Isolation and Characterization of a Freeze-Tolerant Diploid Derivative of an Industrial Baker's Yeast Strain and Its Use in Frozen Doughs

Self Cite

105

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...

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions

Blieck

Toye

Dumortier

et al. 2007

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105

“…Evolution may then be used to improve substrate utilization or to improve substrate utilization under novel conditions. While simpler traits may be directly selected for (20,27), more complex, multigene modifications require an evolutionary approach for stepwise improvement (24). FIG.…”

Section: Discussionmentioning

confidence: 99%

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Sonderegger

Sauer

2003

229

169

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.Over the last decade, metabolic engineering has become a standard procedure for strain improvement and has been very successful when simple cellular traits have been targeted (19,26). Although the genomics age and the associated genomewide analytical technologies provide further impetus for rational approaches, metabolic engineering of more complex cellular systems or cellular systems that are not fully understood remains a challenge (4). Along with random, combinatorial approaches, such as directed evolution in contemporary protein engineering (3), evolutionary approaches are becoming increasingly important for augmenting metabolic engineering of complex phenotypes (21,22,24,36). In certain cases, however, even seemingly simple metabolic systems resist straightforward rational engineering.

“…The strains used in this study were the laboratory strain BY4743 (MATa/MAT␣ his3⌬1/his3⌬1 leu2⌬0/leu2⌬0 ura3⌬0/ura3⌬0) (3) and the industrial strain AT25 (Ϯ diploid, probably aneuploid) (35). In these strains, pYX012 KanMX (vector), pYX012 KanMX/AQY1-1 (pAQY1-1), and pYX012 KanMX/AQY2-1 (pAQY2-1) have previously been integrated at the TPI locus, resulting in geneticin-resistant strains (33).…”

Section: Methodsmentioning

confidence: 99%

Aquaporin-Mediated Improvement of Freeze Tolerance of Saccharomyces cerevisiae Is Restricted to Rapid Freezing Conditions

Tanghe

Dijck

Colavizza

et al. 2004

Self Cite

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporinoverexpressing strains have less potential for use in frozen doughs than originally thought.Although much correlative evidence is available, the determinants of freeze resistance in Saccharomyces cerevisiae are largely unknown (for a review see reference 34). The injury sustained during freezing and thawing is caused by a combination of multiple types of stress imposed on the cells, such as changes in temperature, water content, water state, pH, and free radical, ion, and solute concentrations. The concomitant loss of survival is not attributable to any one form of injury and depends on the cooling and warming rates, the final temperature, the duration of freezing, and the suspending medium (23,24).When a cell suspension is cooled to a temperature of 0°C or less, both the suspending medium and the cells initially supercool. Extracellular ice crystal formation precedes intracellular freezing and is determined by the freezing point of the suspending medium and the presence of ice-nucleating agents. Based only on osmolarity, the freezing point of the cytoplasm is predicted to be approximately Ϫ1°C. Nevertheless, the cell interior typically remains unfrozen until the temperature is Ϫ10 to Ϫ15°C. This intracellular supercooling may be due to prevention of growth of ice into the cell interior by the cell membrane and due to the lack of nucleators of supercooled water within the cell (23, 24).Following external fr...