moderate success, thus the focus of the current study was to re-direct carbon
flux towards trehalose production by moderate over-expression of the TPS1 gene. This gene
encodes trehalose-6-phosphate synthase, which converts glucose 6-phosphate and UDPglucose
to α,α-trehalose 6-phosphate. Previous data have shown that the overproduction of
trehalose restricts hexokinase activity reducing the amount of glucose that enters glycolysis.
Nevertheless, preliminary TPS1 over-expression studies using multiple copy plasmids have
shown some promise, but also indicated significant negative impact on the general fermentation
behaviour of strains. In order to reduce such negative impacts of excessive trehalose
production, a new strategy consisting in increasing the expression of TPS1 only during specific
growth phases and by a relatively minor degree was investigated. Our study employed a lowcopy
number episomal vector to drive moderate over-expression of the TPS1 gene in the widely
used industrial strain VIN13 at different stages during fermentation. The fermentations were
performed in synthetic must with sugar levels representative of those found in real grape must.
This, as well as the use of an industrial yeast strain, makes it easier to relate our results to real
winemaking conditions. A reduction in fermentation capacity was observed for all transformed
strains and controls. Expression profiles suggest that the DUT1 promoter certainly results in
increased TPS1 expression (up to 40%) during early exponential growth phase compared to the
wild type strain (VIN13). TPS1 expression under the control of the GIP2 promoter region
showed increased expression levels during early stationary phase (up to 60%). Chemical
analysis of the yeast and the must at the end after fermentation showed an increase in
trehalose production =in line with the expression data of TPS1. Importantly, glycerol production
was also slightly increased, but without affecting acetic acid levels for the transformed strains.
Although ethanol yield is not significantly lower in the DUT1-TPDS1 strain, s statistically
significantly lower ethanol yield is observed for over-expression under the GIP2 promotor.
Increasing trehalose production during stationary phase appears therefore to be a more
promising approach at lowering ethanol yield and redirecting flux away from ethanol production.
This controlled, growth phase specific over expression suggests a unique approach of lowering
ethanol yield while not impacting on the redox balance. / AFRIKAANSE OPSOMMING: Wêreldwyd is daar ‘n aanvraag na tegnologie wat die etanol vlakke in wyn kan verminder
sonder om wyngehalte te benadeel. Terwyl verskeie chemiese en fisiese metodes ontwikkel is
om etanol in die finale wynproduk te verminder, is die soeke na 'n industrieel gebaseerde
biologiese oplossing tot dusver nie gevind nie. Meeste biologiese strategieë fokus op die
ontwikkeling van nuwe rasse van die primêre fermentatiewe organisme, naamlik
Saccharomyces cerevisiae. Geen modifikasie benaderings het hoofsaaklik gefokus op die
gedeeltelike kanalisering van koolstof metabolisme weg van etanol produksie na gliserol
produksie. Hierdie benadering is net matiglik suksesvol, dus is ons huidige fokus om koolstof te
kanaliseer na trehalose produksie deur gematigde oor-uitdrukking van die TPS1 geen. Hierdie
geen kodeer vir trehalose-6-fosfaat sintase, wat glukose-6-fosfaat en UDP-glukose omskakel na
α, α-trehalose-6-fosfaat. Vorige data het getoon dat die oorproduksie van trehalose hexokinase
aktiwiteit beperk en die hoeveelheid glukose wat glikolise binne gaan. Voorlopige TPS1 ooruitdrukking
studies met behulp van multi-kopie plasmiede toon matige sukses, maar het ook ‘n
negatiewe impak op die algemene fermentasie kapasiteit van die gis. Ten einde so 'n negatiewe
impak van oormatige trehalose produksie te oorkom, is 'n nuwe strategie gevolg wat bestaan uit
die verhoogde uitdrukking van die TPS1 geen slegs gedurende spesifieke groei fases met baie
lae vlakke van oor-uitdrukking. Ons studie gebruik 'n lae-kopie episomale vektor met matige
oor-uitdrukking van die TPS1 geen in die industriële ras VIN13 op verskillende stadiums tydens
fermentasie. Die fermentasie is uitgevoer in sintetiese mos met suiker vlakke
verteenwoordigend van dié van werklike wyn mos. Hierdie, sowel as die gebruik van 'n
industriële gisras, maak dit makliker om ons resultate te vergelyk met regte wyn fermentasie
kondisies. Verlaagde fermentasie kapasiteit is waargeneem vir alle getransformeerde stamme
en hul kontroles. Geen uitdrukkings profiele dui op verhoogde TPS1 uitdrukking (tot 40%) onder
beheer van die DUT1 promotor gedurende die vroeë eksponensiële groeifase wanneer vergelyk
word met die wilde tiepe (VIN13). TPS1 uitdrukking onder die beheer van die GIP2 promotor het
verhoogde uitdrukking van tot 60% gedurende die vroeë stasionêre fase. Chemiese analise van
die gis aan die einde van fermentasie dui op ‘n toename in trehalose produksie wat korreleer
met die uitdrukking profiele van TPS1. Gliserol produksie is ook effens verhoog, maar sonder ‘n
toename in asynsuur vlakke vir die getransformeerde rasse. Alhoewel etanol opbrengs nie
aansienlik laer vir die DUT1-TPS1 ras is nie, is etanol opbrengs vir die oor-uitdrukking onder
beheer van die GIP2 promotor wel laer. Toenemende trehalose produksie gedurende
stasionêre fase blyk dus 'n meer belowende benadering op die verlaging van etanol opbrengs
en her-kanaliseering weg van etanol produksie. Hierdie benadering met die fokus op groeifase
spesifieke oor-uitdrukking dui op 'n unieke strategie vir die verlaging van etanol opbrengs
sonder om die redoks balans te beinvloed. / The NRF, IWBT and Stellenbosch University for funding
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80137 |
| Date | 03 1900 |
| Creators | Heyns, Eva Hutton |
| Contributors | Bauer, F. F., Setati, M. E., Rossouw, D., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 59 p. : ill. |
| Rights | Stellenbosch University |
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