No abstract

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7⌬ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.During wine alcoholic fermentation, yeasts convert most of the glucose and fructose present into alcohol and CO 2 . Grape musts contain equal amounts of glucose and fructose, and the total hexose concentrations typically range from 160 to 300 g/liter. Saccharomyces cerevisiae is the preferred species of yeast for winemaking, and selected strains of S. cerevisiae are used as starters for inoculation of grape musts and for alcoholic fermentation. S. cerevisiae is a glucophilic yeast, preferring glucose to fructose. During fermentation, glucose at a higher rate than fructose, and the proportion of fructose therefore increases as fermentation progresses. Consequently, fructose becomes the main sugar present during the late stages of alcoholic fermentation, and wine yeasts have to ferment this nonpreferred sugar after long periods of starvation and in the presence of large amounts of ethanol. The stress associated with these conditions may be amplified by nutritional imbalances which may alter yeast activity, resulting in sluggish or stuck fermentations (1, 4, 5). In such situations, the low fructose utilization capacity of S. cerevisiae is thought to contribute to the low fermentation rate (9,26,27). The ability of wine yeasts to ferment fructose is therefore critically important for the maintenance of a high rate of fermentation at the end of the process and for fermentation of the must to dryness.The reasons for the difference between the glucose fermentation rate and the fructose fermentation rate are unclear, but one of the first steps in hexose metabolism is generally thought to be involved. Sugar transport across the plasma membrane is the primary step in hexose metabolism. Another potential source of the difference is hexose phosphorylation, as glucose and f...