Different trajectories of polyploidization shape the genomic landscape of the Brettanomyces bruxellensis yeast species.

Polyploidization events are observed across the tree of life and occur in many fungi, plant, and animal species. During evolution, polyploidy is thought to be an important source of speciation and tumorigenesis. However, the origin of polyploid populations is not always clear, and little is known about the precise nature and structure of their complex genome. Using a long-read sequencing strategy, we sequenced 71 strains from the Brettanomyces bruxellensis yeast species, which is found in anthropized environments (e.g., beer, contaminant of wine, kombucha, and ethanol production) and characterized by several polyploid subpopulations. To reconstruct the polyploid genomes, we phased them by using different strategies and found that each subpopulation had a unique polyploidization history with distinct trajectories. The polyploid genomes contain either genetically closely related (with a genetic divergence <1%) or diverged copies (>3%), indicating auto- as well as allopolyploidization events. These latest events have occurred independently with a specific and unique donor in each of the polyploid subpopulations and exclude the known Brettanomyces sister species as possible donors. Finally, loss of heterozygosity events has shaped the structure of these polyploid genomes and underline their dynamics. Overall, our study highlights the multiplicity of the trajectories leading to polyploid genomes within the same species.

Effect of abiotic and biotic factors on Brettanomyces bruxellensis bioadhesion properties.

Biofilms are central to microbial life because of the advantage that this mode of life provides, whereas the planktonic form is considered to be transient in the environment. During the winemaking process, grape must and wines host a wide diversity of microorganisms able to grow in biofilm. This is the case of Brettanomyces bruxellensis considered the most harmful spoilage yeast, due to its negative sensory effect on wine and its ability to colonise stressful environments. In this study, the effect of different biotic and abiotic factors on the bioadhesion and biofilm formation capacities of B. bruxellensis was analyzed. Ethanol concentration and pH had negligible effect on yeast surface properties, pseudohyphal cell formation or bioadhesion, while the strain and genetic group factors strongly modulated the phenotypes studied. From a biotic point of view, the presence of two different strains of B. bruxellensis did not lead to a synergistic effect. A competition between the strains was rather observed during biofilm formation which seemed to be driven by the strain with the highest bioadhesion capacity. Finally, the presence of wine bacteria reduced the bioadhesion of B. bruxellensis. Due to biofilm formation, O. oeni cells were observed attached to B. bruxellensis as well as extracellular matrix on the surface of the cells.

Brettanomyces bruxellensis: Overview of the genetic and phenotypic diversity of an anthropized yeast.

Human-associated microorganisms are ideal models to study the impact of environmental changes on species evolution and adaptation because of their small genome, short generation time, and their colonization of contrasting and ever-changing ecological niches. The yeast Brettanomyces bruxellensis is a good example of organism facing anthropogenic-driven selective pressures. It is associated with fermentation processes in which it can be considered either as a spoiler (e.g., winemaking, bioethanol production) or as a beneficial microorganism (e.g., production of specific beers, kombucha). In addition to its industrial interests, noteworthy parallels and dichotomies with Saccharomyces cerevisiae propelled B. bruxellensis as a valuable complementary yeast model. In this review, we emphasize that the broad genetic and phenotypic diversity of this species is only beginning to be uncovered. Population genomic studies have revealed the coexistence of auto- and allotriploidization events with different evolutionary outcomes. The different diploid, autotriploid and allotriploid subpopulations are associated with specific fermented processes, suggesting independent adaptation events to anthropized environments. Phenotypically, B. bruxellensis is renowned for its ability to metabolize a wide variety of carbon and nitrogen sources, which may explain its ability to colonize already fermented environments showing low-nutrient contents. Several traits of interest could be related to adaptation to human activities (e.g., nitrate metabolization in bioethanol production, resistance to sulphite treatments in winemaking). However, phenotypic traits are insufficiently studied in view of the great genomic diversity of the species. Future work will have to take into account strains of varied substrates, geographical origins as well as displaying different ploidy levels to improve our understanding of an anthropized yeast's phenotypic landscape.

Evaluating the influence of operational parameters of pulsed light on wine related yeasts: focus on inter- and intra-specific variability sensitivity.

In oenology, there is a growing demand by consumers for wines produced with less inputs (such as sulphite, frequently used for microbial control). Emerging control methods for managing microorganisms in wine are widely studied. In this study, the efficiency of pulsed light (PL) treatment was investigated. A drop-platted system was used to evaluate the impact of three PL operational parameters: the fluence per flash, the total fluence and the flash frequency. Fluence per flash appeared to be a key parameter prior to total fluence, thus demonstrating the importance of the effect of peak voltage during PL treatments. The efficiency of PL treatment was assessed on 198 strains distributed amongst fourteen yeast species related to wine environment, and an important variability in PL response was observed. Brettanomyces bruxellensis strains were strongly sensitive to PL, with intraspecific variation. PL was then applied to red wines inoculated with 9 strains of B. bruxellensis, Saccharomyces cerevisiae and Lachancea thermotolerans. Results confirmed interspecific response variability and a higher sensitivity of B. bruxellensis species to PL. Wine treatments with a total fluence of 22.8 J cm-2 resulted in more than 6 log reduction for different B. bruxellensis strains. These results highlight the potential of PL for wine microbial stabilization.

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.

Wine yeast species show strong inter- and intra-specific variability in their sensitivity to ultraviolet radiation.

While the trend in winemaking is toward reducing the inputs and especially sulphites utilization, emerging technologies for the preservation of wine is a relevant topic for the industry. Amongst yeast spoilage in wine, Brettanomyces bruxellensis is undoubtedly the most feared. In this study, UV-C treatment is investigated. This non-thermal technique is widely used for food preservation. A first approach was conducted using a drop-platted system to compare the sensitivity of various strains to UV-C surface treatment. 147 strains distributed amongst fourteen yeast species related to wine environment were assessed for six UV-C doses. An important variability in UV-C response was observed at the interspecific level. Interestingly, cellar resident species, which are mainly associated with wine spoilage, shows higher sensitivity to UV-C than vineyard-resident species. A focus on B. bruxellensis species with 104 screened strains highlighted an important effect of the UV-C, with intra-specific variation. This intra-specific variation was confirmed on 6 strains in liquid red wine by using a home-made pilot. 6624 J.L-1 was enough for a reduction of 5 log10 of magnitude for 5 upon 6 strains. These results highlight the potential of UV-C utilization against wine yeast spoiler at cellar scale.

Metabolic, Organoleptic and Transcriptomic Impact of Saccharomyces cerevisiae Genes Involved in the Biosynthesis of Linear and Substituted Esters.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.

Brettanomyces bruxellensis phenotypic diversity, tolerance to wine stress and wine spoilage ability.

Brettanomyces bruxellensis is a yeast species found in many fermented matrices. A high level of genetic diversity prevails in this species and was recently connected with tolerance to sulfur dioxide, the main preservative used in wine. We therefore examine other phenotypes that may modulate the ability of the species to spoil wine, in a selection of representative strains. The species shows a fairly high homogeneity with respect to the carbohydrates that can support growth, but more diverse behaviors regarding tolerance to low pH or ethanol. Thought no clear link can be drawn with genotype, some strains appear more tolerant than the others, mainly in the AWRI1499 like genetic group. Volatile phenol production is ubiquitous within the species, independent from yeast growth profile and not affected by the nature of the growth substrate. The specific production. n rate of volatile phenol production raises in case of increased aeration. It is little affected by pH decrease until 3.0 or by ethanol concentration increase up to 12% vol, but it decreased in case of increased constraint (pH < 3.0, Ethanol ≥14% vol) or combination of constraints. All the strain studied have thus the ability to spoil wine but some outstanding dangerous strains can even spoil the wine with high level of constrainst.

Vitis species, vintage, and alcoholic fermentation do not drive population structure in Starmerella bacillaris (synonym Candida zemplinina) species.

The yeast species Starmerella bacillaris (synonym Candida zemplinina) is widely associated with oenological ecosystems and is frequently isolated from grape and grape must. Previous work showed that the genetic diversity of this species is high in wine environments and it is shaped by geographic location. Most analysed C. zemplinina strains, however, have been isolated from Vitis vinifera, disregarding the existence of other worldwide-distributed Vitis species used in winemaking. In this work, we address the impact of the Vitis species and geographic location on the genetic diversity of C. zemplinina. Microsatellite genotyping analysis was applied to two remarkable populations of C. zemplinina from Argentina and Portugal (Azores Archipelago), isolated from neighbouring V. vinifera and Vitis labrusca vineyards. The study also included a large population of previously characterized worldwide-isolated C. zemplinina strains. Genetic analyses confirmed that geographic localization significantly shaped the genetic diversity of C. zemplinina. No genetic differentiation on the basis of the Vitis species was recorded, indicating that C. zemplinina populations from neighbouring V. vinifera and V. labrusca vineyards are genetically homogeneous. In addition, no impact of the vintage was found on the C. zemplinina populations being both highly diversified and homogeneous during initial stages of alcoholic fermentation. Altogether, these results confirmed that winemaking-related factors (i.e., vintage, Vitis species, and alcoholic fermentation) do not impact the genetic diversity of C. zemplinina and that only geographic localization significantly shapes this yeast species.

Competition experiments between Brettanomyces bruxellensis strains reveal specific adaptation to sulfur dioxide and complex interactions at intraspecies level.

Recent studies have suggested a strong niche adaptation for Brettanomyces bruxellensis strains according to human-related fermentation environments, including beer, wine and bioethanol. This is further supported by a correlation between B. bruxellensis genetic grouping and tolerance to SO2, the main antimicrobial used in wine. The allotriploid AWRI1499-like cluster, in particular, shows high SO2 tolerance suggesting that the genetic configuration observed for these strains may confer a selective advantage in winemaking conditions. To test this hypothesis, we evaluated the relative selective advantage of representatives of the three main B. bruxellensis genetic groups in presence of SO2. As a proof-of-concept and using recently developed transformation cassettes, we compared strains under different SO2 concentrations using pairwise competitive fitness experiments. Our results showed that AWRI1499 is specifically adapted to environments with high SO2 concentrations compared to other B. bruxellensis wine strains, indicating a potential correlation between allotriploidisation origin and environmental adaptation in this species. Additionally, our findings suggest different types of competition between strains, such as coexistence and exclusion, revealing new insights on B. bruxellensis interactions at intraspecies level.

Sulfur dioxide response of Brettanomyces bruxellensis strains isolated from Greek wine.

Brettanomyces bruxellensis is the most common spoilage wine yeast which can provoke great economic damage to the wine industry due to the production of undesirable odors. The capacity of the species to adapt in various environmental conditions offers a selective advantage that is reflected by intraspecific variability at genotypic and phenotypic level. In this study, microsatellite analysis of 22 strains isolated from Greek wine revealed the existence of distinct genetic subgroups that are correlated with their geographical origin. The response of these strains to increasing levels of sulfur dioxide confirmed the presence of both sensitive and tolerant strains, which belong to distinguished genetic clusters. The genetic categorization of B. bruxellensis strains could be used by the winemakers as a diagnostic tool regarding sulfur dioxide sensitivity.

Many interspecific chromosomal introgressions are highly prevalent in Holarctic Saccharomyces uvarum strains found in human-related fermentations.

In the last two decades, the extensive genome sequencing of strains belonging to the Saccharomyces genus has revealed the complex reticulated evolution of this group. Among the various evolutionary mechanisms described, the introgression of large chromosomal regions resulting from interspecific hybridization has recently shed light on Saccharomyces uvarum species. In this work we provide the de novo assembled genomes of four S. uvarum strains presenting more than 712 kb of introgressed loci inherited from both Saccharomyces eubayanus and Saccharomyces kudriavzevii species. In order to study the prevalence of such introgressions in a large population, we designed multiplexed PCR markers able to survey the inheritance of eight chromosomal regions. Our data confirm that introgressions are widely disseminated in Holarctic S. uvarum populations and are more frequently found in strains isolated from human-related fermentations. According to the origin of the strains (nature or cider- or wine-related processes), some loci are over-represented, suggesting their positive selection by human activity. Except for one locus located on chromosome 7, the introgressions present a low level of heterozygozity similar to that observed for nine neutral markers (microsatellites). Finally, most of the loci tested showed an expected Mendelian segregation after meiosis and can recombine with their chromosomal counterpart in S. uvarum. Copyright © 2017 John Wiley & Sons, Ltd.

New oenological practice to promote non-Saccharomyces species of interest: saturating grape juice with carbon dioxide.

Non-Saccharomyces yeast species, naturally found in grape must, may impact wine quality positively or negatively. In this study, a mixture of five non-Saccharomyces species (Torulaspora delbrueckii, Metschnikowia spp., Starmerella bacillaris (formerly called Candida zemplinina), Hanseniaspora uvarum, Pichia kluyveri), mimicking the composition of the natural non-Saccharomyces community found in grape must, was used for alcoholic fermentation. The impact of CO2 saturation of the grape juice was studied first on this mixture alone, and then in the presence of Saccharomyces cerevisiae. Two isogenic strains of this species were used: the first with a short and the second a long fermentation lag phase. This study demonstrated that saturating grape juice with CO2 had interesting potential as an oenological technique, inhibiting undesirable species (S. bacillaris and H. uvarum) and stimulating non-Saccharomyces of interest (T. delbrueckii and P. kluyveri). This stimulating effect was particularly marked when CO2 saturation was associated with the presence of S. cerevisiae with long fermentation lag phase. The direct consequence of this association was an enhancement of 3-SH levels in the resulting wine.

The complexity of wine: clarifying the role of microorganisms.

The concept of wine complexity has gained considerable interest in recent years, both for wine consumers and wine scientists. As a consequence, some research programs concentrate on the factors that could improve the perceived complexity of a wine. Notably, the possible influence of microbiological factors is particularly investigated. However, wine complexity is a multicomponent concept not easily defined. In this review, we first describe the actual knowledge regarding wine complexity, its perception, and wine chemical composition. In particular, we emphasize that, contrary to expectations, the perception of wine complexity is not related to wine chemical complexity. Then, we review the impact of wine microorganisms on wine complexity, with a specific focus on publications including sensory analyses. While microorganisms definitively can impact wine complexity, the underlying mechanisms and molecules are far from being deciphered. Finally, we discuss some prospective research fields that will help improving our understanding of wine complexity, including perceptive interactions, microbial interactions, and other challenging phenomena.

Combined effect of the Saccharomyces cerevisiae lag phase and the non-Saccharomyces consortium to enhance wine fruitiness and complexity.

Non-Saccharomyces (NS) species that are either naturally present in grape must or added in mixed fermentation with S. cerevisiae may impact the wine's chemical composition and sensory properties. NS yeasts are prevailing during prefermentation and early stages of alcoholic fermentation. However, obtaining the correct balance between S. cerevisiae and NS species is still a critical issue: if S. cerevisiae outcompetes the non-Saccharomyces, it may minimize their impact, while conversely if NS take over S. cerevisiae, it may result in stuck or sluggish fermentations. Here, we propose an original strategy to promote the non-Saccharomyces consortium during the prefermentation stage while securing fermentation completion: the use of a long lag phase S. cerevisiae. Various fermentations in a Sauvignon Blanc with near isogenic S. cerevisiae displaying short or long lag phase were compared. Fermentations were performed with or without a consortium of five non-Saccharomyces yeasts (Hanseniaspora uvarum, Candida zemplinina, Metschnikowia spp., Torulaspora delbrueckii, and Pichia kluyveri), mimicking the composition of natural NS community in grape must. The sensorial analysis highlighted the positive impact of the long lag phase on the wine fruitiness and complexity. Surprisingly, the presence of NS modified only marginally the wine composition but significantly impacted the lag phase of S. cerevisiae. The underlying mechanisms are still unclear, but it is the first time that a study suggests that the wine composition can be affected by the lag phase duration per se. Further experiments should address the suitability of the use of long lag phase S. cerevisiae in winemaking.

A Systems Approach to Elucidate Heterosis of Protein Abundances in Yeast.

Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis.

The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments.

The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

Linking post-translational modifications and variation of phenotypic traits.

Enzymes can be post-translationally modified, leading to isoforms with different properties. The phenotypic consequences of the quantitative variability of isoforms have never been studied. We used quantitative proteomics to dissect the relationships between the abundances of the enzymes and isoforms of alcoholic fermentation, metabolic traits, and growth-related traits in Saccharomyces cerevisiae. Although the enzymatic pool allocated to the fermentation proteome was constant over the culture media and the strains considered, there was variation in abundance of individual enzymes and sometimes much more of their isoforms, which suggests the existence of selective constraints on total protein abundance and trade-offs between isoforms. Variations in abundance of some isoforms were significantly associated to metabolic traits and growth-related traits. In particular, cell size and maximum population size were highly correlated to the degree of N-terminal acetylation of the alcohol dehydrogenase. The fermentation proteome was found to be shaped by human selection, through the differential targeting of a few isoforms for each food-processing origin of strains. These results highlight the importance of post-translational modifications in the diversity of metabolic and life-history traits.

Yeast proteome variations reveal different adaptive responses to grape must fermentation.

Saccharomyces cerevisiae and S. uvarum are two domesticated species of the Saccharomyces sensu stricto clade that diverged around 100 Ma after whole-genome duplication. Both have retained many duplicated genes associated with glucose fermentation and are characterized by the ability to achieve grape must fermentation. Nevertheless, these two species differ for many other traits, indicating that they underwent different evolutionary histories. To determine how the evolutionary histories of S. cerevisiae and S. uvarum are mirrored on the proteome, we analyzed the genetic variability of the proteomes of domesticated strains of these two species by quantitative mass spectrometry. Overall, 445 proteins were quantified. Massive variations of protein abundances were found, that clearly differentiated the two species. Abundance variations in specific metabolic pathways could be related to phenotypic traits known to discriminate the two species. In addition, proteins encoded by duplicated genes were shown to be differently recruited in each species. Comparing the strain differentiation based on the proteome variability to those based on the phenotypic and genetic variations further revealed that the strains of S. uvarum and some strains of S. cerevisiae displayed similar fermentative performances despite strong proteomic and genomic differences. Altogether, these results indicate that the ability of S. cerevisae and S. uvarum to complete grape must fermentation arose through different evolutionary roads, involving different metabolic pathways and duplicated genes.

Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level.

Although many yeasts are useful for food production and beverage, some species may cause spoilage with important economic loss. This is the case of Dekkera/Brettanomyces bruxellensis, a contaminant species that is mainly associated with fermented beverages (wine, beer, cider and traditional drinks). To better control Brettanomyces spoilage, rapid and reliable genotyping methods are necessary to determine the origins of the spoilage, to assess the effectiveness of preventive treatments and to develop new control strategies. Despite several previously published typing methods, ranging from classical molecular methods (RAPD, AFLP, REA-PFGE, mtDNA restriction analysis) to more engineered technologies (infrared spectroscopy), there is still a lack of a rapid, reliable and universal genotyping approach. In this work, we developed eight polymorphic microsatellites markers for the Brettanomyces/Dekkera bruxellensis species. Microsatellite typing was applied to the genetic analysis of wine and beer isolates from Europe, Australia and South Africa. Our results suggest that B. bruxellensis is a highly disseminated species, with some strains isolated from different continents being closely related at the genetic level. We also focused on strains isolated from two Bordeaux wineries on different substrates (grapes, red wines) and for different vintages (over half a century). We showed that all B. bruxellensis strains within a cellar are strongly related at the genetic level, suggesting that one clonal population may cause spoilage over decades. The microsatellite tool now paves the way for future population genetics research of the B. bruxellensis species.