APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2002, p. 5981–5989 Vol. 68, No. 120099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.12.5981–5989.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Aquaporin Expression Correlates with Freeze Tolerance in Baker’sYeast, and Overexpression Improves Freeze Tolerance

in Industrial StrainsAn Tanghe,1 Patrick Van Dijck,1,2 Francoise Dumortier,1 Aloys Teunissen,1†

Stefan Hohmann,3 and Johan M. Thevelein1*Laboratorium voor Moleculaire Celbiologie1 and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB),2

Institute of Botany and Microbiology, Katholieke Universiteit Leuven, B-3001 Leuven-Heverlee, Flanders,Belgium, and Department of Cell and Molecular Biology/Microbiology, Lundberg Laboratory,

Goteborg University, S-405 30 Goteborg, Sweden3

Received 16 April 2002/Accepted 23 August 2002

Little information is available about the precise mechanisms and determinants of freeze resistance inbaker’s yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of dif-ferent freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistanceand the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cellsmore sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression ofthe human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasmamembrane water transport activity in determination of freeze tolerance in yeast. This appears to be the firstclear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically drivenefflux 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 cellsuspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporinoverexpression transformant could be selected based on its improved freeze-thaw resistance without the needfor a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermen-tation 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.

Bread making is one of the oldest food-manufacturing pro-cesses and involves the fermentative capacity of the yeast Sac-charomyces cerevisiae for the leavening of the dough. Specialtypes of dough, such as sweet or sour dough, present specificchallenges to the leavening activity of the yeast, and specificstrains with better performance under such conditions havebeen selected. However, no appropriate strains of yeast areavailable yet for use in frozen doughs, an important recentdevelopment in the bakery industry (2, 33).

The use of frozen doughs is steadily increasing in all indus-trialized countries because it offers great convenience, auto-mation, and economy of scale. However, significant reductionof the leavening capacity during freeze storage is a seriousdrawback. Minimizing this loss requires specialized equipmentfor cold and rapid mixing of the dough which is not available toartisanal bakers. Moreover, these optimized production con-ditions still cannot completely overcome the drop in leaveningactivity during long-term storage.

Conditions for production of baker’s yeast have been opti-mized in the past decades and nowadays allow yeast with a veryhigh stress resistance to be produced. Active dry yeast, for

instance, is guaranteed to maintain its activity during shelfstorage at room temperature for 2 years. However, the prep-aration of frozen doughs presents an unusual challenge. Al-though marketed baker’s yeast is highly stress resistant, itrapidly loses this stress resistance upon the initiation of fer-mentation during the preparation of the dough. Moreover, ashort prefermentation period before freezing of the dough isrequired to obtain an appropriate texture in the bread. Hence,fermentation-induced loss of stress resistance is a central ob-stacle to the production of frozen doughs (28, 35). The rapidloss of stress resistance in the yeast is due to activation of signaltransduction pathways by the nutrients in the flour. In partic-ular, activation of the Ras-cyclic AMP (cAMP)-protein kinaseA pathway by sucrose and glucose causes rapid loss of stressresistance due to mobilization of trehalose, repression of heatshock proteins, and disappearance of other, unknown stressprotection factors (42, 43). Neither the addition of more yeastor of protective additives nor the optimization of dough pro-duction conditions has resulted in a satisfying solution for theloss of rising capacity in frozen doughs.

Yeast strains with improved freeze tolerance have been iso-lated from natural sources, selected from culture collections,or obtained by mutagenesis, hybridization, or protoplast fusionof natural and commercial strains (1, 8, 12, 28, 29). Uponcharacterization of those strains, several correlations havebeen reported between freeze resistance and cellular factorssuch as trehalose content (11, 16, 36, 45), heat shock proteinlevels (14), the lipid composition of the cell membrane (27),

* Corresponding author. Mailing address: Laboratorium voor Mo-leculaire Celbiologie, Institute of Botany and Microbiology, Katho-lieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium. Phone: 32-16-32 15 07. Fax: 32-16-32 1979. E-mail: Johan.Thevelein@bio.kuleuven.ac.be.

† Present address: Department of Pharmacochemistry, Vrije Uni-versiteit Amsterdam, 1081HV Amsterdam, The Netherlands.

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respiratory capacity (31), and accumulation of charged aminoacids (39). However, to date no single factor has been identi-fied which allows reduction or enhancement of freeze toler-ance in baker’s yeast by genetic modification of specific targetgenes in a consistent and predictable way.

Yeast mutants deficient in “fermentation-induced loss ofstress resistance” (fil mutants) have been isolated, and compo-nents of the cAMP-protein kinase A pathway, such as theputative glucose-sensing G-protein-coupled receptor Gpr1(17) and adenylate cyclase (46), have been shown to be affectedin these mutants. Recently, fil mutant AT25, derived from theindustrial strain S47, which is in commercial use worldwide,has been isolated (41a). We have now performed genomewideexpression analysis with this strain and its parent strain S47, aswell as with several freeze-resistant and freeze-sensitive deriv-atives of AT25 and S47, respectively. This has led to the iden-tification of aquaporins as determinants of freeze resistance.

Aquaporins belong to the major intrinsic protein (MIP) fam-ily of membrane proteins. Members of this family are channelproteins with six transmembrane domains. They are involvedin the transport of water and/or small neutral solutes such asglycerol (30). S. cerevisiae contains four genes encoding mem-bers of the MIP family (30): the osmoregulated glycerol facil-itator Fps1 (24, 40), its homologue Yfl054c, with putative glyc-erol transport function, and the two aquaporin water channels,Aqy1 and Aqy2. In most laboratory strains, industrial strains,and natural isolates, the AQY2 open reading frame (ORF) issplit into two overlapping ORFs (YLL052c-YLL053c) as a con-sequence of an 11-bp deletion (AQY2-2). Only in strains withthe �1278b background is an intact, nondisrupted ORF found,encoding a functional Aqy2 water channel (AQY2-1) (20). Forthe AQY1 gene also, functional (AQY1-1) and nonfunctional(AQY1-2) alleles have been identified (20). Both yeast aqua-porins are localized at the plasma membrane (26; F. Sidoux-Walter and S. Hohmann, unpublished data). Aqy1 has beenshown to mediate water transport upon expression in Xenopuslaevis oocytes (3), while Aqy2 has been shown by stopped-flowanalysis to mediate water transport in yeast cell-derived vesi-cles (26). While mammalian and plant aquaporins have impor-tant functions in water homeostasis and osmoregulation ofindividual cells and whole organisms, no well-defined pheno-type indicative of a physiological function for yeast or othermicrobial aquaporins has been described yet. In Escherichiacoli, for instance, a requirement for the water channel AqpZduring rapid growth and osmotic adaptation has been sug-gested, but so far without any direct evidence (5). In baker’syeast, a possible role during yeast spore formation and germi-nation has been attributed to Aqy1, whereas Aqy2 has beensuggested to play a role in water retrieval after hyperosmoticshock. However, these suggestions were based only on theresults of expression analyses (18). The precise physiologicalfunctions of the yeast aquaporins and apparently of other mi-crobial aquaporins as well have remained unknown so far (15).

Here we demonstrate a novel phenotype for yeast strainswith a modification of aquaporin expression. Deletion reducesthe freeze tolerance of the cells, while overexpression enhancesit. We also show that the freeze tolerance of industrial strainscan be improved by aquaporin overexpression without affect-ing growth and fermentation characteristics, making the aqua-

porin genes promising tools for improvement of freeze toler-ance in commercial baker’s yeast.

MATERIALS AND METHODS

Strains, plasmids, and culture conditions. The strains and plasmids used inthis study are listed in Table 1. Cells were routinely grown in molasses medium[0.5% (wt/vol) yeast extract, 0.5% (wt/vol) molasses (Lesaffre Developpement,Lille, France), 0.05% (wt/vol) (NH4)2HPO4 (pH 5.0 to 5.5)] or in YP (1%[wt/vol] yeast extract, 2% [wt/vol] Bacto Peptone) with either 2% glucose (YPD),2% galactose (YPGal), or 0.5% molasses (YPM) at 30°C in an orbital shaker.

AT25 was obtained via UV mutagenesis of the production strain S47 (LesaffreDeveloppement), followed by screening for survival after multiple freeze-thawcycles of small doughs prepared with UV-mutagenized S47 cells, and was sub-sequently characterized as a fil mutant (deficient in fermentation-induced loss ofstress resistance). In addition to its higher freeze tolerance, the commerciallyimportant properties of mutant AT25 are similar to or better than those of theparent strain S47 (41a).

Strains S47 and AT25 were sporulated, and mutual mating of freeze-resistantspores of AT25 and freeze-sensitive spores of S47 resulted in resistant strainsHAT36, HAT43, and HAT44 and the sensitive strain SS1. The idea behind thiswas to concentrate possible positive alleles for freeze resistance in the HATstrains and to diminish their number in strain SS1. The integrative plasmidpYX012 (Novagen) was modified with a dominant marker gene for use inprototrophic strains by cloning the EcoRV/PvuII fragment containing the loxP-KanMX4-loxP cassette from pUG6 (7) in the URA3 marker, resulting in plasmidpYX012 KanMX. The aquaporin ORFs AQY1-1 and AQY2-2 were PCR ampli-fied using genomic DNA of strain 10560-6B (G. R. Fink, Cambridge, Mass.) andW303-1A (44), respectively, and cloned into pYX012 KanMX downstream of theTPI promoter. Likewise, AQY2-1 was subcloned from pYX242/AQY2-1 (26).Integration of pYX012 KanMX/AQY1-1, AQY2-1, and AQY2-2 at the TPI locusresulted in Geneticin-resistant strains of 10560-6B (�1278b background),BY4743 (S288C background) (4), and AT25 overexpressing AQY1-1, AQY2-1,and AQY2-2, respectively. The empty plasmid pYX012 KanMX was routinelyinserted as a control. The TPI1 promoter of pYX012 KanMX was also replacedby the truncated HXT7 promoter (10), resulting in plasmid pYX012 HXT7pKanMX. Subsequently, the aquaporin-encoding genes AQY1-1 and AQY2-1 werePCR amplified and cloned downstream of this strong, constitutive promoter.Correct cloning was verified by sequence analysis. Integration of NdeI-linearizedplasmids at the URA3 locus resulted in Geneticin-resistant strains of AT25 andS47, overexpressing AQY1-1 and AQY2-1, respectively. The empty plasmidpYX012 HXT7p KanMX was routinely inserted as a control. Selection for Ge-neticin resistance was carried out with media supplemented with 150 mg of G418sulfate (Life Technologies)/liter. All strains were checked by PCR on genomicDNA. For use in industrial strains, the loxP-KanMX-loxP cassette from pUG6 (7)was inserted into plasmids pYeDP (32) and pYeDP hAQP1 (19) at the EcoRVrestriction site.

RNA isolation, microarray analysis, and Northern analysis. Strains weregrown for 2.5 days until stationary phase in YPD or YPM at 30°C in an orbitalshaker. Cells were collected and resuspended in YP. After 30 min of incubationat 30°C, glucose was added to a final concentration of 100 mM. Culture samplesfor total RNA isolation were taken 30 min after the resuspension in YP as wellas 30 min after the addition of glucose and were immediately added to ice-coldwater. The cells were washed once with ice-cold water and stored at �70°C. TotalRNA was isolated using the RNApure reagent (GeneHunter Corporation) ac-cording to the manufacturer’s instructions. Microarray analysis was performedusing microarrays containing 6,144 yeast ORFs on nylon membranes (YeastGenefilters Microarrays; Research Genetics) according to the manufacturer’sinstructions. Probes were prepared by reverse transcription-PCR in the presenceof [�-33P]dCTP by using total RNA as a template. Microarray imaging results(Fuji BAS-1000 with MacBAS, version 2.5, software) were compared usingPathways 2.0 software (Research Genetics). Data were normalized against alldata points. This genomewide expression analysis was used as a screeningmethod for candidate genes involved in freeze resistance; therefore, each hy-bridization was performed only once. The reliability and reproducibility of thetechnique in our hands has been tested extensively as described previously (34).It should be noted that the set of genes present on the membranes is incomplete:genes YPR131C through YPR204W and a number of smaller ORFs were notrepresented on the Yeast Genefilters Microarrays. For Northern analysis, totalRNA was separated in denaturing agarose gels and transferred to nylon mem-branes. Generally, probes used for hybridization were �-32P-labeled fragmentsgenerated with Highprime (Boehringer Mannheim) by using PCR-amplifiedORFs as templates. For AQY1 and AQY2, the C-terminal parts of the ORFs and

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part of the terminator sequence were amplified and labeled. Actin was used as aloading standard. Signals were quantified using a phosphorimager (Fuji BAS-1000 with MacBAS, version 2.5, software) and expressed as percentages of theactin messenger level. For the Northern analysis, independent isolations of totalRNA were used.

RGC after freezing. Strains were grown for 2.5 days until stationary phase inYPD or YPGal at 30°C in an orbital shaker. Equal amounts of cells (correspond-ing to 1 ml of culture with an optical density at 600 nm [OD600] of 20 [about 25mg {wet weight}/ml] for laboratory strains and an OD600 of 15 [about 20 mg {wetweight}/ml] for industrial strains) were collected and resuspended in 1 ml of YP.After incubation at 30°C for 30 min, glucose was added to final concentrations of100 mM for industrial strains and 200 mM for laboratory strains. Half of the cellsuspension was immediately cooled on ice (nonfermenting cells), and the otherhalf was incubated at 30°C for either 30 min (industrial strains) or 40 min(laboratory strains) and then cooled on ice (fermenting cells). After being har-vested and resuspended in precooled YP, the cell suspensions were again di-vided: two aliquots were kept on ice, and another two aliquots were frozen. Afterfreezing in an ethanol bath at �30°C for 1 h, followed by frozen storage in afreezer at �30°C for 1 day, 10 volumes of YP containing 33 mM glucose wereadded to the control samples and the thawed samples. After incubation at 30°Cfor either 2.5 h (industrial strains) or 4 h (laboratory strains), the cell suspensionswere centrifuged and the glucose concentration of 4 �l of supernatant wasdetermined using 200 �l of Trinder reagent (Sigma Diagnostics). The residualglucose consumption (RGC) was calculated as the glucose consumption of thetwo frozen samples (FGC) compared to that of the two control samples (initialglucose consumption [IGC]) from both fermenting and nonfermenting cells.

Growth. The length of the lag phase and the maximum growth rate of yeaststrains in YPD and molasses medium were monitored automatically by OD600

measurement with a BioscreenC apparatus (Labsystems). The parameters wereas follows: 250 �l of culture in each well, 30 s of shaking each min (medium

intensity), an OD600 measurement every 30 min. Readings are saturated atOD600s above 1.5.

Frozen doughs. A 100-�l volume of an overnight culture in 3 ml of YPD wasspread out on molasses plates (25 ml) and grown at 30°C for 24 h. Molassesplates were washed with 6 ml of water, and for each strain the same amount ofcells was added to 7.5 g of flour and 0.15 g of salt. The doughs were mixed andkneaded with a spatula, divided into 0.25-g amounts in screw-cap tubes, andfermented for 30 min at 30°C in an incubator. All doughs were put at �30°C inan ethanol bath except for two nonfrozen controls that were analyzed immedi-ately. After 1 h, the samples were either stored in the freezer (�30°C) orsubjected to freeze-thaw cycles in a computer-controlled cryostat (one cycleconsists of 30°C, �30°C, and 30°C in 2 h). For each measuring point (x days inthe freezer or y freeze-thaw cycles), two tubes for each strain were taken out ofthe freezer or cryostat. To analyze survival, 1 ml of TS (1 g of tryptone/liter and9 g of NaCl/liter) and 0.5 ml of glass beads (diameter, 3 mm) were added to thedough and yeast cells were released from the dough by vortexing for 1 min. Thesuspension obtained was diluted and plated on YPD to determine the number ofCFU.

Selection of aquaporin overexpression strains based on freeze resistance.Strain AT25 was transformed with pYX012 KanMX AQY2-1, a recovery periodof 1 h at 30°C in YPD was given, and the transformation mixture was aliquoted(25 aliquots of 15 �l, each containing about 4 � 107 cells). Two aliquots werediluted and plated on YPD plates immediately, and the remaining aliquots wereenriched for the desired recombinants via freeze-thaw cycling in a computer-controlled cryostat (one cycle consists of 30°C, �30°C, and 30°C in 2 h). After sixcycles, all aliquots were diluted and plated on YPD. The resulting colonies weresubcultured three times to ensure removal of all nonintegrated plasmids. Sub-sequently, the surviving strains were tested for the presence of the overexpressionconstruct via PCR analysis using primers complementary to the 5� end of the TPIpromoter and the 3�end of the AQY2-1 gene.

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Description Source or reference

Industrial strainsS47 Polyploid, aneuploid, prototrophic Lesaffre DeveloppementAT25 Polyploid, aneuploid, prototrophic 41aSS1 Polyploid, aneuploid, prototrophic This studyHAT36, -43, -44 Polyploid, aneuploid, prototrophic This studyS47/HXT7p S47/pYX012 HXT7p KanMX This studyS47/HXT7pA1-1 S47/pYX012 HXT7p KanMX AQY1-1 This studyS47/HXT7pA2-1 S47/pYX012 HXT7p KanMX AQY2-1 This studyAT25/HXT7p AT25/pYX012 HXT7p KanMX This studyAT25/HXT7pA1-1 AT25/pYX012 HXT7p KanMX AQY1-1 This studyAT25/HXT7pA2-1 AT25/pYX012 HXT7p KanMX AQY2-1 This studyAT25/TPIIp AT25/pYX012 KanMX This studyAT25/TPIIpA2-1 AT25/pYX012 KanMX AQY2-1 This study

Laboratory strainsBY4743 MATa/MAT� his3DI leu2D0 ura3D0 410560-6B MAT� leu2::hisG trp1::hisG his3::hisG ura3-52 G. FinkYSH 1170 10560-6B aqy1::KanMX4 26YSH 1171 10560-6B aqy2::HIS3 26YSH 1172 10560-6B aqy1::KanMX4 aqy2::HIS3 V. Laize

PlasmidspUG6 Containing loxP-KanMX-loxP cassette 7pYX012 Integrative plasmid with TPI promoter, URA3 marker NovagenpYX012 KanMX pYX012 URA3::loxP-KanMX-loxP This studypYX012 KanMX/AQY2-1 AQY2-1 cloned into pYX012 KanMX This studypYX012 HXT7p KanMX pYX012 KanMX TPI1p replaced by HXT7p This studypYX012 HXT7p KanMX AQY1-1 AQY1-1 cloned into pYX012 HXT7p KanMX This studypYX012 HXT7p KanMX AQY2-1 AQY2-1 cloned into pYX012 HXT7p KanMX This studypYX242/AQY2-1 AQY2-1 cloned into pYX242 26pYeDP 2�m plasmid with GAL10-CYC1 promoter, URA3 marker 32pYeDP hAQP1 hAQP1 (CHIP28) in pYeDP 19pYeDP hAQP1-A73M hAQP1-A73M (CHIP28-A73M) in pYeDP R. BillpYeDP KanMX 2�m plasmid with GAL10-CYC1 promoter, KanMX marker This studypYeDP hAQP1 KanMX KanMX in pYeDP hAQP1 This study

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Reproducibility of the results. All experiments were repeated at least threetimes with reproducible results. Representative results are shown. For glucoseconsumption experiments, the RGCs obtained for the control strains are variablebetween experiments; therefore, mean ratios of the RGCs of the studied strainsto the RGCs of the control strains � errors are reported.

RESULTS

Genomewide gene expression analyses at the onset of fer-mentation reveal upregulation of AQY2 in freeze-resistantstrains. Using microarrays containing 6,144 yeast ORFs onnylon membranes, genomewide gene expression analyses ofdifferent freeze-resistant and freeze-sensitive yeast strains wereperformed. The resistant strains HAT36, HAT43, and HAT44(Fig. 1) are derived from the freeze-resistant mutant AT25(41a), and the sensitive strain SS1 (Fig. 1) is derived from thefreeze-sensitive industrial strain S47 (Lesaffre Developpe-ment). The global gene expression patterns of these strainswere compared at the onset of fermentation, i.e., 30 min afteraddition of glucose to YPM-grown stationary-phase cells, so asto mimic the conditions under which the commercial yeastshould maintain better freeze tolerance.

Six genes showed at least a 2.5-fold-higher or -lower expres-sion in all comparisons between a resistant and a sensitivestrain (see Discussion). However, neither individual overex-pression nor individual deletion of these genes in differentstrain backgrounds resulted in significant effects on freeze tol-erance (data not shown). In addition, expression of the AQY2(YLL052c-YLL053c) gene was higher in the freeze-resistantstrains HAT36 (Fig. 2A), HAT43, and HAT44 than in thefreeze-sensitive strain SS1. Although AQY2 was not among thegenes with the most-pronounced differences in expression, apossible role of a water channel in freeze resistance wasintriguing. Expression of the other water channel, AQY1(YPR192w), was not monitored in the genomewide gene anal-ysis, because it is not represented on the Yeast GenefiltersMicroarrays. The sequence identity between AQY1-2 and

AQY2-2 is 75.5% at the DNA level, which should excludecross-hybridization between the two genes (34). By use ofprobes designed to check specific expression of AQY1 andAQY2 by Northern analysis, the higher expression of AQY2 inthe resistant strains HAT36 (Fig. 2B), HAT43, and HAT44than in the sensitive strain SS1 was confirmed, whereas expres-sion of AQY1 could not be detected 30 min after the onset offermentation in either the freeze-resistant or the freeze-sensi-tive strains (data not shown).

Overexpression of water channel proteins Aqy1-1 andAqy2-1 improves freeze tolerance in laboratory and industrialyeast strains without affecting growth and fermentation rates.Alleles AQY1-1 and AQY2-1, encoding functional Aqy1 andAqy2, respectively, were overexpressed in laboratory strainsBY4743 and 10560-6B as well as in industrial strains S47 andAT25. As determined by diagnostic restriction analysis of thePCR-amplified ORFs according to the work of Laize et al.(20), BY4743 contains no functional endogenous aquaporinalleles, whereas 10560-6B contains functional endogenous al-leles of both aquaporins. AT25, like S47, possibly contains afunctional AQY2-1 allele, and it contains at least one func-tional AQY1-1 allele (data not shown). Freeze tolerance wasdetermined as the difference in glucose consumption betweenfrozen and nonfrozen cells (RGC, expressed as a percentage),which is the most meaningful assay for yeast activity in frozendoughs. Overexpression of AQY1-1 or AQY2-1 clearly im-proved the RGC after prefermentation and freezing for bothlaboratory strains (data not shown) and industrial strains (Fig.3A). Overexpression of the aquaporin genes at the moment of

FIG. 1. Freeze tolerance of freeze-resistant strains (AT25, HAT36,HAT43, HAT44) and freeze-sensitive strains (S47 and SS1) used formicroarray analysis. IGC, FGC, and RGC were determined 30 minafter the onset of fermentation by addition of 100 mM glucose. Thecells were either frozen (for 1 day at �30°C) (FGC) or not frozen (i.e.,cooled on ice) (IGC). After thawing, glucose consumption was mea-sured for 2.5 h to assess residual yeast activity. RGC is calculated as(FGC/IGC) � 100. Representative results are shown. AT25 showed anRGC 2.0 (�0.3) times higher than that of S47. HAT36, HAT43, andHAT44 each showed an RGC 2.9 (�0.1) times higher than that of SS1.

FIG. 2. Differential expression of the AQY2 (YLL052c andYLL053c) gene in the freeze-resistant strain HAT36 and the freeze-sensitive strain SS1 at the onset of fermentation. (A) Microarray anal-ysis. The YLL052c and YLL053c signals are situated at the center ofthe crosshair and are indicated by an arrow. (B) Northern blot analysis.ACT1 and IPP2 were used as loading controls. The HAT36/SS1 ex-pression ratio was 3.5.

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freeze treatment, i.e., 30 min after addition of glucose, wasconfirmed by Northern and Western blot analyses (data notshown). For neither of the two aquaporin genes did overex-pression affect the growth rate, the length of the lag phase inYPD (Fig. 3B) or molasses (data not shown) medium, or theinitial fermentation capacity (IGC) (Fig. 3A). The improve-ment of stress resistance appeared to be specific for freezestress, since no effect of aquaporin overexpression was ob-served on the resistance of AT25 to heat (56°C), cold (4°C),ethanol (7.5%), osmotic stress, or salt (1.5 M NaCl, KCl, sor-bitol) (data not shown).

Deletion of AQY1-1 and AQY2-1 in a laboratory strain re-duces freeze tolerance. The freeze tolerances of AQY1-1 andAQY2-1 single- and double-deletion strains were determined inthe laboratory strain background 10560-6B. Both nonferment-

ing and fermenting cells were tested. Freeze tolerance wasmeasured as RGC in frozen versus nonfrozen cells. In nonfer-menting cells (Fig. 4A), deletion of AQY1-1 reduced freezetolerance, whereas this was not the case for AQY2-1. Thedouble-deletion strain showed a freeze sensitivity similar tothat of the AQY1-1 single-deletion strain. In fermenting cells(Fig. 4B), single deletion of either AQY1-1 or AQY2-1 reducedfreeze tolerance, with the latter producing the largest effect.The double-deletion strain was more freeze sensitive than thesingle-deletion strains. These results appear to fit with themRNA expression patterns of the aquaporin genes at the onsetof fermentation (Fig. 4C). The AQY1-1 gene is highly ex-pressed in nonfermenting cells and poorly expressed in glucosemedium, while expression of AQY2-1 is very low in nonfer-menting cells and increases after the addition of glucose. Thisis in accordance with the findings of recent expression studiesof the two aquaporins using Northern blot analysis (18).

Overexpression of human aquaporin hAQP1 also enhancesfreeze tolerance in yeast, which is only partly the case for thepoorly functional hAQP1-A73M allele. To gain further evi-dence that the water transport capacity of cells is the truedeterminant of freeze resistance, the human aquaporin genehAQP1 was overexpressed in yeast, as was a mutant alleleencoding a water channel with impaired function. Laize et al.have shown that hAQP1 was highly expressed, correctly local-ized, and active upon heterologous expression in yeast underthe control of the inducible GAL10-CYC1 hybrid promoter(19). Essentially the same construct has been made with amutant allele, hAQP1-A73M; this construct is localized in themembrane but is poorly functional (R. Bill and S. Hohmann,unpublished data). hAQP1 and its mutant allele hAQP1-A73Mwere expressed in strain BY4743, and freeze tolerance wasdetermined for cells grown in YPGal, to obtain full inductionof hAQP1, and also in YPD, where the GAL10-CYC1 pro-moter is repressed. Freeze tolerance, as determined by RGC infrozen versus nonfrozen cells, was significantly improved ingalactose-grown cells expressing hAQP1 compared to that incells transformed with an empty plasmid (Fig. 5A). In cellsexpressing the poorly functional hAQP1-A73M allele, only apartial effect was observed (Fig. 5A). In cells grown on glucose,there was no difference among the strains (data not shown).Also, overexpression of hAQP1 in the industrial strains S47and AT25 improved freeze tolerance in comparison with thatof strains transformed with an empty plasmid (Fig. 5B). Sim-ilarly, overexpression of the nonfunctional yeast AQY2-2 allelein several strain backgrounds failed to improve freeze toler-ance (data not shown).

Overexpression of Aqy2-1 also provides protection to yeastin frozen doughs or in doughs submitted to multiple freeze-thaw cycles. Routinely, yeast cell suspensions were used todetermine freeze tolerance. However, to test whether the ob-served improvement of freeze tolerance by overexpression ofaquaporins also applies to yeast in frozen dough conditions,small doughs were prepared with strain AT25 and with strainAT25 overexpressing the AQY2-1 gene and were either storedin frozen form or submitted to freeze-thaw cycles. Freeze tol-erance was determined as the number of CFU with and with-out freezing of the doughs. The results clearly show that thestrain overexpressing aquaporins survives better during storage

FIG. 3. Overexpression of aquaporin-encoding genes improvesfreeze tolerance without affecting growth and initial fermentationrates. (A) IGC, FGC, and RGC were determined for S47 and AT25overexpressing AQY1-1, S47 and AT25 overexpressing AQY2-1, and, asa control, S47 and AT25 with an integrated empty plasmid. The cellswere either frozen (for 1 day at �30°C) (FGC) or not frozen (i.e.,cooled on ice) (IGC) 30 min after the onset of fermentation by addi-tion of 100 mM glucose. After thawing, glucose consumption wasmeasured for 2.5 h to assess residual yeast activity. RGC is calculatedas (FGC/IGC) � 100. Representative results are shown. Compared toAT25 containing an empty plasmid, AT25 AQY1-1 and AQY2-1 over-expression strains showed 1.5 (�0.1)- and 1.4 (�0.1)-times-higherRGCs, respectively. Compared to S47 containing an empty plasmid,S47 AQY1-1 and AQY2-1 overexpression strains showed 9.8 (�0.8)-and 9.0 (�1.2)-times-higher RGCs, respectively. (B) Growth of thesame strains in YPD medium (Bioscreen measurements).

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in frozen doughs (Fig. 6A) as well as during most of thefreeze-thaw cycling of the doughs (Fig. 6B).

Improvement of freeze tolerance as a selection tool for iso-lation of aquaporin transformants. An AT25 transformantoverexpressing AQY2-1 could be isolated directly on the basisof better freeze-thaw survival by using six freeze-thaw cyclesand PCR analysis of the surviving strains. Freeze-thaw selec-tion on 23 aliquots, each containing about 4 � 107 cells, re-sulted in 23 surviving colonies (representing 2.5 � 10�6%survival), among which 1 strain contained the overexpressionconstruct. The freeze resistance of this strain was similar to thefreeze resistance of strain AT25/HXT7pA2-1 shown in Fig. 3A(data not shown). This implies that usage of an antibioticselection marker is not required for the construction of freeze-resistant commercial yeast strains overexpressing aquaporins.

DISCUSSION

By use of genomewide gene expression analysis of differentfreeze-resistant and freeze-sensitive yeast strains, many geneswere identified as differentially expressed (ratio, 2.5 or more)in at least two comparisons of a resistant and a sensitive strain:67 genes were found to be expressed at higher levels, and 15genes were found to be expressed at lower levels, in the resis-tant strains (data not shown). However, only six genes showedat least a 2.5-fold differential expression in all comparisonsbetween a resistant and a sensitive strain (data not shown).Three of these genes were expressed at lower levels in resistantstrains (ERG5, YHB1, and YGR154C), and three were ex-pressed at higher levels in resistant strains (PLB2, CRH1, andCSI2). These differences in expression were confirmed byNorthern analysis (data not shown). Intriguingly, five of the sixgenes are related to the cell membrane or cell wall, organellesthat have always been regarded as the primary targets of freezestress (38). ERG5 encodes a protein that catalyzes an interme-diate step in the biosynthesis of ergosterol (37); PLB2 encodesa lysophospholipase for which it has been shown that overpro-duction causes a modest increase in total phospholipid contentin late growth phase (6); YGR154C is an orphan gene, relatedto ECM4, encoding a protein possibly involved in cell wallstructure or biosynthesis; CRH1 encodes a protein importantfor cell wall maintenance; and CSI2 encodes a protein involvedin chitin synthesis. The other gene, YHB1, encodes a yeastflavohemoglobin. Expression of YHB1 seems to be repressedby a shift to high osmolarity (34), a stress that is inherent to thefreezing process. However, neither individual overexpression(in the industrial strains S47 and AT25) nor individual deletion(in the laboratory strain BY4743) of the six genes resulted insignificant effects on freeze tolerance (data not shown). In spiteof this, the possibility that a particular combination of dele-tion and/or overexpression of several of these genes wouldaffect freeze tolerance cannot be excluded. When aquaporin(AQY2-1) was overexpressed in strains with deletions of one ofthe genes YHB1, ERG5, and YGR154C, which were deter-mined to be expressed at lower levels in the resistant strainsexamined, the effect of aquaporin overexpression was slightlymore pronounced (data not shown). This supports the notionthat freeze tolerance is a multifactorial property and that thepresence or absence of certain gene products influences theeffects of other gene products on freeze tolerance.

FIG. 4. Deletion of aquaporin-encoding genes reduces freeze tol-erance. (A and B) The effects of freezing on glucose consumption weremeasured in nonfermenting and fermenting cells of aquaporin single-and double-deletion mutants in the 10560-6B background. IGC, FGC,and RGC were determined for the wild-type strain, the aqy1 strain,the aqy2 strain, and the aqy1 aqy2 strain. The cells were eitherfrozen (for 1 day at �30°C) (FGC) or not frozen (i.e., cooled on ice)(IGC) 30 min after resuspension of stationary-phase cells in YP (non-fermenting cells) (A) or 40 min after the subsequent addition of 200mM glucose (fermenting cells) (B). After thawing, glucose consump-tion was measured for 4 h to assess residual yeast activity. RGC iscalculated as (FGC/IGC) � 100. Representative results are shown.Compared to the wild-type strain 10560-6B, Aqy1-1, Aqy2-1, and dou-ble-deletion strains showed RGCs that were 0.7 (�0.1), 1.1 (�0.2), and0.3 (�0.1) times higher, respectively, for nonfermenting cells and 0.6(�0.1), 0.4 (�0.1), and 0.2 (�0.1) times higher, respectively, for fer-menting cells. (C) Northern analysis of AQY1 and AQY2 expression innonfermenting and fermenting wild-type 10560-6B cells. ACT1 wasused as a loading control.

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Expression of AQY2 was higher in most of the freeze-resis-tant strains. However, microarray analysis of the freeze-resis-tant mutant AT25 and its freeze-sensitive parent, S47, revealedlower expression of AQY2 in the resistant strain at the onset of

fermentation. Also, when pools of total RNA from severalother freeze-resistant versus freeze-sensitive strains were pre-viously compared for AQY2 expression at the onset of fermen-tation, no clear differential expression was observed (41).Moreover, according to restriction analysis, the AQY2 geneappears to be a nonfunctional gene in the AT25 background(data not shown). This would indicate that the higher freezetolerance of AT25 than of S47 is probably not primarily due todifferential AQY2 expression. However, from the restrictionanalysis, the possibility that a particular AQY2-allele(s) in thesestrains encodes a functional water channel cannot be excluded.Only the cloning of all of the AQY2 alleles present and asubsequent test in X. laevis oocytes for water transport capacitycould answer this question. Expression of AQY1 before theaddition of glucose (Fig. 4C) could perhaps still influence re-sistance 30 min after the onset of fermentation, but no differ-ential expression between AT25 and S47 could be detectedunder nonfermenting conditions (data not shown). Altogether,the possibility that the aquaporin genes have been identified“by accident” in the screening for genes with importance infreeze resistance cannot be excluded. It is very likely that otherfactors in addition to aquaporins also influence freeze toler-ance in yeast at the onset of fermentation.

The aquaporin genes AQY1 and AQY2 were found to beimportant determinants of freeze resistance: overexpressionimproved freeze tolerance in laboratory (data not shown) andindustrial (Fig. 3A) yeast strains, whereas deletion reduced

FIG. 5. Heterologous overexpression of the human aquaporin genehAQP1 improves freeze tolerance. (A) Overexpression of the wild-typegene, but not of the mutant allele hAQP1-A73M, improves freezetolerance in a laboratory strain. IGC, FGC, and RGC were determinedfor strain BY4743 overexpressing either wild-type hAQP1 or the poorlyactive mutant hAQP1-A73M versus strain BY4743 transformed withan empty plasmid. Cells were either frozen (for 1 day at �30°C) (FGC)or not frozen (i.e., cooled on ice) (IGC) 40 min after the onset offermentation by addition of 100 mM glucose. After thawing, glucoseconsumption was measured for 4 h to assess residual yeast activity.RGC is calculated as (FGC/IGC) � 100. Representative results areshown. Compared to that of BY4743 containing an empty plasmid,hAQP1 and hAQP1-A73M expression strains showed RGCs that were2.3 (�0.2) and 1.5 (�0.1) times higher, respectively. (B) Overexpres-sion of the human aquaporin gene hAQP1 improves freeze tolerance inindustrial strains. IGC, FGC, and RGC were determined for strainsAT25 and S47 overexpressing wild-type hAQP1 versus the respectivestrains transformed with an empty plasmid. The procedure describedfor panel A was used, except that cells were frozen or cooled on ice 30min after the onset of fermentation. Representative results are shown.Compared to those of AT25 and S47 containing empty plasmids,hAQP1 expression strains showed RGCs that were 1.5 (�0.1) and 2.0(�0.0) times higher, respectively.

FIG. 6. Overexpression of functional aquaporins improves thefreeze tolerance of yeast in dough. Shown is the survival of strain AT25(open symbols) and that of strain AT25 overexpressing AQY2-1 (solidsymbols) in small doughs during frozen storage (�30°C) (A) or insmall doughs subjected to multiple freeze-thaw cycles (between �30°Cand 30°C) (B). Survival was determined as the number of CFU isolatedfrom the doughs relative to those from nonfrozen controls.

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freeze tolerance in a laboratory strain (Fig. 4A and B). Al-though it has been shown that both Aqy1-1 and Aqy2-1 medi-ate water transport (3, 26), it could in principle not be excludedthat the yeast aquaporins influenced freeze tolerance in a man-ner unrelated to their water transport activity, for instance, byaffecting membrane properties, such as membrane fluidity,that could affect water permeability (21). The fact that over-expression of the nonfunctional yeast AQY2-2 allele in severalstrain backgrounds did not improve freeze tolerance (data notshown) did not reliably exclude this possibility, since propermembrane localization has never been shown for this particu-lar protein. Therefore, the human aquaporin gene hAQP1 wasoverexpressed in yeast, as was a mutant allele encoding a waterchannel with impaired function. Overexpression of the humanaquaporin gene hAQP1 enhanced freeze tolerance in yeast,which was only partly the case for the poorly functionalhAQP1-A73M allele (Fig. 5). These results support the notionthat a rapid, osmotically driven water efflux from the cellsduring the initial freezing process lowers the intracellular wa-ter content and as a result reduces subsequent ice crystal for-mation upon freezing of the protoplasm (25). Higher levels ofaquaporins in the plasma membrane would allow faster waterefflux, especially at freezing temperatures, at which water dif-fusion through the phospholipid layer of the membrane ismuch slower than at higher temperatures. Because reductionof ice crystal formation results in reduced destruction of cel-lular membranes and other components, it allows the cells tomaintain higher activity and viability. This explanation is inaccordance with previous observations that the protective ef-fects of ethanol and methanol against freeze damage correlatewith their stimulating effects on membrane permeability, pre-sumably allowing faster water efflux during freezing (22). Sinceaquaporin-mediated protection was specific for freeze stress(data not shown), the effect can apparently not be attributed toan improvement in general stress tolerance of the cells butappears to be due to a more specific mechanism, such as thestimulation of rapid water efflux from the cells.

The passive diffusion rate of water through membranes is ingeneral relatively rapid (compared to those of other smallhydrophilic molecules), and because of the high surface-to-volume ratios of microorganisms, one would not expect thewater permeability of the plasma membrane to be rate limitingunder most conditions. However, it has already been suggestedthat in microorganisms particular conditions might exist wherewater permeability would be limiting and therefore the pres-ence of water channels would be advantageous (13). No suchcondition has yet been identified, and no well-defined pheno-type indicative of a physiological function of any microbialaquaporin has yet been described. Our results indicate a pos-sible novel function for water channels in microorganisms:aquaporins apparently help to increase the freeze tolerance ofthe cells by supporting rapid water efflux during initial freezing.Such a function would also fit with the apparently low selectiveadvantage of functional aquaporins in yeast under laboratoryconditions (3). Whereas nowadays yeast strains are routinelystored at �80°C in glycerol, in the past yeast strains werestored on agar slants and from time to time were reinoculatedon fresh slants. Hence, laboratory strains normally never ex-perience freeze stress, as strains in nature do under freezingconditions. This might explain why so few laboratory strains

have maintained functional aquaporin alleles. The same ap-plies to industrial yeast strains and even to some natural iso-lates which appear to have lost functional AQY2 alleles (20).There are probably other functions in yeast cells as well thatconfer a selective advantage only under highly specific naturalconditions but not under other conditions, in particular thoseused for laboratory cultivation of yeast. Many laboratorystrains, for instance, carry the same FLO8 mutation causing adefect in flocculation, and the capacity for pseudohyphalgrowth is also known to be deficient in most laboratory strains(23).

Since overexpression of Aqy2-1 also provides protection toyeast in frozen doughs or in doughs submitted to multiplefreeze-thaw cycles (Fig. 6), this modification could be a con-venient way to improve the freeze tolerance of commercialbaker’s yeast strains for use in frozen dough applications. Inthis context it is important that other commercially importantproperties such as the growth rate (Fig. 3B) and initial fermen-tation capacity (Fig. 3A) of the aquaporin overexpressionstrains were not affected. Construction of commercial baker’syeast strains overexpressing aquaporins normally requires theuse of a dominant selection marker to identify the transfor-mants. Generally, antibiotic resistance markers are used forthat purpose. However, the use of antibiotic resistance markersin foodstuffs is controversial (9). We succeeded in isolating anAT25 transformant overexpressing AQY2-1 directly on the ba-sis of better freeze-thaw survival, implying that usage of anantibiotic selection marker is not required for the constructionof commercial yeast strains overexpressing aquaporins. Thiscould facilitate the introduction of such strains on the market.Moreover, our results also imply that overexpression of a yeastaquaporin gene can be used as a selection marker for theconstruction of transformants of industrial yeast strains. Up tonow no phenotype clearly indicative of a physiological functioncould be detected in yeast strains with aquaporin overexpres-sion, except for the improvement of freeze tolerance as re-ported in this paper. Hence, it appears that aquaporin overex-pression is unlikely to interfere with commercially importantproperties of industrial yeast strains.

In conclusion, our results show that genomewide microarrayexpression analysis can be used for the identification of genesrelevant for a specific phenotype. They show that aquaporinexpression influences the freeze tolerance of yeast cells, whichappears to be the first clear physiological function identifiedfor microbial aquaporins. Since aquaporin overexpression sig-nificantly improved the maintenance of viability of industrialyeast strains upon freezing and seems to have little effect onother yeast properties, it appears to be a promising tool forimprovement of freeze tolerance in commercial baker’s yeaststrains.

ACKNOWLEDGMENTS

This work was supported by a fellowship from the Institute forScientific and Technological research (IWT) to An Tanghe and bygrants from the Flemish Interuniversity Institute of Biotechnology(VIB/PRJ2), the Fund for Scientific Research—Flanders, and the Re-search Fund of the Katholieke Universiteit Leuven (Concerted Re-search Actions) to J.M.T. S.H. is a special researcher supported byVetenskapsradet, Stockholm, Sweden. Aquaporin research in S.H.’slaboratory is supported by the European Commission via grants BIO4-CT98-0024, FMRX-CT97-0128, and QLK3-2000-00778.

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We are grateful to Renata Wicik for excellent technical assistance.We also thank Vincent Laize, Roslyn Bill, and Frederic Sidoux-Walterfor kindly providing strains, plasmids, and information, and we thankMarkus Tamas for critical reading of the manuscript.

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