Paper Title
Constitutive Flo1 Over Expression in S. Cerevisiae Strains Bearing Deletions in Cell Wall Biogenesis Related Genes

To date, it has been reported that the five dominant FLO genes in S. cerevisiae encode for a family of glycosylphosphatidylinositol (GPI) linked glycoproteins that are commonly referred to as adhesins, flocculins or cell wall mannoproteins. The adhesion phenotypes that are associated with individual FLO genes have been extensively researched and well characterized. However, far less is understood about the cellular metabolic routes that lead to their biochemical synthesis and incorporation into the yeast cell wall. Additionally our fairly limited current understanding of the fine molecular architecture of these mannoproteins predominantly relies on data generated from in silico predictive research studies. In this research study a genetic engineering approach will be employed to constitutively overexpress the FLO1- encoded mannoprotein in the wild type BY4741 haploid laboratory S. cerevisiae strain and in mutant BY4741 strains bearing a disruptive deletion in either their KNR4 or GPI7 cell wall biogenesis related genes. As such the effect of the gene deletions on the intensity of the flocculation phenotype will shed light on the contribution of the deleted gene products in biochemical processing of FLO1 mannoproteins. Additionally it is envisioned that the transgenic yeast strains overexpressing FLO1 mannoproteins will provide a viable alternative for the large scale isolation and purification of the intact mannoprotein especially if it were to be released into the growth medium by FLO1 overexpressing deletion transgenic strains. This glycoprotein reservoir can be utilised in the structural analysis of FLO1 mannoproteins. In relation to the transgenic wild type BY4741-F1P strain decreased flocculation intensity was observed in the BY4741ΔKNR4-F1P strain. BY4741ΔGPI7- F1P displayed a flocculation intensity that was similar to the BY4741-F1P strain. Interestingly, 10% higher protein concentrations were observed in the spent growth medium of the transgenic BY4741ΔKNR4-F1P strain thereby seemingly suggesting the possibility of the FLO1 mannoprotein being released extracellularly. Keywords - Genetic engineering, Glycoprotein, Flocculation, Overexpression