In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation.

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.

Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function.

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.

Frozen-dough baking potential of psychrotolerant Saccharomyces species and derived hybrids.

Despite Saccharomyces cerevisiae being a synonym for baker's yeast, the species does not perform well in all baking-related conditions. In particular, dough fermentation, or proofing, is compromised by the species' sensitivity to the low and freezing temperatures that are often used in modern bakeries. Here, screening trials that included representatives of all known Saccharomyces species, showed that S. cerevisiae was generally the most sensitive member of the genus with respect to cold and freezing conditions. We hypothesized therefore that the superior cold tolerance of the non-S. cerevisiae yeast would enable their use as frozen-dough baking strains. To test this, the different yeast species were incorporated into doughs, flash frozen and kept in a frozen state for 14 days. During the proofing stage, dough development was lower in doughs that had been frozen, relative to fresh doughs. This reduction in fermentation performance was however most pronounced with S. cerevisiae. The psychrotolerant yeasts S. eubayanus, S. jurei and S. arboricola showed a strong capacity for post-freeze proofing in terms of dough development and duration of lag phase prior to fermentation. The superior proofing power of these species resulted in breads that were significantly softer and less dense than those prepared with S. cerevisiae. A sensory panel could distinguish the S. cerevisiae and non-S. cerevisiae breads based on their physical properties, but aroma and taste were unaffected by the species employed. To further improve frozen dough baking properties, S. eubayanus, S. jurei and S. arboricola were crossed with baker's yeast through rare mating, and hybrids with improved proofing capacities in both fresh and frozen doughs relative to the parents were created. The use of S. jurei and S. arboricola in baking represents the first potential technological application of these species.

Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations.

Hybridisation is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces genus, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in unhopped wort and at low temperatures, qualities necessary in good candidates for fermented bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft brewing and beverage industry in general. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while eliminating the hyper-attenuation characteristic and a more complex flavour profile. This study exploits the genetic diversity of yeasts and shows how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21 h-1 (µcrit), resulting in a Yx/s of 0.76 g/g at 0.21 h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133 h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61 g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83 g/g and 0.68 g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12 h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Saccharomyces bayanus Enhances Volatile Profile of Apple Brandies.

Qualitative and quantitative profiles of volatiles in alcoholic beverages depend mainly on the quality of raw materials, yeasts used for fermentation, and processing technique. Saccharomyces bayanus is a yeast species which is not commonly used for the production of alcoholic beverages, but it is able to produce volatiles that add desirable aroma. Since there is little information regarding the application of that microorganism for the production of apple brandies and how it affects volatile profile of finished products, we decided to address that issue. The aim of the study was to determine the impact of S. bayanus on the profile of volatile compounds and sensory properties of apple spirits obtained from three apple cultivars (Topaz, Rubin, and Elise) in comparison to spirits obtained from fermentation carried out spontaneously or with Saccharomyces cerevisiae. Obtained brandies were analysed using gas chromatography-flame ionization detector (GC-FID), solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and sensorially. In our study, brandies produced from musts fermented by S. bayanus demonstrated the highest concentration of ethyl esters and increased concentrations of isoamyl acetate, 2-phenylethyl acetate, ethyl palmitate and hexanol. Moreover, our results support the hypothesis that non-Saccharomyces yeasts which are present during spontaneous fermentation and demonstrate higher ß-glucosidase activities enhance aroma of alcoholic beverages through releasing aroma compounds from glycosidic forms, e.g., α-phellandrene, (E)-ß-fanesene, (Z,E)-α-farnesene, α-farnesene, and farnesol. Considering results obtained in sensory analysis, we proved that S. bayanus is suitable for the production of apple brandies, improving their flavour. Brandies obtained from musts fermented by S. bayanus obtained the highest average range for "overall note" parameter in sensory analysis.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

The impacts of Schizosaccharomyces on winemaking.

In the past century, yeasts from the genus Saccharomyces represented the only option in fermentation industries, such as winemaking, to produce wine, beer, and other fermented products. However, other genera are currently emerging to solve challenges in modern enology. Schizosaccharomyces pombe is showing promising results in solving specific challenges in northern, cool viticulture regions with highly acidic wines by deacidifying these wines through its malic acid metabolism. In addition, this microorganism is considered beneficial in warm growing regions with challenges such as the control of wine food safety problems such as the presence of biogenic amines, ochratoxin A, or ethyl carbamate. Indeed, the genus Schizosaccharomyces positively influences other important wine quality parameters, such as color and polysaccharide content. However, the main challenge of using this genus remains the selection of proper strains that alleviate problems such as the production of high acetate concentrations. Industries other than wine production such as ginger fermentation, apple wine, Kei-apple fermentation, plum wine, sparkling wine, and bilberry fermentation industries have also started to study Schizosaccharomyces species as an alternative tool for solving specific related problems. The review discusses the influence of Schizosaccharomyces on different fermentation quality parameters and its main applications in different industries.

Fungal bio-sorption potential of chromium in Norkrans liquid medium by shake flask technique.

In this study, the myco-reduction potential of fungi isolated from soil was ascertained by Norkrans shake flask experiment contaminated with chromium(VI). Fungal tolerance assay and induced tolerance training of the fungi were also carried out. Aspergillus niger, Penicillium, and Saccharomyces strains were isolated from the soil samples using culture based technique. Norkrans samples were collected and analyzed for Cr(VI) concentration using diphenylcarbazide spectrophotometric method. Penicillium strain was observed to be most effect at Cr(VI) concentrations of 16.1 and 8.1 mg L-1 since it was able to reduce Cr(VI) more than Saccharomyces strain and A. niger on day 20. Bio-sorption kinetics for this study was better described by pseudo second order model while Langmuir isotherm model fitted better to the equilibrium data. There was virtually steady increase in fungal growth for all the treatments through-out the experimental period. Significant negative correlation (p < 0.05) was observed between fungal growth and Cr(VI) reduction rate. The results from the induced tolerance training showed that Penicillium had the highest tolerance index (TI) values at 18, 20, and 25 mg L-1 concentrations of Cr(VI) compared to A. niger and Saccharomyces strain. These results demonstrated that these fungi have the potential to bio-absorb Cr(VI) and if properly harnessed, could be used in place of conventional remediation technology to clean-up the Cr(VI) contaminant in the field.

Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.

How structured yeast multicellular communities live, age and die?

Yeasts, like other microorganisms, create numerous types of multicellular communities, which differ in their complexity, cell differentiation and in the occupation of different niches. Some of the communities, such as colonies and some types of biofilms, develop by division and subsequent differentiation of cells growing on semisolid or solid surfaces to which they are attached or which they can penetrate. Aggregation of individual cells is important for formation of other community types, such as multicellular flocs, which sediment to the bottom or float to the surface of liquid cultures forming flor biofilms, organized at the border between liquid and air under specific circumstances. These examples together with the existence of more obscure communities, such as stalks, demonstrate that multicellularity is widespread in yeast. Despite this fact, identification of mechanisms and regulations involved in complex multicellular behavior still remains one of the challenges of microbiology. Here, we briefly discuss metabolic differences between particular yeast communities as well as the presence and functions of various differentiated cells and provide examples of the ability of these cells to develop different ways to cope with stress during community development and aging.

Trans-regulation and localization of orthologous maltose transporters in the interspecies lager yeast hybrid.

In the interspecies lager yeast hybrid there are MAL loci involved in maltose and maltotriose utilization derived from each parent (Saccharomyces cerevisiae and Saccharomyces eubayanus). We show that trans-regulation across hybrid subgenomes occurs for MAL genes. However, gene expression is less efficient with non-native activators (trans-activation) compared to native activators (cis-activation). MAL genes were induced by maltose and repressed by glucose irrespective of host. Despite the strong expression of S. cerevisiae-type genes in the S. eubayanus host, a very low amount of transporter protein was actually observed in cells. This suggests that proper formation and configuration of the S. cerevisiae transporters is not efficient in S. eubayanus. The S. eubayanus-type Malx1 transporter was present in the plasma membrane in high amounts in all hosts (S. cerevisiae, S. eubayanus and Saccharomyces pastorianus) at all times. However, the S. cerevisiae-type transporters appeared sequentially in the plasma membrane; scMalx1 was localized in the plasma membrane during early to late linear growth and subsequently withdrawn to intracellular compartments. In contrast, the scAgt1 transporter was found in the plasma membrane mainly in the stationary phase of growth. Different localization patterns may explain why certain transporter orthologues in natural S. pastorianus strains were lost to mutation.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history.

Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier.

Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-ß-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.

Inheritance of brewing-relevant phenotypes in constructed Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids.

BACKGROUND: Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity. RESULTS: Here we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn't produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose. CONCLUSIONS: Our study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.

Exploration of genetic and phenotypic diversity within Saccharomyces uvarum for driving strain improvement in winemaking.

The selection and genetic improvement of wine yeast is an ongoing process, since yeast strains should match new technologies in winemaking to satisfy evolving consumer preferences. A large genetic background is the necessary starting point for any genetic improvement programme. For this reason, we collected and characterized a large number of strains belonging to Saccharomyces uvarum. In particular, 70 strains were isolated from cold-stored must samples: they were identified and compared to S. uvarum strains originating from different collections, regarding fermentation profile, spore viability and stress response. The results demonstrate a large biodiversity among the new isolates, with particular emphasis to fermentation performances, genotypes and high spore viability, making the isolates suitable for further genetic improvement programmes. Furthermore, few of them are competitive with Saccharomyces cerevisiae and per se, suitable for wine fermentation, due to their resistance to stress, short lag phase and fermentation by-products.

The interactive effect of fungicide residues and yeast assimilable nitrogen on fermentation kinetics and hydrogen sulfide production during cider fermentation.

BACKGROUND: Fungicide residues on fruit may adversely affect yeast during cider fermentation, leading to sluggish or stuck fermentation or the production of hydrogen sulfide (H2 S), which is an undesirable aroma compound. This phenomenon has been studied in grape fermentation but not in apple fermentation. Low nitrogen availability, which is characteristic of apples, may further exacerbate the effects of fungicides on yeast during fermentation. The present study explored the effects of three fungicides: elemental sulfur (S0 ) (known to result in increased H2 S in wine); fenbuconazole (used in orchards but not vineyards); and fludioxonil (used in post-harvest storage of apples). RESULTS: Only S0 led to increased H2 S production. Fenbuconazole (≥0.2 mg L-1 ) resulted in a decreased fermentation rate and increased residual sugar. An interactive effect of yeast assimilable nitrogen (YAN) concentration and fenbuconazole was observed such that increasing the YAN concentration alleviated the negative effects of fenbuconazole on fermentation kinetics. CONCLUSION: Cidermakers should be aware that residual fenbuconazole (as low as 0.2 mg L-1 ) in apple juice may lead to stuck fermentation, especially when the YAN concentration is below 250 mg L-1 . These results indicate that fermentation problems attributed to low YAN may be caused or exacerbated by additional factors such as fungicide residues, which have a greater impact on fermentation performance under low YAN conditions. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic relationship and biological status of the industrially important yeast Saccharomyces eubayanus Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.

Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

Metabolic Engineering of Probiotic Saccharomyces boulardii.

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii.