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Applied and Environmental Microbiology, August 2007, p. 4824-4831, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.02651-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Overexpression of the Calcineurin Target CRZ1 Provides Freeze Tolerance and Enhances the Fermentative Capacity of Baker's Yeast

Joaquín Panadero, Maria José Hernández-López, José Antonio Prieto, and Francisca Randez-Gil^*

Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), P.O. Box 73, E-46100-Burjassot, Valencia, Spain

Received 13 November 2006/ Accepted 26 May 2007

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Recent years have shown a huge growth in the market of industrial baker's yeasts (Saccharomyces cerevisiae), with the need for strains affording better performance in prefrozen dough. Evidence suggests that during the freezing process, cells can suffer biochemical damage caused by osmotic stress. Nevertheless, the involvement of ion-responsive transcriptional factors and pathways in conferring freeze resistance has not yet been examined. Here, we have investigated the role of the salt-responsive calcineurin-Crz1p pathway in mediating tolerance to freezing by industrial baker's yeast. Overexpression of CRZ1 in the industrial HS13 strain increased both salt and freeze tolerance and improved the leavening ability of baker's yeast in high-sugar dough. Moreover, engineered cells were able to produce more gas during fermentation of prefrozen dough than the parental strain. Similar effects were observed for overexpression of TdCRZ1, the homologue to CRZ1 in Torulaspora delbrueckii, suggesting that expression of calcineurin-Crz1p target genes can alleviate the harmful effects of ionic stress during freezing. However, overexpression of STZ and FTZ, two unrelated Arabidopsis thaliana genes encoding Cys₂/His₂-type zinc finger proteins, also conferred freeze resistance in yeast. Furthermore, experiments with cnb1 and crz1 mutants failed to show a freeze-sensitive phenotype, even in cells pretreated with NaCl. Overall, our results demonstrate that overexpression of CRZ1 has the potential to be a useful tool for increasing freeze tolerance and fermentative capacity in industrial strains. However, these effects do not appear to be mediated through activation of known salt-responding pathways.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability of a commercial baker's yeast (Saccharomyces cerevisiae) strain to withstand environmental stress conditions is an industrially relevant trait (1, 30). Resistance to freezing is particularly important due to the growing demand for prefrozen dough products. Nowadays, no suitable industrial baker's yeast is available with this property, and it is unlikely that classical breeding programs could provide significant improvements of this characteristic (31). Our limited knowledge of physiological and genetic determinants of freeze tolerance suggests that this trait is controlled by multiple genes and complex regulatory mechanisms. Moreover, freezing is a multifaceted stress in which different stressors and stress responses appear to play important roles (31).

At subzero temperatures, the deleterious effects on yeast cells depend on the cooling rate during the freezing process. At high cooling rates, intracellular ice is formed, leading to macromolecules and membrane denaturation (25). Ultrastructural examination of these cells shows discontinuous nuclear membranes, disappearance of vacuoles, and DNA spreads all over the cells (15). When the cooling rate is low, osmotic shrinkage of the cell and frozen extracellular water are observed. In this case, the cells become exposed to toxic levels of solutes and equilibrate by water movement across the membranes (47). Thus, ion tolerance might be an important factor influencing freezing injury in yeast. However, the existence of such a relationship is unclear and the involvement of ion-responsive transcriptional factors and pathways in conferring freeze resistance has not yet been examined.

Up-regulation of salt-responsive genes appears to be mainly dependent on two well-characterized molecular signaling pathways, the HOG (high-osmolarity glycerol) pathway (46), one of the five mitogen-activated protein kinase pathways known to occur in S. cerevisiae (7, 13, 38), and the calcineurin/Crz1p pathway (3, 33). While signaling through the HOG cascade is triggered by both osmotic and saline stress, the calcineurin/Crz1p pathway appears to be specifically implicated in the salt response. Calcineurin, a highly conserved Ca²⁺-calmodulin-dependent type 2B Ser/Thr protein phosphatase identified in a huge range of eukaryotic organisms, from yeast to mammalian (5, 9), is essential for salt tolerance, since mutants lacking the catalytic subunits Cna1p and Cna2p or the regulatory subunit encoded by CNB1 (4) are sensitive to Na⁺ and Li⁺ (23, 26). However, constitutively activated calcineurin confers NaCl and LiCl resistance (24). Calcineurin mediates the transcriptional response via the Crz1p transcription factor, which is translocated to the nucleus upon dephosphorylation by calcineurin (20, 42). Crz1p binds specifically to the calcineurin-dependent response element (CDRE) consensus sequence, located in the promoters of most salt-responsive genes (22, 42, 48). Activation of the calcineurin/Crz1p pathway induces the expression of ENA1 (19), the specific P-type ATPase that mediates the active efflux of Na⁺ (8), and PMC1 and PMR1, which encode Ca²⁺ ATPases (20), and activates the high-affinity state of the K⁺ uptake system (23).

Homologues to S. cerevisiae Crz1p have been identified in Schizosaccharomyces pombe (12), Candida albicans (27), and the salt-tolerant yeast Torulaspora delbrueckii (11). Overproduction of T. delbrueckii TdCrz1p enhanced the salt tolerance of S. cerevisiae wild-type cells and suppressed the sensitivity phenotypes of cnb1 and crz1 mutants to monovalent and divalent cations (11). Similarly, two cDNA clones from Arabidopsis thaliana, referred to as STO and STZ, have been isolated for their abilities to complement the salt sensitivity phenotypes of yeast calcineurin mutants (18). STZ encodes a member of the Cys₂/His₂-type zinc finger protein family (34, 43, 44), which is believed to function as a transcription repressor (36). Expression of STZ is induced under drought, cold, and high-salinity conditions (16, 35, 36), and its overexpression confers stress tolerance (36).

In this work, we hypothesized that ionic injury is an important factor in determining yeast cell survival upon freezing/thawing and analyzed whether engineering of ion tolerance may provide a successful approach for increasing baker's yeast performance in prefrozen dough. To elucidate this, we examined the functional role of the calcineurin-Crz1p pathway in the freeze tolerance of wild-type cells of S. cerevisiae and the effects of overexpression of CRZ1 and CRZ1-like genes in the response of industrial baker's yeast to freezing stress. Additionally, we analyzed whether these effects could be mediated by enhanced expression of CDRE-regulated target genes. Overall, our data indicate that increased dosage of CRZ1 provides freeze tolerance in baker's yeast. However, it is unclear whether the calcineurin-Crz1p pathway plays a role in the yeast freeze protective mechanisms.

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Strains, culture media, and general methods.
The industrial and laboratory S. cerevisiae strains used throughout this work are described in Table 1. Escherichia coli strain DH10B was used as the host for plasmid construction. Yeast cells were cultured at 30°C in YPD (1% yeast extract, 2% peptone, 2% glucose) or SD [0.2% yeast nitrogen base without amino acids (DIFCO, BD Diagnostics, Sparks, MD), 0.5% (NH₄)₂SO₄, 2% glucose] supplemented with the appropriate auxotrophic requirements (40). Yeast biomass for technological experiments was prepared by cultivating cells (2.7 mg cells [dry weight]) on solid SD or solid molasses (0.5% beet molasses [49% sucrose], 0.05% ammonium phosphate, 2.6% agar, and 20 µg of biotin per liter, adjusted to a final pH of 5.0) plates (140-mm diameter) for 20 h at 30°C. E. coli was grown in Luria-Bertani (LB) medium (1% peptone, 0.5% yeast extract, 0.5% NaCl) supplemented with ampicillin (50 mg/liter). Yeasts were transformed by the lithium acetate method (14). E. coli was transformed by using a model 2510 Eppendorf electroporator (Eppendorf AG, Hamburg, Germany).

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TABLE 1. S. cerevisiae strains used in this study

Freeze tolerance assays.
SD plate-grown cells of the industrial HS13 strain were recovered by washing them out with 20 ml of distilled water, and the resulting yeast suspension was poured into a tube. After centrifugation (3,000 x g, 2 min, 4°C), the yeast cake was washed twice with distilled water (4°C), resuspended in distilled water, and vortexed and the optical density at 600 nm (OD₆₀₀) measured. The final yeast concentration was adjusted to 30 mg (dry weight) per ml (an OD₆₀₀ of 1 equals 0.35 mg cells [dry weight]/ml). Five milliliters of the yeast suspension was mixed with the same volume of liquid dough (LD), and 100-µl aliquots were shifted immediately to –20°C. At various times, samples were thawed at 30°C for 10 min and diluted and cells plated onto solid YPD. After 2 days at 30°C, colonies were counted. Viability is expressed as logarithm numbers of CFU per ml. The LD model system was prepared as previously reported (28).

For freeze tolerance experiments with the laboratory strain BY4741 and its derivatives, YPD-grown cells were diluted (OD₆₀₀ = 0.3) in the same medium or in YPD containing 0.8 M NaCl, incubated for 1 h at 30°C, harvested by centrifugation (3,000 x g, 2 min, 4°C), and resuspended in YPD (final OD₆₀₀ = 10.0) and then 100-µl aliquots were shifted to –20°C. After frozen storage for different periods, cell samples were thawed at 30°C for 10 min and diluted and cells plated onto standard YPD-agar plates for analysis of cell viability.

Dough preparation and CO₂ production measurements.
A commercial wheat bread flour (moisture, 14%; W, 113.5 x 10^–4 J [Alveograph]; P/L, 0.26 [Alveograph]) was used throughout this work. Bread dough was prepared using the following (with the amount of flour as the basis for the percentages): flour (100%), yeast (3.75% [dry weight]), salt (2%), and tap water (50.5%). Molasses plate-grown yeast samples were prepared as described above. Sweet dough also contained 20, 25, or 30% sucrose (flour basis). Ingredients were mixed for 13 min in a Chopin Alveograph mixer (Chopin Technologies, France), and the resulting dough was divided into 35-g pieces. One piece was analyzed immediately for gassing power (unfrozen control). The rest of the pieces were molded by hand to 0.5-cm thicknesses, placed in aluminum salves, and wrapped in plastic bags. Dough was quickly frozen at –80°C for 1 h and stored at –20°C for different times. After each storage stage, the frozen dough was left to thaw at 30°C for 30 min before gassing power was measured.

CO₂ production was measured at 20-min intervals by using a homemade fermentometer (Chittick apparatus) as described previously (28). Values are expressed as ml of CO₂ per mg of yeast cells and were normalized to the initial dry weight of the yeast sample tested.

Plasmids.
Plasmid pAMS435 (42), containing the S. cerevisiae CRZ1 gene in the LEU2-based plasmid pRS315 (41), was a gift from M. Cyert. Plasmid YEpCRZ1 was created by inserting a BamHI/SalI fragment from pAMS435 into the respective sites of YEplac195 (URA3). Plasmid YEpTdCRZ1 (URA3), which allows high-copy-number expression of the CRZ1 gene from T. delbrueckii (TdCRZ1), was constructed in a previous work (11). The Arabidopsis thaliana genes designated STZ (18) (GenBank accession no. AY034998) and FTZ (this study) (GenBank accession no. AY054225) were amplified by PCR using the primers AtSTZ-1 (5'-AGAAATCCTCTAGAATCTTT-3' [XbaI site underlined]) and AtSTZ-2 (5'-TTACGCCAAGATCGATATTA-3' [ClaI site underlined]) and AtFTZ-1 (5'-CAAGTAAG[TCTAGA]TCACGC-3') and AtFTZ-2 (5'-CCTACTATTGTAG[ATCGAT]ATTT-3'), respectively, and plasmids pda00509 and pda05670 (RIKEN Genomic Sciences Center, GSC, Yokohama, Japan), which contain the corresponding full-length cDNA clones (37, 39). The amplified sequences were subcloned into the pGEM-T Easy vector (Promega, Madison, WI) and treated sequentially with XbaI, the Klenow fragment (to obtain a blunt end), and ClaI. The released DNA fragments were accommodated into the plasmid pBS-P_ACTGS5T_PGK (29), previously digested with the same set of enzymes, yielding the plasmids pBS-P_ACTSTZT_PGK and pBS-P_ACTFTZT_PGK, which contain the STZ and FTZ sequences flanked by the S. cerevisiae ACT1 promoter P_ACT (positions –497 to +11) and the PGK1 terminator T_PGK (positions +1064 to +1544). Finally, the STZ and FTZ expression cassettes were accommodated into the vector YEplac195 or YCplac33 (6) by digestion with BamHI and SalI, yielding the plasmids YEpSTZ and YEpFTZ or YCpSTZ and YCpFTZ, respectively.

Plasmids pEMBL-STZ and pEMBL-FTZ, which contain the STZ and FTZ sequences under the regulation of the S. cerevisiae GAL10-CYC1 hybrid promoter (2), were constructed by digesting the plasmids YEpSTZ and YEpFTZ with XbaI and HindIII and cloning them into the pEMBLyex4 vector (2), previously treated with the same set of enzymes.

ß-Galactosidase assay.
Exponentially SD-growing cells (OD₆₀₀ = 0.4) were collected, washed twice with distilled water, resuspended in YP medium containing 2% galactose as the sole carbon source, and incubated at 30°C and 200 rpm. After 4 h, NaCl was added (0.8 M final concentration) and the cell suspension was again incubated for 45 min. Then, cells were recovered by centrifugation and the ß-galactosidase activity was determined as described previously (11).

Statistical analyses.
Data were subjected to a single analysis of variance, using the Statgraphics Plus v. 2 software suite (STSC, Inc., Rockville, MD). Differences were considered significant when the P value was <0.05.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Overexpression of CRZ1 confers freeze tolerance to industrial baker's yeast cells.
We tested whether the overexpression of CRZ1 might be a useful approach for improving freeze tolerance in industrial strains of S. cerevisiae. To address this, cells of the HS13 (ura3) baker's yeast strain were transformed with the plasmid YEpCRZ1, which affords high-copy-number expression of CRZ1, and transformants were tested under salt stress conditions (Fig. 1A). YEpTdCRZ1 (11) transformants, carrying the T. delbrueckii CRZ1 gene, the homologue to the transcriptional factor Crz1p, were also included in the analysis. As shown in Fig. 1A, overexpression of either CRZ1 or TdCRZ1 conferred enhanced salt tolerance, indicating that the plasmids were functional in the industrial host strain. Next, we tested the behavior of the recombinant baker's yeast strains under freezing. For this, SD-grown cells were transferred to LD medium and analyzed for cell viability after freezing and frozen storage at –20°C. The LD model system mimics the nutritional status and stress conditions encountered by baker's yeast in real dough, and isolated cells could easily be collected (28). As can be seen in Fig. 1B, overexpression of either CRZ1 or TdCRZ1 increased the freezing resistance of the industrial strain. Thus, after 12 days of frozen storage, Crz1p- and TdCrz1p-overproducing cells survived at rates about 10-fold higher than those for control cells.

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FIG. 1. Overexpression of CRZ1 or TdCRZ1 is functional in baker's yeast and confers increased freeze tolerance. (A) Cells of the S. cerevisiae industrial strain HS13 (wild type; ura3) were transformed with the plasmid YEplac195 (control; empty plasmid), YEpCRZ1, or YEpTdCRZ1, the latter two of which contain the S. cerevisiae or T. delbrueckii gene CRZ1 or TdCRZ1, respectively, and assayed for NaCl resistance. Cells were grown in SD liquid medium at 30°C until early exponential phase, and the cultures were adjusted to an OD600 of 0.3. Then, serial dilutions (1:10–4) of the cell suspensions were spotted (3 µl) onto standard YPD-agar plates lacking or containing NaCl at the indicated concentration. Plates were inspected after 2 to 5 days at 30°C. (B) Cells of the wild-type (white circles), CRZ1-overexpressing (black circles), and TdCRZ1-overexpressing (gray squares) strains were grown in SD plates at 30°C, harvested, and resuspended in an LD model system (7.5 mg cells [dry weight]/ml). Then, 100-µl aliquots of the cell suspensions were transferred to –20°C. At various times, samples were thawed at 30°C for 10 min and diluted and cells plated onto solid YPD. After 2 days at 30°C, colonies were counted. Viability is expressed as logarithm numbers of CFU per ml. Unfrozen samples were used as a control (0 days). Values represent the means for at least three independent experiments. The errors associated with the points were calculated by using the formula , where n is the number of measurements. P values were 0.0023 and 0.0009 for log CFU/ml of the wild-type strain compared to log CFU/ml of the TdCRZ1- and CRZ1-overexpressing strains after 12 days at –20°C.

Fermentative capacity.
We tested whether overexpression of CRZ1 or TdCRZ1 determines a change in the gassing abilities of baker's yeast cells in unfrozen bread dough. Both unsugared (lean) and sweet-dough preparations were tested. As shown in Fig. 2A, no effects on CO₂ production could be detected in lean dough. However, high-copy-number expression of TdCRZ1, and especially of CRZ1, increased the gassing power of osmotically stressed baker's yeast cells in sweet dough, the effect increasing in line with sucrose concentration. A common experiment for testing the total production of CO₂ by YEpTdCRZ1 or YEpCRZ1 transformants in 30% sucrose-containing samples gave production values 29% and 77% higher than those found in the control strain (Fig. 2A).

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FIG. 2. CRZ1-overexpressing cells display increased leavening activities in both unfrozen and frozen high-sugar dough. (A) Molasses plate-grown cells of the HS13 baker's yeast strain transformed with the plasmids YEplac195 (white bars), YEpCRZ1 (black bars), and YEpT dCRZ1 (gray bars) were used to prepare lean (unsugared)- and sweet-dough samples containing 20, 25, or 30% sucrose (flour basis) as described in Materials and Methods. Thirty-five-gram pieces were incubated at 30°C with low shaking (80 rpm), and the amounts of CO2 evolved were measured by using a homemade fermentometer (Chittick apparatus). Values are expressed as ml of CO2 produced after 180 min of dough fermentation and represent the means for at least three independent experiments. The errors were calculated as described in the legend to Fig. 1. *, P values of <0.05 for gas production of the mutant strain compared to gas production of the wild-type strain at the same sucrose concentration. (B) Sucrose (30%)-containing dough samples prepared with the above-mentioned transformants, YEplac195 (white circles), YEpCRZ1 (black circles), and YEpTdCRZ1 (gray squares), were quickly frozen at –80°C for 1 h and stored at –20°C for 2 weeks. Then, the frozen dough was left to thaw at 30°C for 30 min and CO2 production was recorded at 20-min intervals as described above. Results for a representative experiment are shown. In all cases (A, B), values for CO2 were normalized to the initial dry weight (dw) of the yeast sample tested.

As expected, the effects of both gene products on gassing power were more evident in prefrozen dough and in particular in sweet dough, where exposure of yeast cells to salt, sucrose, and freezing strongly compromises cell viability and fermentative performance. For instance, CO₂ production attained by overexpression of TdCRZ1 in 14-day-prefrozen sweet (30% sucrose) dough fermented for 180 min (0.064 ± 0.003 ml CO₂/mg yeast [dry weight]) was about 80% higher than that observed with the wild-type strain (0.035 ± 0.002 ml CO₂/mg yeast [dry weight]) (P = 0.0001). Compared with this, the improvement hardly reached 18% in prefrozen lean dough (data not shown). Again, the effects were more pronounced in cells overexpressing the S. cerevisiae gene (0.176 ± 0.005 ml CO₂/mg yeast [dry weight]; P = 0.00001). As the gas production profile clearly illustrates (Fig. 2B), both gassing rate and total CO₂ were remarkably higher in samples prepared with the CRZ1-overexpressing strain.

Effect of the expression of STZ and FTZ in baker's yeast.
We decided to investigate whether enhanced freeze tolerance in baker's yeast might be attained by overexpression of other zinc finger proteins. In particular, we tested two A. thaliana proteins, which, like Crz1p, include Cys₂/His₂-type zinc fingers, STZ, and a novel protein that we named FTZ (freezing tolerance zinc finger). STZ has previously been characterized for its ability to rescue the salt-sensitive phenotypes of yeast calcineurin mutants (18), whereas FTZ was identified by screening the TIGR A. thaliana database (available at http://www.tigr.org), using a C-terminal partial amino acid sequence of the Crz1 protein that contains the conserved zinc finger C₂H₂-type domains. The full-length FTZ cDNA encodes a predicted protein of 324 amino acids with six C₂H₂-type zinc finger domains (positions 8 to 30, 37 to 60, 129 to 151, 161 to 184, 219 to 242, and 250 to 274). However, sequence similarity between Crz1p and FTZ (51%) is, as expected, entirely restricted to the two C-terminal C₂H₂-type domains (positions 193 to 279).

High-level expression of STZ and FTZ was achieved by placing each gene under the control of the galactose-inducible GAL10-CYC1 promoter. Strains carrying these expression plasmids grew well on medium containing glucose which represses expression from the GAL10-CYC1 promoter (Fig. 3A, top). However, production of either of the plant proteins reduced the growth capacities of the recombinant strains on galactose (Fig. 3A, bottom), suggesting that overexpression of STZ and FTZ is somewhat toxic. The functionalities of the expression cassettes were further assayed by using a 4x-CDRE::lacZ reporter, which contains four tandem copies of the 24-base-pair CDRE (42). As can be seen in Table 2, galactose-induced production of STZ or FTZ in cells of the S. cerevisiae YPH499 wild-type strain increased the expression of the lacZ reporter gene in the presence of salt. Nevertheless, STZ- and FTZ-dependent activation of the CDRE-driven expression required a functional calcineurin-Crz1p pathway. Thus, overproduction of STZ or FTZ in a crz1-null background increased only 2-fold the ß-galactosidase activity in response to Na⁺, while in the wild-type strain, induction was about 37-fold or 60-fold, respectively (Table 2). Moreover, no differential ß-galactosidase activity could be detected in the absence of Cnb1p (Table 2). We also observed that transcription of the lacZ reporter under noninducing conditions (control) was unregulated in the wild-type strain overexpressing the homologous CRZ1 gene, with 480.5 units/mg protein (Table 2). However, in pEMBL-STZ and pEMBL-FTZ transformants, values for ß-galactosidase activity remain low, at 17.0 and 5.5 units/mg protein, respectively. Hence, STZ and FTZ are able to activate the CDRE-driven expression in S. cerevisiae under inducing conditions. However, our results suggest that production of the two plant proteins does not replace the function of the calcineurin-Crz1p pathway in regulating the expression of CDRE-dependent target genes.

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FIG. 3. Enhanced freeze tolerance in baker's yeast can be attained by overexpression of Crz1p-unrelated C2H2-type zinc finger proteins. (A) Cells of the wild-type baker's yeast HS13 were transformed with the plasmids pEMBLyex4 (pEMBL; control), pEMBL-STZ (-STZ), and pEMBL-FTZ (-FTZ), the latter two of which contain the A. thaliana STZ and FTZ genes under the control of the S. cerevisiae GAL10-CYC1 hybrid promoter, respectively, and tested for growth on solid SD medium containing glucose or galactose as the sole carbon source. Plates were incubated at 30°C for 2 days. (B) SD plate-grown cells of the pEMBLyex4 (control; circles), pEMBL-STZ (triangles), and pEMBL-FTZ (squares) transformants were harvested, resuspended in SD liquid medium containing galactose as the carbon source (7.5 mg cells [dry weight]/ml), and incubated at 30°C for 4 h. Then, 100-µl aliquots of the cell suspensions were transferred to –20°C, and at different times, cell viability was determined as described in the legend to Fig. 1. Values represent the means for at least three independent experiments. The errors associated with the points were calculated by using the formula , where n is the number of measurements. P values were 0.0034 and 0.0038 for log CFU/ml of the wild-type strain compared to log CFU/ml of the STZ- and FTZ-overexpressing strains after 12 days at –20°C.

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TABLE 2. Overexpression of STZ and FTZ allows the calcineurin-dependent induction of a 4x-CDRE-lacZ gene fusion in response to Na+

Next, we analyzed the effect of STZ and FTZ expression on freeze tolerance of the industrial strain HS13. Glucose-grown cells were shifted to SD medium containing galactose as the sole carbon source, incubated at 30°C for 4 h, and then transferred to –20°C and analyzed for cell viability during different periods of frozen storage. Again, it was evident that wild-type cells harboring a plasmid that carried either STZ or FTZ were more resistant to freezing than cells of the same strain harboring a control plasmid (Fig. 3B).

A functional calcineurin-Crz1p pathway is not essential for freeze resistance.
We were interested in determining whether the calcineurin-Crz1p pathway plays a role in the ability of yeast cells to cope with freeze stress. YPD cultures of the BY4741 wild-type and cnb1 and crz1 mutant strains (Table 1) were transferred from 30°C to –20°C, thawed after 1, 4, 7, or 14 days, and analyzed for cell viability. As shown in Fig. 4 (top graph, –NaCl), cnb1 and crz1 mutant cells of S. cerevisiae were as sensitive as wild-type cells to freezing/thawing and frozen storage. For instance, the P values were 0.2402 and 0.2016 for log CFU/ml of the wild-type strain compared to log CFU/ml of the mutant strains cnb1 and crz1, respectively, after 14 days of frozen storage. Next, we investigated the freeze protection conferred by pretreatment of cells with salt. YPD-grown cells were exposed to 0.8 M NaCl at 30°C for 1 h and then transferred to –20°C (Fig. 4, bottom graph, +NaCl). As can be seen, exposure of yeast cells to salt stress afforded higher freeze resistance. For instance, 7.8 x 10⁵ cells/ml of the NaCl-treated wild-type strain survived 14 days of freezing, compared with 1.4 x 10⁵ cells/ml in the untreated sample (P = 0.0365). Differences were found to be even larger when NaCl-treated and untreated samples of the cnb1 and crz1 mutant strains were compared, with 1.5 x 10⁶ and 1.8 x 10⁶ cells/ml versus 0.9 x 10⁵ (P = 0.0097) and 1.1 x 10⁵ (P = 0.0006) cells/ml, respectively. However, no significant differences were found in the degrees of sensitivity to freezing when NaCl-treated wild-type cells were compared with cnb1 (P = 0.4383 at day 14 of freezing) and crz1 (P = 0.2990 at day 14 of freezing) cells. Hence, the salt-sensitive calcineurin-Crz1p pathway appears to play no role in protecting yeast cells against freeze damage.

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FIG. 4. The calcineurin-Crz1p pathway plays no apparent role in providing freeze tolerance. Exponentially growing cells of the S. cerevisiae BY4741 wild-type (circles), cnb1 mutant (squares), and crz1 mutant (triangles) strains were examined for freeze tolerance. YPD-grown cells were diluted (OD600 = 0.3) into YPD (–NaCl graph) or YPD containing 0.8 M salt (+NaCl graph), incubated for 1 h at 30°C (pretreatment), harvested by centrifugation (3,000 x g, 2 min, 4°C), and resuspended in YPD (final OD600 = 10.0), and then 100-µl aliquots were shifted to –20°C. At the indicated times, cell samples were thawed at 30°C for 10 min, and cell viability was determined as described in the legend to Fig. 1. Values represent the means for at least three independent experiments. The P values for the F test were greater than or equal to 0.05 for log CFU/ml of the wild-type strain compared to log CFU/ml of the mutant strains at all time points (0, 1, 4, 7, and 14 days) and treatments (–NaCl, +NaCl) analyzed. Differences in viability were found to be significant only when untreated and NaCl-treated samples were compared (see main text).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Overexpression of CRZ1 and CRZ1-like genes has enabled us to examine the functional role of these C₂H₂-type zinc finger proteins in the stress response of baker's yeast cells. As demonstrated, transformants of the industrial HS13 strain in which the CRZ1 gene from S. cerevisiae or T. delbrueckii had been introduced in high copy numbers exhibited higher salt tolerance. Sodium ions are particularly toxic to S. cerevisiae cells, and regulation of their intracellular content by the calcineurin-Crz1p pathway is essential for survival (3). More importantly, enhanced Na⁺ tolerance increased the metabolic activities of baker's yeast cells in sweet dough. These bakery products often contain salt (around 2% [flour basis]) combined with up to 30 to 40% added sucrose or glucose-fructose syrup as a sweetener, which strongly reduces the water activity of the dough (1, 30, 31). Consequently, the effective concentrations of Na⁺ in these products increase, leading to harmful effects on baking performance that are not observed in lean dough (10). Na⁺ resistance should therefore be considered one of the traits that a commercial baker's yeast strain should exhibit, and the improvement of this trait is an important biotechnological challenge within this field (10, 31). Therefore, from a technological viewpoint, the finding that coordinated expression of Na⁺ detoxification systems improves the leavening ability of baker's yeast cells in high-sugar dough is of relevance.

Engineered strains overexpressing either CRZ1 or TdCRZ1 also exhibited higher freeze resistance and were able to produce more gas during fermentation of prefrozen dough than the parental strain. An obvious explanation for the behavior of this phenotype is that a downward shift in temperature below 0°C exposes yeast cells to ionic stress. Indeed, ionic imbalance caused by ice crystal formation is an important factor determining freeze injury in all living organisms (47). Thus, enhanced expression of calcineurin-Crz1p-controlled target genes would allow yeast cells to alleviate the harmful effects of ionic stress during freezing. We tried to confirm this idea using different approaches. First, we analyzed freeze tolerance in yeast cells overexpressing STZ and FTZ, two A. thaliana genes encoding Cys₂/His₂-type zinc finger proteins. STZ had been identified in a wide screening of Arabidopsis cDNA clones that rescue the Na⁺ and Li⁺ sensitivity phenotypes of yeast calcineurin mutants (18). Nevertheless, neither STZ nor FTZ is related to Crz1p, and these proteins share only nucleic acid-binding protein structures. Indeed, it was suggested that this gene product was involved in regulating ion adaptation by calcineurin-independent mechanisms, since STZ expression was unable to compensate for a subset of calcineurin deficiency-associated phenotypes (18). As we showed, overexpression of either STZ or FTZ activated the CDRE-mediated expression of a lacZ reporter. The CDRE (42), a consensus DNA sequence (21) found in the promoters of most salt-responsive genes (48), has been proposed to function by targeting the transcriptional activator Crz1p. However, neither of these two proteins was able to fully compensate for the absence of calcineurin or Crz1p in the ability to activate CDRE-mediated transcription. Moreover, increased expression of Crz1p-regulated targets by expression of STZ or FTZ was evident only after exposure of cells to salt stress, while salt-untreated transformants showed a clear improvement in freeze tolerance. Hence, our results suggested that the activity of the calcineurin-Crz1p pathway might not be relevant in conferring freeze resistance in yeast. Furthermore, the effects of STZ and FTZ and, by extension, of Crz1p on freeze tolerance in yeast may not be a consequence of increased ion resistance.

This was further supported by experiments with a cnb1 mutant, which failed to show a freeze-sensitive phenotype, even though lack of the protein phosphatase has dramatic effects on salt sensitivity in S. cerevisiae (23, 26). Thus, a downward shift in temperature below 0°C does not appear to activate a protective response mediated by calcineurin. Nevertheless, we hypothesized that Crz1p may act to directly regulate a subset of calcineurin-independent, salt-responsive genes required for freeze protection. However, no freeze-associated phenotype could be detected by the absence of the transcription factor. Moreover, we found that pretreatment of wild-type cells with NaCl increased cell survival during freezing and frozen storage, which was to be expected. Indeed, the existence of cross-protection mechanisms in S. cerevisiae is well documented (18). However, absence of Crz1p or Cnb1p led to approximately the same loss of viability after freezing in NaCl-treated cells. Hence, the effects of high dosage of CRZ1 on freeze tolerance do not appear to be mediated through activation of calcineurin-Crz1p-dependent salt protective mechanisms.

Alternatively, Crz1p might be involved in the transcriptional activations of unknown essential genes in determining cell survival upon freezing. However, the observation of similar effects caused by expression of unrelated plant Cys₂-His₂-type zinc finger proteins suggests that these nuclear factors play an unspecific role in regulating freeze tolerance. Overexpression of DNA-binding proteins may affect the activity of the transcriptional machinery, leading to increased expression levels of a great many yeast genes. This altered expression could affect broad traits, like robustness, which appear to be controlled by hundreds of genes and positively influence stress resistance (1, 30, 31). However, more work is needed to confirm this idea and establish the functional connections, should they exist, between ion and freeze tolerance.

Enhanced freeze tolerance has previously been engineered in industrial baker's yeast by overexpression of genes coding for aquaporins (45), antifreezing peptides (29), or oleate ¹² desaturases (32). In our work, overexpression of CRZ1 not only provided a protective role upon freezing and frozen storage but also increased the gas production capacities of yeast cells in high-sugar dough. Presently, the so-called sweet goods represent 25 to 30% of the sales of baked products worldwide, and demand is expected to grow in the near future, especially for those products derived from modern frozen-dough technology.

 
We thank A. Blasco for technical assistance and J. Salinas for providing us with the plant cDNA clones and for helpful discussions. We also thank C. Collar for her contribution in the statistical analysis and the anonymous referees for valuable suggestions and comments on the manuscript. The English text was revised by F. Barraclough.

This research was funded by CICYT projects (AGL2001-1203 and AGL2004-00462) from the Ministry of Education and Science of Spain. J.P. and M.J.H.-L. were supported by FPI and CSIC-EPO fellowships, respectively.

 
* Corresponding author. Mailing address: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos, Consejo Superior de Investigaciones Científicas, P.O. Box 73, E-46100-Burjassot, Valencia, Spain. Phone: 34-963900022. Fax: 34-963636301. E-mail: randez{at}iata.csic.es

Published ahead of print on 8 June 2007.

Present address: Instituto de Biología Molecular y Celular de Plantas, IBMCP (CSIC), Valencia, Spain.

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Applied and Environmental Microbiology, August 2007, p. 4824-4831, Vol. 73, No. 15
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