Over-expression of FLO genes in Saccharomyces cerevisiae BY4742 strains bearing a deletion in genes related to cell wall biogenesis.
Mhlongo, Sizwe Innocent.
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Mannoproteins form the outermost layer of the cell wall in Saccharomyces cerevisiae. These glycoproteins are first synthesized in the endoplasmic reticulum and undergo posttranslational modification before they are transported through the secretory pathway. Some of the glycosylphosphatidyl inositol (GPI) anchored proteins are incorporated into the cell wall where their GPI-anchor is first trimmed off before they are anchored into the β-glucan network in the cell wall. The yeast cells are constantly faced with different environmental conditions and the cell surface mannoproteins are responsible for different morphological transitions that allow the cell to survive harsh conditions. The Flo proteins or adhesins encoded for by the family of FLO genes are known to confer adhesion to biotic and abiotic surfaces, hydrophobicity, biofilm and pseudohyphal filamentation. These phenotypes are suggested to be passive mechanisms employed by the cells to escape from stress or to prevent being washed away. The adhesion properties conferred by the adhesins are important in biotechnological processes. Identification of genes that have the potential to release more adhesins into the culture media will facilitate studies on the fine structural details and functional domains in these glycoproteins. The knowledge will also help in the formulation of fungal drugs since the adhesion of fungal pathogens to host such as humans is known to be the first step of infection. In this study, yeast strains with a deletion in KNR4 or GPI7, which are genes related to the biogenesis of the cell wall were employed to over-express FLO genes. Flocculation intensity and hydrophobicity of cells in the stationary phase were used as a measure of phenotypic changes of the cell surface. The effects of these deletions on the cell surface phenotypes in transgenic strains over-expressing FLO genes were assessed. We found that the KNR4 deletion resulted in a 50% decrease in cell-cell adhesion compared to the wild type. The deletion in GPI7 was found to have no effect in flocculation or the cell initiated a response that resulted in the expression of other genes to compensate for the loss of GPI7. The ability of the yeast cells to invade agar surfaces was not affected by the deletion of GPI7 or KNR4. The observed flocculation intensity was found to correlate with cell surface hydrophobicity. A decrease in the level of flocculation was also accompanied by a decrease in cell surface hydrophobicity. The results of this study indicate that deletion of the KNR4 gene affects the adhesins more than the deletion of the GPI7 gene. A screen of other genes related to cell wall biosynthesis will allow for a selection of genes with the potential to release adhesins to the cell culture medium.