Isolation and characterisation of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting.

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines suggesting that yeast cell walls may be applied for haze protection. Here we present a high throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and Fluorescence-Activated Cell Sorting of cells labelled with either GFP-tagged chitinase or with Calcofluor White. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, Saccharomyces cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.