Constitutive over-expression of FL01 encoded mannoproteins.

Abstract

The ability of yeast cell wall-derived mannoproteins (glycoproteins) to positively contribute to oenological properties has been a key factor that stimulates research initiatives into these industrially important glycoproteins. In addition, and from a fundamental research perspective, yeast cell wall glycoproteins are involved in a wide range of biological interactions. To date, and to the best of our knowledge, biochemical insight into the fine molecular structure of these mannoproteins is fairly limited. The amino acid sequences of their protein moieties have been established from structural and functional in silico analysis of the genomic sequence of Saccharomyces cerevisiae whilst far less structural details are available on the glycosyl moieties of these mannoproteins. In an attempt to generate significant quantities of cell wall-attached glycoproteins that may be employed for the purpose of biochemical structural analysis, a novel strategy was envisaged that encompasses the genetic manipulation of S. cerevisiae strains to over-express and release cell wall-associated glycoproteins into the liquid growth medium. To this end, the FLO1-encoded cell wall mannoprotein was overexpressed in haploid S. cerevisiae laboratory strains bearing a specific cell wall biosynthesis-related gene deletion that has been previously shown to promote extracellular hyper-secretion of cell wall-based glycoproteins that would otherwise be attached to the cell wall in the feral strain. A polymerase chain reaction (PCR)-based cloning stratagem was employed to yield transgenic strains in which the native FLO1 open reading frame (ORF) is placed under transcriptional control of the constitutive PGK1 promoter. The data reveals that the strategy employed resulted in the expected constitutive expression pattern of the FLO1 ORF in all transgenic strains created in this study. A modified Helm’s flocculation assay was employed to assess flocculation intensities of the three FLO1-based transgenic strains. This assay confirmed that the flocculation phenotypes observed were indeed of the Flo1-type in that they were Ca²⁺-dependent, sensitive to ethylenediaminetetraacetic acid (EDTA) and inhibited in the presence of mannose. Interestingly, the KNR4 deletion-based transgenic yeast strain displayed a visibly weaker different adhesion behaviour in terms of weaker flocculation intensity as evidenced by noticeably smaller floc aggregate size. In addition a greater quantity of proteins was detected at statistically significant levels in the spent culture medium of the KNR4 deletion-based transgenic yeast strain. Although yet to be purposefully optimised, the data seemingly suggests that PGK1 promoter-based mediated overexpression of the FLO1 ORF in a KNR4 deletion-based genetic background has the potential to release the desired mannoprotein into the culture medium.