Abstract
The high concentration of sugars in Icewine juice causes formidable stress for the fermenting Saccharomyces cerevisiae, causing cells to lose water and shrink in size. Yeast can combat this stress by increasing the internal concentration of glycerol by activating the high osmolarity glycerol response to synthesize glycerol and by actively transporting glycerol into the cell from the environment. The H+/glycerol symporter, Stl1p, has been previously characterized as being glucose repressed and inactivated, despite osmotic stress induction. To further investigate the role of Stl1p in Icewine fermentations, we developed a rapid single plasmid CRISPR-Cas9-based genome editing method to construct a strain of the common Icewine yeast, S. cerevisiae K1-V1116, that lacks STL1. In an Icewine fermentation, the ∆STL1 strain had reduced fermentation performance, and elevated glycerol and acetic acid production compared to the parent. These results demonstrate that glycerol uptake by Stl1p has a significant role during osmotically challenging Icewine fermentations in K1-V1116 despite potential glucose downregulation.
Highlights
Icewine is a signature sweet dessert wine that is fermented from the juice of naturally frozen grapes
We developed a rapid and effective CRISPR-Cas9 genome editing method for the wine yeast S. cerevisiae strain, K1-V1116, which is commonly used for Icewine production
Our work demonstrates that CRISPR-Cas9 genome editing is an effective tool for studying the role of glycerol uptake by Stl1p in the commercial wine yeast, K1-V1116
Summary
Icewine is a signature sweet dessert wine that is fermented from the juice of naturally frozen grapes. Previous gene expression analysis on the short-term hyperosmotic stress response in wine yeast to grape must inoculation revealed that STL1 was highly expressed between the first 10 and 30 minutes, before rapid downregulation to near undetectable levels [14]. We developed a rapid and effective CRISPR-Cas genome editing method for the wine yeast S. cerevisiae strain, K1-V1116, which is commonly used for Icewine production. The reduced fermentation performance, along with increased glycerol and acetic acid production per gram of sugar consumed, of the ∆STL1 strain, provides further evidence that Stl1p contributes to the osmotolerance of K1-V1116 during Icewine fermentations, despite the high concentration of glucose
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