Increased mannoprotein content in wines produced by Saccharomyces kudriavzevii × Saccharomyces cerevisiae hybrids

Several wine quality aspects are influenced by yeast mannoproteins on account of aroma compounds retention, lactic-acid bacterial growth stimulation, protection against protein haze and astringency reduction. Thus selecting a yeast strain that produces high levels of mannoproteins is important for the winemaking industry. In this work, we observed increased levels of mannoproteins in S. cerevisiae × S. kudriavzevii hybrids, compared to the S. cerevisiae strain, in wine fermentations. Furthermore, the expression of a key gene related to mannoproteins biosynthesis, PMT1, increased in the S. cerevisiae × S. kudriavzevii hybrid. We showed that artificially constructed S. cerevisiae × S. kudriavzevii hybrids also increased the levels of mannoproteins. This work demonstrates that either natural or artificial S. cerevisiae × S. kudriavzevii hybrids present mannoprotein overproducing capacity under winemaking conditions, a desirable physiological feature for this industry. These results suggest that genome interaction in hybrids generates a physiological environment that enhances the release of mannoproteins.R. Pérez-Torrado was supported by the JAEDOC postdoctoral program. L. Pérez-Través was supported by an I3P fellowship from the CSIC. This work was supported by CICYT grants (ref. AGL2012-39937-CO2-01 and AGL2012-39937-CO2-02 and AGL2015-67504-C3-1-R) from the Spanish Ministry of Education and Science and FEDER, and by grant PROMETEO (Project PROMETEO/2009/019) from the Generalitat Valenciana.Peer reviewe

Stabilization process in Saccharomyces intra- and interspecific hybrids in fermentative conditions

We evaluated the genetic stabilization of artificial intra-(Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30–50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively. [Int Microbiol 2014; 17(4):213-224]Keywords: Saccharomyces cerevisiae · Saccharomyces kudriavzevii · rare-mating in yeast · molecular markers · DNA content evaluation · stabilization of genome

Las levaduras cerveceras como biocatalizadores: variedad de estilos de cerveza y bioflavoring

La biocatálisis, comprendida como el uso de catalizadores naturales en procesos químicos, es responsable de numerosos procesos biotecnológicos, entre ellos la elaboración de cerveza. El presente trabajo, realizado en el marco de una tesis doctoral, compara las capacidades tecnológicas de un panel de cepas Saccharomyces sp. y no sacaromicéticas para catalizar la transformación de mosto de cebada en cerveza. Catorce levaduras fueron aisladas a partir de lodos residuales de fermentación cervecera y muestras de starters comerciales e identificadas a nivel de cepa por secuenciación del fragmento ITS1-ITS2 y RFLP del ADN mitocondrial

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: β-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.EEA MendozaFil: Perez, Maria Dolores. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentina.Fil: Perez, Maria Dolores. Consejo Superior de Investigación Científica (CSIC). Instituto de Agroquímica y Tecnología de los Alimentos (IATA). Departamento de Biotecnología de los Alimentos, Grupo de Biología de Sistemas en Levaduras de Interés Biotecnológico; EspañaFil: Denat, Marie. Universidad de Zaragoza. Department of Analytical Chemistry. Laboratory for Aroma Analysis and Enology. Instituto Agroalimentario de Aragón; EspañaFil: Pérez-Través, Laura. Consejo Superior de Investigación Científica (CSIC). Instituto de Agroquímica y Tecnología de los Alimentos (IATA). Departamento de Biotecnología de los Alimentos, Grupo de Biología de Sistemas en Levaduras de Interés Biotecnológico; EspañaFil: Heras, José María. Lallemand Bio S.L.; EspañaFil: Guillamón, José Manuel. Consejo Superior de Investigación Científica (CSIC). Instituto de Agroquímica y Tecnología de los Alimentos (IATA). Departamento de Biotecnología de los Alimentos, Grupo de Biología de Sistemas en Levaduras de Interés Biotecnológico; EspañaFil: Ferreira, Vicente. Universidad de Zaragoza. Department of Analytical Chemistry. Laboratory for Aroma Analysis and Enology. Instituto Agroalimentario de Aragón; EspañaFil: Querol, Amparo. Consejo Superior de Investigación Científica (CSIC). Instituto de Agroquímica y Tecnología de los Alimentos (IATA). Departamento de Biotecnología de los Alimentos, Grupo de Biología de Sistemas en Levaduras de Interés Biotecnológico; Españ

Improving the Cryotolerance of Wine Yeast by Interspecific Hybridization in the Genus Saccharomyces

Fermentations carried out at low temperatures (10–15°C) enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. Notwithstanding, as Saccharomyces cerevisiae is the main species responsible for alcoholic fermentation, other species of the genus Saccharomyces, such as cryophilic species Saccharomyces eubayanus, Saccharomyces kudriavzevii and Saccharomyces uvarum, are better adapted to low-temperature fermentations during winemaking. In this work, a Saccharomyces cerevisiae × S. uvarum hybrid was constructed to improve the enological features of a wine S. cerevisiae strain at low temperature. Fermentations of white grape musts were performed, and the phenotypic differences between parental and hybrid strains under different temperature conditions were examined. This work demonstrates that hybridization constitutes an effective approach to obtain yeast strains with desirable physiological features, like low-temperature fermentation capacity, which genetically depend on the expression of numerous genes (polygenic character). As this interspecific hybridization approach is not considered a GMO, the genetically improved strains can be quickly transferred to the wine industry

On the complexity of the Saccharomyces bayanus taxon: hybridization and potential hybrid speciation

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99¿100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus. Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains

Enological characterization of Spanish Saccharomyces kudriavzevii strains, one of the closest relatives to parental strains of winemaking and brewing Saccharomyces cerevisiae × S. kudriavzevii hybrids

Wine fermentation and innovation have focused mostly on Saccharomyces cerevisiae strains. However, recent studies have shown that other Saccharomyces species can also be involved in wine fermentation or are useful for wine bouquet, such as Saccharomyces uvarum and Saccharomyces paradoxus. Many interspecies hybrids have also been isolated from wine fermentation, such as S. cerevisiae × Saccharomyces kudriavzevii hybrids. In this study, we explored the genetic diversity and fermentation performance of Spanish S. kudriavzevii strains, which we compared to other S. kudriavzevii strains. Fermentations of red and white grape musts were performed, and the phenotypic differences between Spanish S. kudriavzevii strains under different temperature conditions were examined. An ANOVA analysis suggested striking similarity between strains for glycerol and ethanol production, although a high diversity of aromatic profiles among fermentations was found. The sources of these phenotypic differences are not well understood and require further investigation. Although the Spanish S. kudriavzevii strains showed desirable properties, particularly must fermentations, the quality of their wines was no better than those produced with a commercial S. cerevisiae. We suggest hybridization or directed evolution as methods to improve and innovate wine.D.P. acknowledges the Spanish Government for its Ministry of Science and Innovation (FPI) fellowship. L.P. acknowledges the CSIC and the Spanish Ministry of Education and Science (MEC) for an I3P fellowship. This work has been supported by grants AGL2012-39937-C02-01 from the Spanish Government, FEDER and Generalitat ValencianaPROMETEOII/2014/042 to AQ.Peer reviewe

Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae × S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters).Fil: Pérez Través, Laura. Consejo Superior de Investigaciones Cientificas. Instituto de Agroquimica y Tecnologia de Alimentos; EspañaFil: Lopes, Christian Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación y Desarrollo En Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina. Consejo Superior de Investigaciones Cientificas. Instituto de Agroquimica y Tecnologia de Alimentos; España. Universidad Nacional del Comahue; ArgentinaFil: González, Ramón. Consejo Superior de Investigaciones Cientificas; EspañaFil: Barrio, Eladio. Consejo Superior de Investigaciones Cientificas. Instituto de Agroquimica y Tecnologia de Alimentos; España. Universidad de Valencia; EspañaFil: Querol. Amparo. Consejo Superior de Investigaciones Cientificas. Instituto de Agroquimica y Tecnologia de Alimentos; Españ

Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity hybrids with improved fermentation performance and mannoprotein release capacity

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae×. S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters).This work has been supported by grants AGL2012-39937-CO2 (01 and 02), and AGL2009-07327 from the Spanish Government and FEDER to A. Querol, E. Barrio and R. Gonzalez, respectively and to PROMETEO grant (Project PROMETEOII/2014/042) from the Generalitat Valenciana to A.Q. L. P-T. and C. L. and wish to acknowledge the CSIC and the Spanish Ministry of Education and Science (MEC) for an I3P fellowship and a postdoctoral contract, respectively.Peer Reviewe