Analysis of endo-polygalacturonase activity in a recombinant yeast containing a reconstituted PGU1 gene

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dc.contributor.advisor Divol, Benoit
dc.contributor.author Van Wyk, Herine
dc.contributor.other University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
dc.date.accessioned 2009-02-20T09:15:23Z en_ZA
dc.date.accessioned 2010-06-01T08:26:54Z
dc.date.available 2009-02-20T09:15:23Z en_ZA
dc.date.available 2010-06-01T08:26:54Z
dc.date.issued 2009-03
dc.identifier.uri http://hdl.handle.net/10019.1/1538
dc.description Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009.
dc.description.abstract The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes. The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol. The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types. This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene. en
dc.language.iso en
dc.publisher Stellenbosch : University of Stellenbosch
dc.subject PGU1 en
dc.subject Endo-polygalacturonase en
dc.subject Dissertations -- Wine biotechnology en
dc.subject Theses -- Wine biotechnology en
dc.subject.lcsh Wine and wine making en
dc.subject.lcsh Fermentation en_ZA
dc.subject.lcsh Yeast -- Genetics en
dc.subject.lcsh Wine -- Flavor and odor en
dc.subject.other Viticulture and Oenology en_ZA
dc.title Analysis of endo-polygalacturonase activity in a recombinant yeast containing a reconstituted PGU1 gene en
dc.type Thesis
dc.rights.holder University of Stellenbosch


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