Identifying and assessing the impact of wine acid-related genes in yeast.

Saccharomyces cerevisiae strains used for winemaking show a wide range of fermentation phenotypes, and the genetic background of individual strains contributes significantly to the organoleptic properties of wine. This strain-dependent impact extends to the organic acid composition of the wine, an important quality parameter. However, little is known about the genes which may impact on organic acids during grape must fermentation. To generate novel insights into the genetic regulation of this metabolic network, a subset of genes was identified based on a comparative analysis of the transcriptomes and organic acid profiles of different yeast strains showing different production levels of organic acids. These genes showed significant inter-strain differences in their transcription levels at one or more stages of fermentation and were also considered likely to influence organic acid metabolism based on existing functional annotations. Genes selected in this manner were ADH3, AAD6, SER33, ICL1, GLY1, SFC1, SER1, KGD1, AGX1, OSM1 and GPD2. Yeast strains carrying deletions for these genes were used to conduct fermentations and determine organic acid levels at various stages of alcoholic fermentation in synthetic grape must. The impact of these deletions on organic acid profiles was quantified, leading to novel insights and hypothesis generation regarding the role/s of these genes in wine yeast acid metabolism under fermentative conditions. Overall, the data contribute to our understanding of the roles of selected genes in yeast metabolism in general and of organic acid metabolism in particular.

Exploring the phenotypic space of non-Saccharomyces wine yeast biodiversity.

Tremendous microbial diversity exists in vineyards, and the potential to harness this diversity for novel mixed or pure starter cultures for wine fermentation has received significant attention in recent years. However, most studies are limited to a small subset of strains and species. Here we present data from a systematic screen of 91 yeast isolates from South African grape must and vineyard samples for oenologically relevant traits. One focus area was finding non-Saccharomyces isolates showing both reduced ethanol yields, as well as improved aromatic characteristics. Of the 91 isolates evaluated initially, 21 showed lower ethanol yields when compared to commercial wine yeast strain controls. Collectively, the metabolic data (primary fermentation and secondary aroma compounds) highlight the enormity of the 'phenotypic space' of yeast communities in South African vineyards. The data also emphasise intraspecies variability, challenging our concept of species typicity. Of particular oenological interest was the ability of several isolates to produce high levels of terpenoid compounds. A few strains were ultimately found which showed a substantial reduction (>1.5%) in the final ethanol content of sequential fermentations, as well as unique aroma compound production profiles. Four of these strains were selected for comprehensive wine trials in both red and white grape musts, complete with microbial, chemical and sensory analyses of the red wines. This presents, for the first time, a full bench-to-bottle characterisation of non-Saccharomyces strains showing the most potential for commercial application. The findings of this study enlarge the potential range of oenological applications for non-Saccharomyces yeast, while also suggesting the potential usefulness of several yeast species that have previously not been considered for winemaking applications.

The effect of scale on gene expression: commercial versus laboratory wine fermentations.

Molecular and cellular processes that are responsible for industrially relevant phenotypes of fermenting microorganisms are a central focus of biotechnological research. Such research intends to generate insights and solutions for fermentation-based industries with regards to issues such as improving product yield or the quality of the final fermentation product. For logistical reasons, and to ensure data reproducibility, such research is mostly carried out in defined or synthetic media and in small-scale fermentation vessels. Two questions are frequently raised regarding the applicability of this approach to solve problems experienced in industrial fermentations: (1) Is synthetic medium a sufficiently accurate approximation of the generally more complex natural (and frequently highly variable) substrates that are employed in most fermentation-based industries, and (2) can results obtained in small-scale laboratory fermentations be extrapolated to large-scale industrial environments? Here, we address the second question through a comparative transcriptomic approach by assessing the response of an industrial wine yeast strain fermenting a natural grape juice in small-scale laboratory and large-scale industrial conditions. In yeast, transcriptome analysis is arguably the best available tool to holistically assess the physiological state of a population and its response to changing environmental conditions. The data suggest that scale does indeed impact on some environmental parameters such as oxygen availability. However, the data show that small-scale fermentations nevertheless accurately reflect general molecular processes and adaptations during large-scale fermentation and that extrapolation of laboratory datasets to real industrial processes can be justified.

The impact of co-inoculation with Oenococcus oeni on the trancriptome of Saccharomyces cerevisiae and on the flavour-active metabolite profiles during fermentation in synthetic must.

Co-inoculation of commercial yeast strains with a bacterial starter culture at the beginning of fermentation of certain varietal grape juices is rapidly becoming a preferred option in the global wine industry, and frequently replaces the previously dominant sequential inoculation strategy where bacterial strains, responsible for malolactic fermentation, are inoculated after alcoholic fermentation has been completed. However, while several studies have highlighted potential advantages of co-inoculation, such studies have mainly focused on broad fermentation properties of the mixed cultures, and no data exist regarding the impact of this strategy on many oenologically relevant attributes of specific wine yeast strains such as aroma production. Here we investigate the impact of co-inoculation on a commercial yeast strain during alcoholic fermentation by comparing the transcriptome of this strain in yeast-only and in co-inoculated fermentations of synthetic must. The data show that a significant number of genes are differentially expressed in this strain in these two conditions. Some of the differentially expressed genes appear to respond to chemical changes in the fermenting must that are linked to bacterial metabolic activities, whereas others might represent a direct response of the yeast to the presence of a competing organism.

Comparative transcriptomic and proteomic profiling of industrial wine yeast strains.

The geno- and phenotypic diversity of commercial Saccharomyces cerevisiae wine yeast strains provides an opportunity to apply the system-wide approaches that are reasonably well established for laboratory strains to generate insight into the functioning of complex cellular networks in industrial environments. We have previously analyzed the transcriptomes of five industrial wine yeast strains at three time points during alcoholic fermentation. Here, we extend the comparative approach to include an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis of two of the previously analyzed wine yeast strains at the same three time points during fermentation in synthetic wine must. The data show that differences in the transcriptomes of the two strains at a given time point rather accurately reflect differences in the corresponding proteomes independently of the gene ontology (GO) category, providing strong support for the biological relevance of comparative transcriptomic data sets in yeast. In line with previous observations, the alignment proves to be less accurate when assessing intrastrain changes at different time points. In this case, differences between the transcriptome and proteome appear to be strongly dependent on the GO category of the corresponding genes. The data in particular suggest that metabolic enzymes and the corresponding genes appear to be strongly correlated over time and between strains, suggesting a strong transcriptional control of such enzymes. The data also allow the generation of hypotheses regarding the molecular origin of significant differences in phenotypic traits between the two strains.

Enforced Mutualism Leads to Improved Cooperative Behavior between Saccharomyces cerevisiae and Lactobacillus plantarum.

Saccharomyces cerevisiae and Lactobacillus plantarum are responsible for alcoholic and malolactic fermentation, respectively. Successful completion of both fermentations is essential for many styles of wine, and an understanding of how these species interact with each other, as well as the development of compatible pairings of these species, will help to manage the process. However, targeted improvements of species interactions are difficult to perform, in part because of the chemical and biological complexity of natural grape juice. Synthetic ecological systems reduce this complexity and can overcome these difficulties. In such synthetic systems, mutualistic growth of different species can be enforced through the reciprocal exchange of essential nutrients. Here, we implemented a novel approach to evolve mutualistic traits by establishing a co-dependent relationship between S. cerevisiae BY4742Δthi4 and Lb. plantarum IWBT B038 by omitting different combinations of amino acids from a chemically defined synthetic medium simulating standard grape juice. After optimization, the two species were able to support the growth of each other when grown in the absence of appropriate combinations of amino acids. In these obligatory mutualistic conditions, BY4742Δthi4 and IWBT B038 were co-evolved for approximately 100 generations. The selected evolved isolates showed improved mutualistic growth and the growth patterns under non-selective conditions indicate the emergence of mutually beneficial adaptations independent of the synthetic selection pressure. The combined use of synthetic ecology and co-evolution is a promising strategy to better understand and biotechnologically improve microbial interactions.

Comparative transcriptomic approach to investigate differences in wine yeast physiology and metabolism during fermentation.

Commercial wine yeast strains of the species Saccharomyces cerevisiae have been selected to satisfy many different, and sometimes highly specific, oenological requirements. As a consequence, more than 200 different strains with significantly diverging phenotypic traits are produced globally. This genetic resource has been rather neglected by the scientific community because industrial strains are less easily manipulated than the limited number of laboratory strains that have been successfully employed to investigate fundamental aspects of cellular biology. However, laboratory strains are unsuitable for the study of many phenotypes that are of significant scientific and industrial interest. Here, we investigate whether a comparative transcriptomics and phenomics approach, based on the analysis of five phenotypically diverging industrial wine yeast strains, can provide insights into the molecular networks that are responsible for the expression of such phenotypes. For this purpose, some oenologically relevant phenotypes, including resistance to various stresses, cell wall properties, and metabolite production of these strains were evaluated and aligned with transcriptomic data collected during alcoholic fermentation. The data reveal significant differences in gene regulation between the five strains. While the genetic complexity underlying the various successive stress responses in a dynamic system such as wine fermentation reveals the limits of the approach, many of the relevant differences in gene expression can be linked to specific phenotypic differences between the strains. This is, in particular, the case for many aspects of metabolic regulation. The comparative approach therefore opens new possibilities to investigate complex phenotypic traits on a molecular level.

A sparse PLS for variable selection when integrating omics data.

Recent biotechnology advances allow for multiple types of omics data, such as transcriptomic, proteomic or metabolomic data sets to be integrated. The problem of feature selection has been addressed several times in the context of classification, but needs to be handled in a specific manner when integrating data. In this study, we focus on the integration of two-block data that are measured on the same samples. Our goal is to combine integration and simultaneous variable selection of the two data sets in a one-step procedure using a Partial Least Squares regression (PLS) variant to facilitate the biologists' interpretation. A novel computational methodology called ;;sparse PLS" is introduced for a predictive analysis to deal with these newly arisen problems. The sparsity of our approach is achieved with a Lasso penalization of the PLS loading vectors when computing the Singular Value Decomposition. Sparse PLS is shown to be effective and biologically meaningful. Comparisons with classical PLS are performed on a simulated data set and on real data sets. On one data set, a thorough biological interpretation of the obtained results is provided. We show that sparse PLS provides a valuable variable selection tool for highly dimensional data sets.

Comparing the transcriptomes of wine yeast strains: toward understanding the interaction between environment and transcriptome during fermentation.

System-wide "omics" approaches have been widely applied to study a limited number of laboratory strains of Saccharomyces cerevisiae. More recently, industrial S. cerevisiae strains have become the target of such analyses, mainly to improve our understanding of biotechnologically relevant phenotypes that cannot be adequately studied in laboratory strains. Most of these studies have investigated single strains in a single medium. This experimental layout cannot differentiate between generally relevant molecular responses and strain- or media-specific features. Here we analyzed the transcriptomes of two phenotypically diverging wine yeast strains in two different fermentation media at three stages of wine fermentation. The data show that the intersection of transcriptome datasets from fermentations using either synthetic MS300 (simulated wine must) or real grape must (Colombard) can help to delineate relevant from "noisy" changes in gene expression in response to experimental factors such as fermentation stage and strain identity. The differences in the expression profiles of strains in the different environments also provide relevant insights into the transcriptional responses toward specific compositional features of the media. The data also suggest that MS300 is a representative environment for conducting research on wine fermentation and industrially relevant properties of wine yeast strains.

Linking gene regulation and the exo-metabolome: a comparative transcriptomics approach to identify genes that impact on the production of volatile aroma compounds in yeast.

BACKGROUND: 'Omics' tools provide novel opportunities for system-wide analysis of complex cellular functions. Secondary metabolism is an example of a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, is not well understood with regards to its physiological roles and genetic and biochemical regulation. Many of the metabolites produced by this network such as higher alcohols and esters are significant aroma impact compounds in fermentation products, and different yeast strains are known to produce highly divergent aroma profiles. Here, we investigated whether we can predict the impact of specific genes of known or unknown function on this metabolic network by combining whole transcriptome and partial exo-metabolome analysis. RESULTS: For this purpose, the gene expression levels of five different industrial wine yeast strains that produce divergent aroma profiles were established at three different time points of alcoholic fermentation in synthetic wine must. A matrix of gene expression data was generated and integrated with the concentrations of volatile aroma compounds measured at the same time points. This relatively unbiased approach to the study of volatile aroma compounds enabled us to identify candidate genes for aroma profile modification. Five of these genes, namely YMR210W, BAT1, AAD10, AAD14 and ACS1 were selected for overexpression in commercial wine yeast, VIN13. Analysis of the data show a statistically significant correlation between the changes in the exo-metabome of the overexpressing strains and the changes that were predicted based on the unbiased alignment of transcriptomic and exo-metabolomic data. CONCLUSION: The data suggest that a comparative transcriptomics and metabolomics approach can be used to identify the metabolic impacts of the expression of individual genes in complex systems, and the amenability of transcriptomic data to direct applications of biotechnological relevance.

Modifying Saccharomyces cerevisiae Adhesion Properties Regulates Yeast Ecosystem Dynamics.

Physical contact between yeast species, in addition to better-understood and reported metabolic interactions, has recently been proposed to significantly impact the relative fitness of these species in cocultures. Such data have been generated by using membrane bioreactors, which physically separate two yeast species. However, doubts persist about the degree that the various membrane systems allow for continuous and complete metabolic contact, including the exchange of proteins. Here, we provide independent evidence for the importance of physical contact by using a genetic system to modify the degree of physical contact and, therefore, the degree of asexual intraspecies and interspecies adhesion in yeast. Such adhesion is controlled by a family of structurally related cell wall proteins encoded by the FLO gene family. As previously shown, the expression of specific members of the FLO gene family in Saccharomyces cerevisiae dramatically changes the coadhesion patterns between this yeast and other yeast species. Here, we use this differential aggregation mediated by FLO genes as a model to assess the impact of physical contact between different yeast species on the relative fitness of these species in simplified ecosystems. The identity of the FLO gene has a marked effect on the persistence of specific non-Saccharomyces yeasts over the course of extended growth periods in batch cultures. Remarkably, FLO1 and FLO5 expression often result in opposite outcomes. The data provide clear evidence for the role of physical contact in multispecies yeast ecosystems and suggest that FLO gene expression may be a major factor in such interactions.IMPORTANCE The impact of direct (physical) versus indirect (metabolic) interactions between different yeast species has attracted significant research interest in recent years. This is due to the growing interest in the use of multispecies consortia in bioprocesses of industrial relevance and the relevance of interspecies interactions in establishing stable synthetic ecosystems. Compartment bioreactors have traditionally been used in this regard but suffer from numerous limitations. Here, we provide independent evidence for the importance of physical contact by using a genetic system, based on the FLO gene family, to modify the degree of physical contact and, therefore, the degree of asexual intraspecies and interspecies adhesion in yeast. Our results show that interspecies contact significantly impacts population dynamics and the survival of individual species. Remarkably, different members of the FLO gene family often lead to very different population outcomes, further suggesting that FLO gene expression may be a major factor in such interactions.

Co-Flocculation of Yeast Species, a New Mechanism to Govern Population Dynamics in Microbial Ecosystems.

Flocculation has primarily been studied as an important technological property of Saccharomyces cerevisiae yeast strains in fermentation processes such as brewing and winemaking. These studies have led to the identification of a group of closely related genes, referred to as the FLO gene family, which controls the flocculation phenotype. All naturally occurring S. cerevisiae strains assessed thus far possess at least four independent copies of structurally similar FLO genes, namely FLO1, FLO5, FLO9 and FLO10. The genes appear to differ primarily by the degree of flocculation induced by their expression. However, the reason for the existence of a large family of very similar genes, all involved in the same phenotype, has remained unclear. In natural ecosystems, and in wine production, S. cerevisiae growth together and competes with a large number of other Saccharomyces and many more non-Saccharomyces yeast species. Our data show that many strains of such wine-related non-Saccharomyces species, some of which have recently attracted significant biotechnological interest as they contribute positively to fermentation and wine character, were able to flocculate efficiently. The data also show that both flocculent and non-flocculent S. cerevisiae strains formed mixed species flocs (a process hereafter referred to as co-flocculation) with some of these non-Saccharomyces yeasts. This ability of yeast strains to impact flocculation behaviour of other species in mixed inocula has not been described previously. Further investigation into the genetic regulation of co-flocculation revealed that different FLO genes impact differently on such adhesion phenotypes, favouring adhesion with some species while excluding other species from such mixed flocs. The data therefore strongly suggest that FLO genes govern the selective association of S. cerevisiae with specific species of non-Saccharomyces yeasts, and may therefore be drivers of ecosystem organisational patterns. Our data provide, for the first time, insights into the role of the FLO gene family beyond intraspecies cellular association, and suggest a wider evolutionary role for the FLO genes. Such a role would explain the evolutionary persistence of a large multigene family of genes with apparently similar function.

Transcriptional regulation and the diversification of metabolism in wine yeast strains.

Transcription factors and their binding sites have been proposed as primary targets of evolutionary adaptation because changes to single transcription factors can lead to far-reaching changes in gene expression patterns. Nevertheless, there is very little concrete evidence for such evolutionary changes. Industrial wine yeast strains, of the species Saccharomyces cerevisiae, are a geno- and phenotypically diverse group of organisms that have adapted to the ecological niches of industrial winemaking environments and have been selected to produce specific styles of wine. Variation in transcriptional regulation among wine yeast strains may be responsible for many of the observed differences and specific adaptations to different fermentative conditions in the context of commercial winemaking. We analyzed gene expression profiles of wine yeast strains to assess the impact of transcription factor expression on metabolic networks. The data provide new insights into the molecular basis of variations in gene expression in industrial strains and their consequent effects on metabolic networks important to wine fermentation. We show that the metabolic phenotype of a strain can be shifted in a relatively predictable manner by changing expression levels of individual transcription factors, opening opportunities to modify transcription networks to achieve desirable outcomes.

Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation.

Suspension cultures were used as a model system to investigate sucrose metabolism in four sugarcane (Saccharum spp. interspecific hybrids) cell lines transformed with antisense neutral invertase (NI) constructs. Throughout a 14-day growth cycle two cell lines in which the antisense sequence was under the control of a tandem CaMV-35S: maize ubiquitin promoter showed a strong reduction in NI activity, as well as reduced hexose and increased sucrose concentrations in comparison to the control line. In lines where the antisense NI sequence was under the control of the weaker CaMV-35S promoter alone, changes in enzyme activity and sugar concentrations were intermediate to those of the more strongly inhibited lines and the control. In comparison to the control line, a higher sucrose to hexose ratio, i.e. increased purity, was obtained in all the lines with reduced NI activity. The in vivo rate of sucrose hydrolysis was reduced in the transgenic lines, suggesting a concomitant reduction in the flux through the 'futile cycle' of sucrose breakdown and re-synthesis. Differences between the transgenic cultures and the control were most pronounced during the early stages of the growth cycle and tapered off as the cultures matured. The transgenic cultures displayed impaired growth characteristics suggesting that the growth rate of these cells was retarded because of the reduced availability of hexoses for respiration.