Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.

Centre for Botany Applied to Agriculture Instituto Superior de Agronomia University of Lisbon Lisbon Portugal

Department of Membrane Transport Institute of Physiology AS CR v v i Prague Czech Republic

Department of Membrane Transport Institute of Physiology AS CR v v i Prague Czech Republic Centre for Botany Applied to Agriculture Instituto Superior de Agronomia University of Lisbon Lisbon Portugal

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Martorell P, Stratford M, Steels H, Fernández-Espinar MT, Querol A. 2007. Physiological characterization of spoilage strains of Zygosaccharomyces bailii and Zygosaccharomyces rouxii isolated from high sugar environments. Int. J. Food Microbiol. 114:234–242. 10.1016/j.ijfoodmicro.2006.09.014. PubMed DOI

Dakal TC, Solieri L, Giudici P. 2014. Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii. Int. J. Food Microbiol. 185:140–157. 10.1016/j.ijfoodmicro.2014.05.015. PubMed DOI

Deák T. 2007. Yeasts in specific types of foods, p 117–201 In Deák T. (ed), Handbook of food spoilage yeasts, 2nd ed. CRC Press, Boca Raton, FL.

Solieri L, Giudici P. 2008. Yeasts associated to traditional balsamic vinegar: ecological and technological features. Int. J. Food Microbiol. 125:36–45. 10.1016/j.ijfoodmicro.2007.06.022. PubMed DOI

Emmerich W, Radler F. 1983. The anaerobic metabolism of glucose and fructose by Saccharomyces bailii. Microbiology 129:3311–3318. 10.1099/00221287-129-11-3311. DOI

Sols A. 1956. Selective fermentation and phosphorylation of sugars by sauternes yeast. Biochim. Biophys. Acta 20:62–68. 10.1016/0006-3002(56)90263-3. PubMed DOI

Eddy AA, Barnett JA. 2007. A history of research on yeasts. 11. The study of solute transport: the first 90 years, simple and facilitated diffusion. Yeast 24:1023–1059. 10.1002/yea.1572. PubMed DOI

Saier MH., Jr 2000. Families of transmembrane sugar transport proteins. Mol. Microbiol. 35:699–710. 10.1046/j.1365-2958.2000.01759.x. PubMed DOI

Leandro MJ, Fonseca C, Gonçalves P. 2009. Hexose and pentose transport in ascomycetous yeasts: an overview. FEMS Yeast Res. 9:511–525. 10.1111/j.1567-1364.2009.00509.x. PubMed DOI

Sousa-Dias S, Gonçalves T, Leyva JS, Peinado JM, Loureiro-Dias MC. 1996. Kinetics and regulation of fructose and glucose transport systems are responsible for fructophily in Zygosaccharomyces bailii. Microbiology 142:1733–1738. 10.1099/13500872-142-7-1733. DOI

Sousa-Dias S. 2000. Ph.D. thesis Technical University of Lisbon, Lisbon, Portugal.

Pina C, Gonçalves P, Prista C, Loureiro-Dias MC. 2004. Ffz1, a new transporter specific for fructose from Zygosaccharomyces bailii. Microbiology 150:2429–2433. 10.1099/mic.0.26979-0. PubMed DOI

Leandro MJ, Sychrová H, Prista C, Loureiro-Dias MC. 2011. The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters. Microbiology 157:601–608. 10.1099/mic.0.044446-0. PubMed DOI

Lee DH, Kim SJ, Seo JH. 2014. Molecular cloning and characterization of two novel fructose-specific transporters from the osmotolerant and fructophilic yeast Candida magnoliae JH110. Appl. Microbiol. Biotechnol. 98:3569–3578. 10.1007/s00253-013-5225-y. PubMed DOI

Mira NP, Münsterkötter M, Dias-Valada F, Santos J, Palma M, Roque FC, Guerreiro JF, Rodrigues F, Sousa MJ, Leão C, Guldener U, Sá-Correia I. 2014. The genome sequence of the highly acetic acid-tolerant Zygosaccharomyces bailii-derived interspecies hybrid strain ISA1307, isolated from a sparkling wine plant. DNA Res. 21:299–313. 10.1093/dnares/dst058. PubMed DOI PMC

Galeote V, Bigey F, Devillers H, Neuveglise C, Dequin S. 2013. Genome sequence of the food spoilage yeast Zygosaccharomyces bailii CLIB 213T. Genome Announc. 1:e00606–13. 10.1128/genomeA.00606-13. PubMed DOI PMC

Leandro MJ, Sychrová H, Prista C, Loureiro-Dias MC. 2013. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii. PLoS One 8:e68165. 10.1371/journal.pone.0068165. PubMed DOI PMC

Pribylová L, Straub ML, Sychrová H, de Montigny J. 2007. Characterisation of Zygosaccharomyces rouxii centromeres and construction of first Z. rouxii centromeric vectors. Chromosome Res. 15:439–445. 10.1007/s10577-007-1136-z. PubMed DOI

Pribylová L, Sychrová H. 2003. Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation. J. Microbiol. Methods 55:481–484. 10.1016/S0167-7012(03)00197-0. PubMed DOI

Pribylová L, de Montigny J, Sychrová H. 2007. Tools for the genetic manipulation of Zygosaccharomyces rouxii. FEMS Yeast Res. 7:1285–1294. 10.1111/j.1567-1364.2007.00308.x. PubMed DOI

Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24:2519–2524. 10.1093/nar/24.13.2519. PubMed DOI PMC

Marešová L, Sychrová H. 2007. Applications of a microplate reader in yeast physiology research. Biotechniques 43:667–672. 10.2144/000112620. PubMed DOI

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Hoffman CS, Winston F. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272. 10.1016/0378-1119(87)90131-4. PubMed DOI

Zaragoza O. 2003. Generation of disruption cassettes in vivo using a PCR product and Saccharomyces cerevisiae. J. Microbiol. Methods 52:141–145. 10.1016/S0167-7012(02)00154-9. PubMed DOI

Kinclová-Zimmermannová O, Zavrel M, Sychrová H. 2005. Identification of conserved prolyl residue important for transport activity and the substrate specificity range of yeast plasma membrane Na+/H+ antiporters. J. Biol. Chem. 280:30638–30647. 10.1074/jbc.M506341200. PubMed DOI

Jones GV, White MA, Cooper OR, Storchmann K. 2005. Climate change and global wine quality. Climatic Change 73:319–343. 10.1007/s10584-005-4704-2. DOI

Berthels NJ, Cordero Otero RR, Bauer FF, Thevelein JM, Pretorius IS. 2004. Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains. FEMS Yeast Res. 4:683–689. 10.1016/j.femsyr.2004.02.005. PubMed DOI

Bisson LF. 1999. Stuck and sluggish fermentations. Am. J. Enol. Vitic. 50:107–119.

Salmon JM. 1989. Effect of sugar transport inactivation in Saccharomyces cerevisiae on sluggish and stuck enological fermentations. Appl. Environ. Microbiol. 55:953–958. PubMed PMC

Palma M, Seret ML, Baret PV. 2009. Combined phylogenetic and neighbourhood analysis of the hexose transporters and glucose sensors in yeasts. FEMS Yeast Res. 9:526–534. 10.1111/j.1567-1364.2009.00511.x. PubMed DOI

Yu JH, Lee DH, Oh YJ, Han KC, Ryu YW, Seo JH. 2006. Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer. Appl. Biochem. Biotechnol. 131:870–879. 10.1385/ABAB:131:1:870. PubMed DOI

Yu JH, Lee DH, Park YC, Lee MG, Kim DO, Ryu YW, Seo JH. 2008. Proteomic analysis of fructophilic properties of osmotolerant Candida magnoliae. J. Microbiol. Biotechnol. 18:248–254. PubMed

Guillaume C, Delobel P, Sablayrolles JM, Blondin B. 2007. Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation. Appl. Environ. Microbiol. 73:2432–2439. 10.1128/AEM.02269-06. PubMed DOI PMC