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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{triangledown}

Joaquín Panadero, Maria José Hernández-López,{dagger} 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

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 {Delta}cnb1 and {Delta}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.

* 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

{triangledown} Published ahead of print on 8 June 2007.

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

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.





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