Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The wine yeast Saccharomyces cerevisiae has the ability to utilize several different
nitrogenous compounds to fulfill its metabolic requirements. Based upon different
growth rates of the yeast in a particular nitrogen source, nitrogen compounds have
been classified as either good or poor nitrogen sources. In an environment which
contains different quality nitrogen sources, such as grape must, the yeast first utilizes
good and then the poor nitrogen sources. This discrimination between good and
poor nitrogen sources is referred to as nitrogen catabolite repression (NCR).
Examples of good nitrogen sources are ammonia, glutamine and asparagine.
Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and proline
are poor quality nitrogen sources.
Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCR
in S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitive
genes. All these proteins, except Ure2p, bind cis-acting elements in the promoters
of genes that are responsible for degradation of poor nitrogen sources. Gln3p is an
activator of NCR sensitive genes in the absence of good nitrogen sources. The
predominant mechanism by which NCR functions is by using Ure2p to inactivate the
activator Gln3p in the presence of a good nitrogen source.
Several research groups have studied the Ure2p, mainly due to its prion-like
characteristics. The Ure2p has two domains: a prion inducing domain located in the
N-terminal region and a NCR regulatory domain located in the C-terminal domain.
The aims of this study were (i) to determine the part of the C-terminal domain which
is responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethyl
carbamate during wine fermentations and (iii) if NCR functions in industrial yeast
strains. Nested deletions of the URE2 gene revealed that the NCR regulatory
domain resides in the last ten amino acids of the Ure2p. This was established by
Northern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes.
Ethyl carbamate in wine is produced by spontaneous chemical reaction between
urea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolism of arginine. Arginine is degraded to ornithine and urea by arginase, the product of
the CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by urea
amidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subject
to NCR. This study investigated if a ure2 mutant strain produced less ethyl
carbamate during wine fermentations.
Wine fermentations were conducted with diploid laboratory strains: a ure2 mutant
strain and its isogenic wild type strain. GC/MS analysis of the wine revealed that the
ure2 mutant produced less ethyl carbamate but more ethanol than the wild type
strain when arginine, di-ammoniumphosphate, asparagine or glutamine were added
as nitrogen sources, in combinations and separately. There was no significant
difference between the wild type fermentation and the ure2 mutant fermentation
when no nitrogen was added. It was found that a combination between the deletion
of URE2 and the addition of a good nitrogen source resulted in lower levels of ethyl
carbamate.
High density micro array analysis done on an industrial strain wine yeast in
Chardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes,
responsible for transport and metabolism of arginine and degradation of urea, are
NCR sensitive. These data strongly suggest that NCR functions in industrial yeast
strains. / AFRIKAANSE OPSOMMING: Die wyngis Saccharomyces cerevisiae kan verskillende stikstofbronne gebruik om in
sy stikstofbehoeftes te voldoen. Stikstofbronne word as goeie of swak stikstofbronne
geklassifiseer op grond van die groeitempo van die gis op die betrokke stikstofbron.
'n Goeie stikstofbron laat die gis vinniger groei as wat dit op 'n swak stikstofbron sou
groei. In omgewings soos druiwemos waar daar 'n verskeidenheid van
stikstofbronne teenwoordig is, sal die gis eers die goeie bronne en daarna die swak
bronne benut. Stikstofbronne soos ammonium, asparagien en glutamien word
geklassifiseer as goeie bronne. Allantoïen, y-amino-butaraat (GABA), prolien en
arginien word as swak stikstofbronne geklassifiseer. Die meganisme waarmee S.
cerevisiae tussen die stikstofbronne onderskei, staan as stikstof kataboliet
onderdrukking (NCR) bekend.
Die proteïene wat vir verantwoordelik is NCR naamlik Ure2p, Gln3p, Gat1 p, Dal80p
en Deh1 p, bind met die uitsondering van Ure2p, almal aan cis-werkende elemente in
die promoters van NCR-sensitiewe gene. Die trans-werkende faktore reguleer die
transkripsie van NCR-sensitiewe gene. NCR werk hoofsaaklik deur die inhibering
van Gln3p deur Ure2p in die teenwoordigheid van 'n goeie stikstofbron. Die oorgrote
meerderheid NCR-sensitiewe gene word deur Gln3p in die afwesigheid van 'n goeie
stikstofbron geaktiveer.
Heelwat navorsing is op die prionvormings vermoë van Ure2p gedoen. Ure2p het
twee domeine: 'n N-terminale domein wat vir prionvorming verantwoordelik is en die
C-terminale domein waar die NCR funksie van Ure2p gesetel is. Die doel van die
studie was (i) om te bepaal waar in die C-terminale domein van Ure2p die NCR
regulering geleë is, (ii) of ure2 delesie mutante minder etielkarbamaat tydens
wynfermentasies produseer en (iii) of NCR in industriële gisrasse funksioneel is.
Delesie analises van URE2 het getoon dat die NCR regulerings domein in die laaste
tien aminosure gesetel is. Dit is vas gestel m.b.v. noordlike klad tegniek analises op
die OALS, CAN1 en GAP1 gene.Etielkarbamaat in wyn word deur die spontane chemiese reaksie tussen ureum en
alkohol geproduseer. Ureum word gedurende die metabolisme van arginien in S.
cerevisiae geproduseer. Arginien word deur arginase, produk van die CAR1 geen,
na ornitien en ureum afgebreek. Die bi-funksionele ureum amidoliase, gekodeer
deur die DUR1,2 geen, breek ureum na CO2 en NH/ af. As gevolg van die NCRsensitiwiteit
van dié gene is ondersoek ingestel na In ure2 mutant se vermoë om
minder etielkarbamaat tydens wynfermentasies te produseer. Chardonnay
druiwemos is met In diploiede laboratorium ras en die isogeniese ure2 mutant
gefermenteer. GC/MS analise op die wyn het getoon dat die ure2 mutant minder
etielkarbamaat, maar meer alkohol in vergelyking met die wilde tipe gis produseer,
as arginien, di-ammoniumfosfaat, asparagien en glutamien, afsonderlik of
gesamentlik byvoeg is. Daar was egter nie In merkwaardige verskil tussen die
fermentasies waar geen stikstof bygevoeg is nie. Dit dui daarop dat In kombinasie
van In URE2 delesie en die byvoeging van stikstof etielkarbamaat vlakke verlaag.
Mikro-skyfie analise van In industriële gis in Chardonnay mos het getoon dat die
GAP1, CAN1, CAR1 en DUR1,2 gene wat verantwoordelik is vir die transport en
metabolisme van arginien en degradasie van ureum, wel NCR-sensitief is. Dit dui
daarop dat NCRwel in industriële gisrasse funksioneel is.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51671 |
| Date | 12 1900 |
| Creators | Erasmus, Daniel J. |
| Contributors | Van Vuuren, H. J. J., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 103 p. : ill. |
| Rights | Stellenbosch University |
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