Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in
winemaking processes and wine quality, including the organoleptic quality and the physical,
biochemical and microbial stability of wine. The production of premium wines depends on the
oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine
components to produce wine with optimum colour and flavour.
Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during
alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The
degree of L-malic acid degradation, however, varies from strain to strain. Some strains of
Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the
underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon
have been partially revealed during preliminary transcriptional regulation research during this study.
In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic
pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative
conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces
cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of
L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of
extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower
substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which
contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial
location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme,
suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of
fermentative metabolism.
The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore
cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae
3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of
S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid
was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent
production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of
S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of
industrial wine yeast strains of Saccharomyces for commercial winemaking.
A prerequisite for becoming an inherited component of yeast is the stable integration of the
malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine
yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of
heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable
markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the
yeast genome is, however, not acceptable for commercial application due to the absence of long-term
risk assessment and consumer resistance.
A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the
incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing
the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2
open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces
bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After
initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the
phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After
correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in
synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete
degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and
expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the
fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay
wines indicated an improvement in wine flavour compared to the control wines, without the
production of any off-flavours. / AFRIKAANSE OPSOMMING: L-Appelsuur en wynsteensuur is die mees prominente organiese sure in wyn en speel 'n kritiese rol in
die wynbereidingsproses en organoleptiese wynkwaliteit, insluitende die fisiese, biochemiese en
mikrobiese stabiliteit van wyn. Die produksie van hoë-kwaliteit wyne berus op die vermoë van 'n
wynmaker om die suurinhoud korrek aan te pas om sodoende 'n gebalanseerde produk met optimale
geur en kleur te produseer.
Saccharomyces rasse kan gewoonlik nie appelsuur volledig tydens alkoholiese gisting benut nie en
dra dus nie noemenswaardig tot 'n verlaging van die totale suurinhoud van wyn by nie. Die mate van
appelsuur afbraak deur Saccharomyces wissel egter van ras tot ras. Sekere Saccharomyces rasse kan
'n groter persentasie appelsuur afbreek, maar die onderliggende rede vir hierdie verskynsel is
onbekend. Die onderliggende meganismes vir hierdie verskynsel is gedurende hierdie studie uitgelig
na afloop van voorlopige transkripsionele regulerings studies op die malaatensiemgeen. In
teenstelling hiermee kan S. pombe tot 29 gIl appelsuur via die malo-alkoholiese padweg afbreek
waartydens appelsuur na pirodruiwesuur en CO2, en uiteindelik na alkoholonder fermentatiewe
toestande omgeskakel word. Verskeie redes vir die swak afbraak van appelsuur deur
Saccharomyces cerevisiae is voorgestel. Eerstens beskik S. cerevisiae nie oor 'n meganisme vir die
aktiewe transport van appelsuur, soos in die geval van S. pombe nie, en is aangewese op die stadige
opname van appelsuur deur eenvoudige diffusie. Tweedens het die S. cerevisiae malaatensiem 'n baie
laer substraataffiniteit vir appelsuur (Km = 50 mM) in vergelyking met die van S. pombe (Km =
3.2 mM), wat verder bydra tot die swak afbraak van appelsuur in S. cerevisiae. Laastens dra die
mitochondriale ligging van die S. cerevisiae malaatensiem in teenstelling met die sitoplasmiese
ligging van die S. pombe malaatensiem, verder by tot die swak afbraak van appelsuur, aangesien die
mitochondria onder fermentatiewe toestande negatief gereguleer word.
Die malaatpermease geen (maely en malaatensiem geen (mae2) van S. pombe is gevolglik gekloneer
en heteroloog in 'n laboratoriumras van S. cerevisiae onder die beheer van die konstitutiewe
3-fosfogliseraat kinase (PGKI) promoter- en termineerdervolgordes uitgedruk. 'n Sterk
malo-alkoholiese fenotipe was duidelik tydens fermentasies met die rekombinante gis in sintetiese en
Chardonnay druiwemos, met 'n gepaardgaande verhoging in alkoholvlakke. Funksionele uitdrukking
van die malo-alkoholiese gene van S. pombe in 'n S. cerevisiae laboratoriumras het die weg vir die
genetiese modifisering van industriële wynrasse van S. cerevisiae vir kommersiële wynfermentasie
gebaan.
Om 'n integrale deel van die gis te word, moet die malo-alkoholiese gene stabiel in die genoom van
industriële wynrasse geïntegreer word. Genetiese manipulering van industriële wynrasse word egter
bemoeilik deur die homotalliese, multi-ploïediese en prototrofiese aard van industriële Saccharomyces rasse. Transformasie en integrasie van heteroloë gene in industriële Saccharomyces
rasse vereis die gebruik van dominante merkers, bv. weerstandbiedendheid teen antibiotika of ander
gifstowwe. Integrasie van hierdie merkers in die gisgenoom is egter nie vir kommersiële toepassing
aanvaarbaar nie weens die afwesigheid van langtermyn risikobepalings en verbruikersweerstand.
Tydens hierdie studie is daar dus gepoog om industriële wynrasse met 'n unieke strategie geneties te
verbeter sodat slegs gis-DNA tydens die integrasie van die S. pombe mae1 en mae2
uitdrukkingskassette in die gisgenoom opgeneem word. Die Malo-alkoholiese integrasiekasset wat
slegs die S. pombe mae1, mae2 oopleesrame en die S. cerevisiae PGK1 promoter en
termineerdervolgordes bevat, is in die URA3 lokus van Saccharomyces bayanus ECll18 geïntegreer
tydens parallelle transformasie met 'n 'phleomycin' weerstandbiedendheidsplasmied. Na seleksie van
transformante op 'phleomycin' -bevattende media, is die S. bayanus EC 1118 transformante in nieselektiewe
kondisies opgegroei sodat verlies van die 'phleomycin' plasmied kon plaasvind. Integrasie
van die mae1 en mae2 uitdrukkingskassette is bevestig en kleinskaalse fermentasies in sintetiese en
druiwemos het 'n vinnige en doeltreffende afbraak van appelsuur in die vroeë fases van die
alkoholiese fermentasie getoon. Integrasie en uitdrukking van die malo-alkoholiese gene in
S. bayanus ECl118 het geen nadelige effek op die fermentasievermoë van die gis getoon nie, terwyl
sensoriese en chemiese ontleding van die Chardonnay wyne 'n verbetering in aroma relatief tot die
kontrole wyne getoon het, met die afwesigheid van enige afgeure.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52728 |
| Date | 12 1900 |
| Creators | Volschenk, Heinrich |
| Contributors | Van Vuuren, H. J. J., Bloom, M., 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 | 185 p. : ill. |
| Rights | Stellenbosch University |
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