Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

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

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.     

Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.     

Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo)

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.     

Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs.

From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained. To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies. By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them. Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast. Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained. These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.     

Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications.

The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The delta ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the delta ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the delta ath1 strain may make this strain useful in commercial applications, including baking and brewing.     

Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。     

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions

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 aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.     

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

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 aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.     

Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells.

The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance. Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight. Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose. Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation. We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation. This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions. Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs. Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.     

Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26

Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.     

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.     

Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu

In our previous studies, it was found that Saccharomycopsis fibuligera sdu cells could accumulate 18.0% (gg(-1)) trehalose from soluble starch in SSY medium. However, the yeast strain contained high activities of acid and neutral trehalases, which were reported to mobilize trehalose accumulated by the cells during fermentation. In order to enhance the yield of trehalose, it is necessary to remove the trehalase activities from the cells. By mutagenesis of ethylmethanesulfonate, one mutant that assimilated trehalose very slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In Biostat B2 2-1 fermentation, trehalose accumulation of the mutant was much higher than that of the wild type when grown in YPS medium containing starch. The activities of acid and neutral trehalases of this mutant were much lower than those of the wild type, respectively. We think the reduction of acid and neutral trehalase activities is considered to be responsible for the increased yield of trehalose accumulated by the mutant.     

Aquaporin expression correlates with freeze tolerance in baker's yeast,and overexpression improves freeze tolerance in industrial strains

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.