A FLO1 variant which yields a NewFlo phenotype

Flocculation is often utilised as means of separation of yeast cells from the product in alcoholic beverage production. Brewery type strains generally start to flocculate towards the end of the fermentation process, when sugars in the wort are depleted. In Saccharomyces cerevisiae, flocculation is governed by the FLO gene family, with FLO1 generally being the main contributor to strong, Flo1 phenotype, flocculation. S. cerevisiae CCUG 53310, isolated from a spent sulphite liquor plant, has high tolerance to fermentation inhibitors typically present in lignocellulose hydrolysates (Westman et al. 2012). Furthermore, CCUG 53310 flocculates constitutively with a Flo1 phenotype that is only marginally affected by the presence of high concentrations of mannose (see figure: circles).Using primers designed for FLO1, we isolated a flocculin gene from the genome of CCUG 53310. However, constitutive expression of the gene in the otherwise non-flocculating S. cerevisiae CEN.PK 113-7D, resulted in a strain with NewFlo phenotype flocculation, being inhibited by various sugars (see figure: squares, triangles, diamonds and stars). Nonetheless, the protein was phylogenetically closely related to Flo1p and by inverse PCR we could also show that the gene is a paralog of FLO1. Homology modelling of the N-terminal part of the protein structure revealed high structural similarities to the reported structure of the Flo5p N-terminal domain. Closer examination revealed differences in certain positions that have been reported to be important for carbohydrate binding by flocculins. Not previously reported, but of special interest due to its position in a loop flanking the carbohydrate binding site, was a glutamate residue that in the corresponding position in Flo1, 5 and 9p is a glycine. We hypothesise that this glutamate residue contributes to the observed NewFlo phenotype flocculation