To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated betterperforming variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22°Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.Today's need to produce quality beers in a short time and in the least-expensive way has led most breweries to switch to high-gravity brewing. Traditionally, brewing worts of 12°Plato (12°P) (i.e., 12 g extract per 100 g liquid) are fermented to produce beers of 5% (vol/vol) ethanol. However, substantial cost savings can be realized by increasing the wort density (called "gravity") from 12°P to 18°P and diluting the 7.5% (vol/vol) ethanol-containing product in order to obtain beer with regular ethanol content (5%). The use of this technology increases the brewery capacity by 50% (for 18°P wort) without the need for any investments, reduces the costs in energy and labor (because liquid volumes are reduced), improves the stability of the beer, and enhances the recovery of ethanol per unit of fermentable sugar ("extract") (15, 44). However, the technology is limited by the stress resistance and fermentation performance of the brewer's lager yeast Saccharomyces pastorianus. S. cerevisiae (used for ale and wine fermentations) can cope much better with such stress conditions. Wine must typically contains up to 25% (wt/wt) sugar, but a major difference from beer production is that the yeast is only used once in wine fermentation whereas it is used multiple times in beer fermentation. In addition, wine m...
The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an α-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of α-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.
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