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

Obtención, estabilización y selección de levaduras híbridas de Saccharomyces de interés enológico.

El género Saccharomyces se compone de 7 especies: S. arboricolus, S. cerevisiae, S. eubayanus, S. kudriavzevii, S. mikatae, S. paradoxus y S. uvarum. Además, en el género Saccharomyces podemos encontrar dos grandes grupos de híbridos S. pastorianus (S. cerevisiae x S. uvarum x S. eubayanus) y S. bayanus (S. uvarum x S. eubayanus), así como un número menor de híbridos que poseen porciones de S. kudriavzevii. Entre estas, se encuentran las especies de levaduras más importantes implicadas en procesos fermentativos. Aunque uno de los mecanismos más interesantes observados en la adaptación de las levaduras a procesos industriales es la formación de híbridos entre especies de este grupo. En los últimos años se han descrito híbridos entre S. cerevisiae, S. uvarum, S. eubayanus y S. kudriavzevii, presentes en la producción de cerveza, vino y sidra. La gran diversidad de híbridos dentro del género indica que este proceso es más frecuente de lo esperado. Pese a que los híbridos están menos adaptados que sus parentales a condiciones ambientales específicas, pueden adaptarse mejor a condiciones fluctuantes intermedias, lo que les proporciona una ventaja selectiva. Por otro lado, los híbridos adquieren propiedades fisiológicas de ambos parentales. Toda levadura comercial debe poseer una buena capacidad fermentativa y producir vinos con cierta calidad organoléptica. En la industria, cada vez más, se buscan aquellas levaduras que posean características fisiológicas que permitan resolver alguna de las exigencias actuales de las bodegas. Una de las principales demandas está asociada a resolver los problemas planteados con el cambio climático. El disponer de levaduras con un menor rendimiento en etanol, o que incrementen el contenido en glicerol en los vinos pueden ser buenas alternativas para resolver este tipo de problemas. Además, las levaduras también deben adaptarse a las actuales prácticas enológicas (como por ejemplo, las fermentaciones a bajas temperaturas o la adición de manoproteínas en el vino). Dado el carácter multigénico de las características a mejorar en las cepas vínicas (tales como el vigor fermentativo, el perfil de temperaturas de crecimiento, el rendimiento y tolerancia al etanol o la producción de glicerol), se tomó la hibridación como un buen mecanismo para abordar esta mejora. En primer lugar se estudió en profundidad la antigua especie S. bayanus (ahora dividida en S. eubayanus, S. uvarum e híbridos llamados S. bayanus) por ser la especie parental de los híbridos naturales que presentaba una mayor problemática. Posteriormente se estudiaron varios mecanismos de generación de híbridos entre las especies de levaduras del género Saccharomyces, como la conjugación de esporas, el rare mating o la fusión de protoplastos (esta última a modo de comparativa ya que se consideran GMOs los individuos obtenidos mediante esta técnica). Se obtuvieron híbridos tanto intra como interespecíficos, se analizó la estabilidad de estos y se estudió el proceso mediante el cual se da la estabilización; en todo momento se compararon los procesos ocurridos entre especies y entre cepas de la misma especie. Finalmente se seleccionó el mejor híbrido del cruce intraespecífico, mejorado en cuanto a su capacidad fermentativa y a la producción de manoproteínas

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

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

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

Sustained release of prostaglandin E2 in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca2+-mobilization

The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2is involved in the regulation of P2Y signaling in these cells.This work was supported by Grants BFU2011-24760 and BFU2011-24743 from MINECO, S2010/BMD-2378 from Comunidad de Madrid, Red de Investigación Cardiovascular, RIC, RD12/0042/0019, and Fundación Marcelino Botín (to María Teresa Miras-Portugal). RIC and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Molecular and enological characterization of a natural Saccharomyces uvarum and Saccharomyces cerevisiae hybrid

Saccharomyces cerevisiae plays a main role in the winemaking process, although other species, like Saccharomyces uvarum or Saccharomyces paradoxus, have been associated with must fermentations. It has been reported in recent years, that yeast hybrids of different Saccharomyces species might be responsible for wine productions. Although S. cerevisiae × Saccharomyces kudriavzevii hybrids have been well studied, very little attention has been paid to S. cerevisiae × S. uvarum hybrids. In this work we characterized the genomic composition of S6U, a widely used commercial S. cerevisiae × S. uvarum yeast hybrid isolated in wine fermentations containing one copy of the genome of each parental species, which suggests a relatively recent hybridization event. We also studied its performance under diverse enological conditions. The results show enhanced performance under low temperature enological conditions, increased glycerol production, lower acetic acid production and increased production of interesting aroma compounds. We also examined the transcriptomic response of the S6U hybrid strain compared with the reference species under enological conditions. The results show that although the hybrid strain transcriptome is more similar to S. uvarum than to S. cerevisiae, it presents specifically regulated genes involved in stress response, lipids and amino acid metabolism. The enological performance and aroma profile of this S. cerevisiae × S. uvarum hybrid makes it a good candidate for participating in winemaking, especially at low temperatures

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