Christian A. Lopes

Natural hybrids of S. cerevisiae × S. kudriavzevii share alleles with European wild populations of Saccharomyces kudriavzevii

Saccharomyces kudriavzevii, a yeast species described from a pair of strains isolated from decayed leaves in Japan, has recently been isolated from oak barks in Portugal. Some data suggest that these European S. kudriavzevii populations could be closely related to the S. kudriavzevii genetic background present in natural hybrids isolated from wines and beers in different regions of Europe. However, a more exhaustive study of European S. kudriavzevii natural populations is necessary to confirm this observation. In this study, new S. kudriavzevii isolates were recovered from oak trees in different areas in Spain, and identified and characterized according to their molecular and physiological features. The restriction analysis of 35 nuclear genes amplified by PCR demonstrated that all Spanish S. kudriavzevii isolates exhibit three gene alleles different from those present in the Japanese S. kudriavzevii type strain, but identical to the alleles previously reported for natural S. cerevisiaexS. kudriavzevii hybrids. These results clearly support the hypothesis of a European origin for these natural hybrids.

Comparative genomics among Saccharomyces cerevisiae × Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins

BackgroundInterspecific hybrids between S. cerevisiae × S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae × S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids.ResultsComparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae × S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains.ConclusionOur data suggest that wine and beer S. cerevisiae × S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions.

Patagonian wines: the selection of an indigenous yeast starter.

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.

Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking

Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains. In this work we evaluated the usefulness of three different hybridization methods: spore to spore mating, rare-mating and protoplast fusion for the generation of intra- and inter-specific stable hybrids, being the first report about the comparison of different methods to obtain artificial hybrids to be used in fermentations. Spore to spore mating is an easy but time-consuming method; hybrids generated with this technique could lack some of the industrially relevant traits present in the parental strains because of the segregation occurred during meiosis and spore generation prior to hybridization. Hybrids obtained by protoplast fusion get the complete information of both parents but they are currently considered as genetically modified organisms (GMOs). Finally, hybrids obtained by rare-mating are easily obtained by the optimized methodology described in this work, they originally contain a complete set of chromosomes of both parents and they are not considered as GMOs. Hybrids obtained by means of the three methodological approaches showed a high genetic variability; however, a loss of genetic material was detected in most of them. Based on these results, it became evident that a last crucial aspect to be considered in every hybridization program is the genetic stabilization of recently generated hybrids that guarantee its invariability during future industrial utilization. In this work, a wine yeast genetic stabilization process was developed and vegetatively stable hybrids were obtained.

Yeast biocontrol of fungal spoilage of pears stored at low temperature.

To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at -1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.

Production of volatile phenols by Pichia manshurica and Pichia membranifaciens isolated from spoiled wines and cellar environment in Patagonia.

In order to detect spoilage yeast species in wines showing off-odors, different yeast isolation protocols were evaluated. Independently of the isolation method, only Saccharomyces cerevisiae and Pichia manshurica were detected. The spoilage capacity of P. manshurica regional isolates was evaluated in red wine and the production of volatile phenols was evidenced. To evaluate the possible source of contamination, yeasts from both grapes and cellar surfaces were obtained. Hanseniaspora uvarum and Zygoascus hellenicus were detected in both sound and damaged grapes from sunny areas. The most frequent species in cellar surfaces was Candida boidinii, Pichia membranifaciens and P. manshurica were detected in filters. The intra-specific genetic characterization of the P. manshurica isolates by mtDNA-RFLP demonstrated that the same strain was detected in both wine and filter. Most P. membranifaciens isolates produced 4-EP (maximum level of 1.895 mg/L) and particularly high levels of 4-EG (maximum level of 10.260 mg/L) were produced by P. manshurica isolates in synthetic wine-like medium. In this work the capacity of P. manshurica and P. membranifaciens species to produce volatile phenols was shown for the first time.

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.

Reconstruction of the Evolutionary History of Saccharomyces cerevisiae x S. kudriavzevii Hybrids Based on Multilocus Sequence Analysis

In recent years, interspecific hybridization and introgression are increasingly recognized as significant events in the evolution of Saccharomyces yeasts. These mechanisms have probably been involved in the origin of novel yeast genotypes and phenotypes, which in due course were to colonize and predominate in the new fermentative environments created by human manipulation. The particular conditions in which hybrids arose are still unknown, as well as the number of possible hybridization events that generated the whole set of natural hybrids described in the literature during recent years. In this study, we could infer at least six different hybridization events that originated a set of 26 S. cerevisiae x S. kudriavzevii hybrids isolated from both fermentative and non-fermentative environments. Different wine S. cerevisiae strains and European S. kudriavzevii strains were probably involved in the hybridization events according to gene sequence information, as well as from previous data on their genome composition and ploidy. Finally, we postulate that these hybrids may have originated after the introduction of vine growing and winemaking practices by the Romans to the present Northern vine-growing limits and spread during the expansion of improved viticulture and enology practices that occurred during the Late Middle Ages.

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.

Physiological characterization of Saccharomyces uvarum and Saccharomyces eubayanus from Patagonia and their potential for cidermaking

A diversity of yeast strains belonging to the cryotolerant fermentative species S. uvarum and S. eubayanus have been recovered from natural habitats and traditional fermentations in North Patagonia. The aim of this work was to evaluate the most relevant physiological features in a set of Patagonian strains belonging to S. uvarum and S. eubayanus, in order to analyze their potentiality to be used as starter cultures for cidermaking elaborated at low temperature. We evidenced that S. uvarum strains isolated from natural habitats (Araucaria araucana bark) showed similar physiological features to S. eubayanus strains obtained from the same habitat, and different from S. uvarum strains from fermentative environments (apple chichas). We also confirm the capacity of S. uvarum to produce high glycerol levels, low acetic acid and elevated production of the higher alcohol 2-phenylethanol and 2-phenylethyl acetate and demonstrated similar properties in S. eubayanus. Finally, we evidenced for the first time the antagonistic activity of S. eubayanus and selected three strains (two S. uvarum and one S. eubayanus) bearing the best combination of features to be used as a starter culture in cidermaking.