Deletion analysis of the Ure2p in Saccharomyces cerevisiae and effect of NCR on the production of ethyl carbamate during wine fermentations

Erasmus, Daniel J. (2000-12)

Thesis (MSc)--University of Stellenbosch, 2000.

Thesis

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.

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