South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa.
FEMS Yeast Res. 2024 Jan 9;24. doi: 10.1093/femsyr/foae028.
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 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, S. 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.
酵母细胞壁几丁质已被证明可以结合葡萄致病相关几丁酶，而几丁酶是葡萄酒蛋白浑浊的主要原因，这表明酵母细胞壁可能被用于防止浑浊。在这里，我们提出了一种高通量筛选方法，使用反复富集策略和 GFP 标记的几丁酶或 Calcofluor white 标记的细胞荧光激活细胞分选，来鉴定具有高细胞壁几丁质的酵母菌株。为了评估该策略的有效性，我们首先使用了酿酒酵母的 pooled deletion strain library。该策略富集了先前被描述为对几丁质水平有影响的基因缺失突变体。还鉴定了以前与几丁质生物合成或沉积无关的基因。这些基因参与细胞壁维护和/或膜运输功能。然后将该策略应用于商业葡萄酒酵母菌株 S. cerevisiae EC1118 的诱变群体。富集的突变菌株的细胞壁几丁质明显高于野生型，并且显著降低了合成模型葡萄酒中几丁酶的活性，表明这些菌株可能能够减少葡萄酒中的浑浊形成。
