Masters Degrees (Institute for Wine Biotechnology)

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Browsing Masters Degrees (Institute for Wine Biotechnology) by Author "Domingo, Jody L. (Jody Lawren)"

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    Stationary phase-specific expression of dominant flocculation genes for controlled flocculation of yeast
    (Stellenbosch : Stellenbosch University, 2003-04) Domingo, Jody L. (Jody Lawren); Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology and Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid environment. This aggregation of cells, also referred to as "floc formation", will in most cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast strains, the floes can move to the surface of the liquid growth substrate to form a thin layer, called a "velum", that has been compared to other microbial biofilms. The factors that trigger flocculation can be divided into two groups, physical/chemical (e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that are either directly or indirectly involved in flocculation). In top-fermenting yeast strains, several physical and chemical factors that trigger the process have been described, including ethanol concentration, the presence of organic solvents, the absence of molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors, no specific genes have thus far been associated with flocculation in top fermenting yeast strains. In bottom-fermenting yeast strains, the physical and chemical factors that affect the process are similar to the ones described for top-fermenting yeast strains, but include, more specifically, the concentration of hexoses in the media (mannose or glucose), which may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type (inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various genes have been associated with the flocculation of bottom-fermenting yeast strains. Through genetic analysis, the genes have been categorised into dominant genes, semidominant genes and recessive genes. In order to better understand the role of some of the proteins responsible for flocculation in S. cerevisiae, and to create strains whose flocculation properties would correspond to those wanted in the wine and beer industries, three of the dominant flocculation genes, FL01, FL05 and FL011, were placed under the control of the promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved by replacing the native promoters of the flocculation genes with the heterologous promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent due to the absence of the transcription factor that is required for flocculation, F108p,was used as a model system. Some of the transformed strains showed high flocculation, especially when the genes were placed under control of the ADH2 promoter. In addition to this, the strains carrying a modified FL011 gene showed increased adhesion to solid agar media and were able to invade the growth substrate. These strains also showed an increased velum-forming ability when grown in media containing only non-fermentable carbon sources.