Understanding the transcriptomic response of Lactiplantibacillus pentosus LPG1 during Spanish-style green table olive fermentations.

Lactiplantibacillus pentosus (Lbp. pentosus) is a species of lactic acid bacteria with a great relevance during the table olive fermentation process, with ability to form non-pathogenic biofilms on olive epidermis. The objective of this work is to deepen into the genetic mechanisms of adaptation of Lpb. pentosus LPG1 during Spanish-style green table olive fermentations, as well as to obtain a better understanding of the mechanisms of adherence of this species to the fruit surface. For this purpose, we have carried out a transcriptomic analysis of the differential gene expression of this bacterium during 60 days of fermentation in both brine and biofilms ecosystems. In brines, it was noticed that a total of 235 genes from Lpb. pentosus LPG1 were differentially expressed during course of fermentation and grouped into 9 clusters according to time-course analysis. Transport and metabolism of carbohydrates and amino acids, energy production, lactic acid and exopolysaccharide synthesis genes increased their expression in the planktonic cells during course of fermentation. On the other hand, expression of genes associated to stress response, bacteriocin synthesis and membrane protein decreased. A total of 127 genes showed significant differential expression between Lpb. pentosus LPG1 planktonic (brine) and sessile (biofilms) cells at the end of fermentation process (60 days). Among the 64 upregulated genes in biofilms, we found genes involved in adhesion (strA), exopolysaccharide production (ywqD, ywqE, and wbnH), cell shape and elongation (MreB), and well as prophage excision. Deeping into the genetic bases of beneficial biofilm formation by Lpb. pentosus strains with probiotic potential will help to turn this fermented vegetable into a carrier of beneficial microorganisms to the final consumers.

Reduction of ethanol content in wine with an improved combination of yeast strains and process conditions.

One interesting strategy to address the increasing alcohol content of wines, associated with climate change, is to reduce the ethanol yield during fermentation. Within this strategy, the approach that would allow the clearest reduction in alcohol content is the respiration of part of the grape sugars by yeasts. Non-Saccharomyces species can be used for this purpose but suffer from a limited ability to dominate the process and complete fermentation. In turn, Saccharomyces cerevisiae shows a high production of acetic acid under the growth conditions required for respiration. Previously proposed procedures used combinations of non-Saccharomyces and S. cerevisiae starters, or a strain of S. cerevisiae (PR1018), with unique metabolic properties. In both cases, precise management of oxygen availability was required to overcome the acetic acid problem. In this work, we have developed a laboratory scale process to take advantage of the properties of PR1018 and a strain of Metschnikowia pulcherrima. This process is more robust than the previous ones and does not rely on strict control of oxygenation or even the use of this particular strain of S. cerevisiae. Aeration can be interrupted instantly without impairing the volatile acidity. Under the selected conditions, an ethanol reduction of around 3% (v/v) was obtained compared to the standard fermentation control.

Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions.

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.

Saccharomyces cerevisiae responds similarly to co-culture or to a fraction enriched in Metschnikowia pulcherrima extracellular vesicles.

The recent introduction of non-conventional yeast species as companion wine starters has prompted a growing interest in microbial interactions during wine fermentation. There is evidence of interactions through interference and exploitation competition, as well as interactions depending on physical contact. Furthermore, the results of some transcriptomic analyses suggest interspecific communication, but the molecules or biological structures involved in recognition are not well understood. In this work, we explored extracellular vesicles (EVs) as possible mediators of interspecific communication between wine yeasts. The transcriptomic response of Saccharomyces cerevisiae after 3 h of contact with a fraction enriched in EVs of Metschnikowia pulcherrima was compared with that induced by active M. pulcherrima cells. Interestingly, there is a high level of overlap between the transcriptomic profiles of yeast cells challenged by either M. pulcherrima whole cells or the EV-enriched fraction. The results indicate an upregulation of yeast metabolism in response to competing species (in line with previous results). This finding points to the presence of a signal, in the EV-enriched fraction, that can be perceived by the yeast cells as a cue for the presence of competitors, even in the absence of metabolically active cells of the other species.

Identifying the Main Drivers in Microbial Diversity for Cabernet Sauvignon Cultivars from Europe to South Africa: Evidence for a Cultivar-Specific Microbial Fingerprint.

Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome.

Protein content of the Oenococcus oeni extracellular vesicles-enriched fraction.

Malolactic fermentation is essential for the quality of red wines and some other wine styles. Spontaneous malolactic fermentation is often driven by Oenococcus oeni, and commercial starters for this purpose are also often of this species. The increasing number of microbial species and inoculation strategies in winemaking has prompted a growing interest in microbial interactions during wine fermentation. Among other interaction mechanisms, extracellular vesicles have been hypothesized to play a role in this context. Extracellular vesicles have already been described and analysed for several wine yeast species. In this work, the production of extracellular vesicles by O. oeni is reported for the first time. The protein content of these extracellular vesicles is also characterised. It shows differences and similarities with the recently described protein content of Lactiplantibacillus plantarum, a bacterial species also capable of performing malolactic fermentation of wine (and used sometimes as an alternative starter). This work further contributes to the development of the field of extracellular vesicles in food biotechnology.

Exploring the suitability of Saccharomyces cerevisiae strains for winemaking under aerobic conditions.

Aerobic fermentation was previously proposed to reduce the ethanol content of wine. The main constraint found for Saccharomyces cerevisiae to be used under these conditions was the high levels of acetic acid produced by all S. cerevisiae strains previously tested. This work addressed the identification of S. cerevisiae wine yeast strains suitable for aerobic fermentation and the optimization of fermentation conditions to obtain a reduced ethanol yield with acceptable volatile acidity. This approach unveiled a great diversity in acetic acid yield for different S. cerevisiae strains under aerobic conditions, with some strains showing very low volatile acidity. Three strains were selected for further characterization in bioreactors, with natural grape must, under aerobic and anaerobic conditions. Ethanol yields were lower under aerobic than under anaerobic conditions for all strains, and acetic acid levels were low for two of them. Strain-dependent changes in volatile compounds were also observed between aerobic and anaerobic conditions. Finally, the process was optimized at laboratory scale for one strain. This is the first report of S. cerevisiae wine strains showing low acetic acid production under aerobic conditions and paves the way for simplified aerobic fermentation protocols aimed to reducing the alcohol content of wines.

Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation.

One of the most prominent consequences of global climate warming for the wine industry is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the several approaches to address this important issue, this review focuses on biotechnological solutions, mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields. Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for this purpose. Genetic improvement is also playing a role in the development of biotechnological tools to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to research, but its contribution to the advancement of the field is still relevant. Furthermore, genetic improvement by non-GMO approaches is providing some interesting results, and will probably result in the development of commercial yeast strains with a lower alcohol yield in the near future. The optimization of fermentation processes using natural isolates is, anyway, the most probable source of advancement in the short term for the production of wines with lower alcohol contents.

Mechanisms Involved in Interspecific Communication between Wine Yeasts.

In parallel with the development of non-Saccharomyces starter cultures in oenology, a growing interest has developed around the interactions between the microorganisms involved in the transformation of grape must into wine. Nowadays, it is widely accepted that the outcome of a fermentation process involving two or more inoculated yeast species will be different from the weighted average of the corresponding individual cultures. Interspecific interactions between wine yeasts take place on several levels, including interference competition, exploitation competition, exchange of metabolic intermediates, and others. Some interactions could be a simple consequence of each yeast running its own metabolic programme in a context where metabolic intermediates and end products from other yeasts are present. However, there are clear indications, in some cases, of specific recognition between interacting yeasts. In this article we discuss the mechanisms that may be involved in the communication between wine yeasts during alcoholic fermentation.

Proteomic Characterization of EVs in Non-pathogenic Yeast Cells.

Most research on extracellular vesicles (EVs) from non-pathogenic fungi has been conducted in S. cerevisiae, taking advantage of the tools available for this model organism; but a few studies on EVs from yeasts of biotechnological interest are also available. Proteomic analyses in EVs from different yeast species and under different culture conditions are consistent in the identification of proteins related to glycolysis and cell wall biogenesis. Consequently, cell wall metabolism and biosynthesis appear as major functions of EVs. Additional functions have been proposed attending to the known biological activities identified on EVs proteomes, including interspecific antagonism, protection against antimicrobial agents, or clearance of aggregates of misfolded proteins (e.g. prion-like proteins). However, caution should be taken since some of these proteins might play a different role in the intracellular space or EVs (including some well known moonlighting proteins). It is also possible that many proteins appear in EVs as an indirect consequence of cellular metabolism and protein traffic, not related to a specific role in the extracellular space. These considerations become especially relevant in the context of the increasing detection power of proteomic technologies, leading in some cases to the identification of thousands of different proteins in the EVs proteome. Mutations in different secretory pathways have been related to differences in protein cargo of EVs, but no mutation has been found completely abolishing the production of EVs. Further work on the composition and biogenesis of EVs is required to better understand their biological significance.

Metschnikowia pulcherrima represses aerobic respiration in Saccharomyces cerevisiae suggesting a direct response to co-cultivation.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics.

Proteomic characterization of extracellular vesicles produced by several wine yeast species.

In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with Saccharomyces cerevisiae, to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species (S. cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans, Hanseniaspora uvarum, Candida sake and Metschnikowia pulcherrima) in synthetic grape must. Proteomic analysis of EV-enriched fractions from S. cerevisiae and T. delbrueckii showed enrichment in glycolytic enzymes and cell-wall-related proteins. The most abundant protein found in S. cerevisiae, T. delbrueckii and L. thermotolerans EV-enriched fractions was the enzyme exo-1,3-ß-glucanase. However, this protein was not involved in the here-observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology.

Autophagy is required for sulfur dioxide tolerance in Saccharomyces cerevisiae.

Sulfiting agents are among the most widely used preservatives in the food and beverages industries, including winemaking, and one of their main functions is inhibition of spoilage microorganisms. We have used a whole genome quantitative fitness analysis in order to improve our knowledge on yeast tolerance to sulfites. Apart from the contribution of sulfite efflux to tolerance, results point to vesicle-mediated transport, autophagy and vacuolar activity as the main cellular functions required to survive sulfite challenges. The involvement of autophagic and vacuolar functions in sulfite tolerance was further confirmed by pairwise competition using a newly constructed atg2-defective strain, as well as by showing induction of ATG8 expression by sulfite. Autophagy is required for the turnover of proteins and subcellular structures damaged by sulfite. In addition, the requirement for vacuolar functions might be related to its role in intracellular pH homeostasis. Finally, the involvement of the sulfite pump Ssu1 and the transcription factor Fzf1 in sulfite tolerance by Saccharomyces cerevisiae was confirmed; a result that validates the experimental approach used in this work. These findings have relevance for understanding sulfite toxicity and tolerance, as well as for the eventual design of strategies aiming to control yeast spoilage.

Evolution of a Yeast With Industrial Background Under Winemaking Conditions Leads to Diploidization and Chromosomal Copy Number Variation.

Industrial wine yeast strains show genome particularities, with strains showing polyploid genomes or chromosome copy number variations, being easier to identify. Although these genomic structures have classically been considered transitory steps in the genomic adaptation to new environmental conditions, they may be more stable than thought. These yeasts are highly specialized strains able to cope with the different stresses associated with the fermentation process, from the high osmolarity to the final ethanol content. In this work, we use adaptive laboratory evolution, focusing on the initial steps of the fermentation process, where growth rate is maximum, to provide new insights into the role of the different genomic and chromosomic rearrangements that occur during adaptation to wine conditions, and providing an understanding of the chronology of the different evolutionary steps.

Low Phenotypic Penetrance and Technological Impact of Yeast [GAR +] Prion-Like Elements on Winemaking.

[GAR +] prion-like elements partially relieve carbon catabolite repression in Saccharomyces cerevisiae. They have been hypothesized to contribute to wine yeast survival and alcohol level reduction, as well as communication with bacteria and stuck fermentation. In this work, we selected [GAR +] derivatives from several genetic backgrounds. They were characterized for phenotypic penetrance, heritability and confirmed as prion-like through curing by desiccation. In terms of fermentation kinetics, the impact of the prion on anaerobic wine fermentation (natural grape juice) was either neutral or negative, depending on the genetic background. Likewise, residual sugars were higher or similar for [GAR +] as compared to the cognate [gar -] strains. The prions had little or no impact on glycerol and ethanol yields; while acetic acid yields experienced the highest variations between [GAR +] and [gar -] strains. Strains analyzed under aerobic conditions followed the same pattern, with either little or no impact on fermentation kinetics, ethanol or glycerol yield; and a clearer influence on volatile acidity. Although no clear winemaking advantages were found for [GAR +] strains in this work, they might eventually show interest for some combinations of genetic background or winemaking conditions, e.g., for reducing acetic acid yield under aerated fermentation.

Aroma profiling of an aerated fermentation of natural grape must with selected yeast strains at pilot scale.

The use of non-Saccharomyces strains in aerated conditions has proven effective for alcohol content reduction in wine during lab-scale fermentation. The process has been scaled up to 20 L batches, in order to produce lower alcohol wines amenable to sensory analysis. Sequential instead of simultaneous inoculation was chosen to prevent oxygen exposure of Saccharomyces cerevisiae during fermentation, since previous results indicated that this would result in increased acetic acid production. In addition, an adaptation step was included to facilitate non-Saccharomyces implantation in natural must. Wines elaborated with Torulaspora delbrueckii or Metschnikowia pulcherrima in aerated conditions contained less alcohol than control wine (S. cerevisiae, non-aerated). Sensory and aroma analysis revealed that the quality of mixed fermentations was affected by the high levels of some yeast amino acid related byproducts, which suggests that further progress requires a careful selection of non-Saccharomyces strains and the use of specific N-nutrients.

Different Non-Saccharomyces Yeast Species Stimulate Nutrient Consumption in S. cerevisiae Mixed Cultures.

The growing interest of the winemaking industry on the use of non-Saccharomyces starters has prompted several studies about the physiological features of this diverse group of microorganisms. The fact that the proposed use of these new starters will almost invariably involve either simultaneous or sequential inoculation with Saccharomyces cerevisiae has also driven the attention to the potential biological interactions between different starters during wine fermentation. Our current understanding is that alternative yeast starters will affect wine features by both direct and indirect mechanisms (through metabolic or other types of interactions with S. cerevisiae). There are still few studies addressing the question of yeast-yeast interactions in winemaking by a transcriptomic approach. In a previous report, we revealed early responses of S. cerevisiae and Torulaspora delbrueckii to the presence of each other under anaerobic conditions, mainly the overexpression of genes related with sugar consumption and cell proliferation. We have now studied the response, under aerobic conditions, of S. cerevisiae to other two non-Saccharomyces species, Hanseniaspora uvarum and Candida sake, keeping T. delbrueckii as a reference; and always focusing on the early stages of the interaction. Results point to some common features of the way S. cerevisiae modifies its transcriptome in front of other yeast species, namely activation of glucose and nitrogen metabolism, being the later specific for aerobic conditions.

Transcriptomic analysis of Saccharomyces cerevisiae x Saccharomyceskudriavzevii hybrids during low temperature winemaking.

BACKGROUND: Although Saccharomyces cerevisiae is the most frequently isolated species in wine fermentation, and the most studied species, other species and interspecific hybrids have greatly attracted the interest of researchers in this field in the last few years, given their potential to solve new winemaking industry challenges. S. cerevisiae x S. kudriavzevii hybrids exhibit good fermentative capabilities at low temperatures, and produce wines with smaller alcohol quantities and larger glycerol quantities, which can be very useful to solve challenges in the winemaking industry such as the necessity to enhance the aroma profile. METHODS: In this study, we performed a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids in low temperature winemaking conditions. RESULTS: The results revealed that the hybrids have acquired both fermentative abilities and cold adaptation abilities, attributed to S. cerevisiae and S. kudriavzevii parental species, respectively, showcasing their industrially relevant characteristics. For several key genes, we also studied the contribution to gene expression of each of the alleles of S. cerevisiae and S. kudriavzevii in the S. cerevisiae x S. kudriavzevii hybrids. From the results, it is not clear how important the differential expression of the specific parental alleles is to the phenotype of the hybrids. CONCLUSIONS: This study shows that the fermentative abilities of S. cerevisiae x S. kudriavzevii hybrids at low temperatures do not seem to result from differential expression of specific parental alleles of the key genes involved in this phentoype.

Hypoxia and iron requirements are the main drivers in transcriptional adaptation of Kluyveromyces lactis during wine aerobic fermentation.

The respiratory metabolism of yeast species alternative to Saccharomyces cerevisiae has been explored in recent years as a tool to reduce ethanol content in grape wine. The efficacy of this strategy has been previously proven for mixed cultures of non-Saccharomyces and S. cerevisiae strains. In this work, we perform a transcriptomic analysis of the Crabtree-negative yeast Kluyveromyces lactis under tightly controlled growth conditions in order to better understand physiology of non-Saccharomyces yeasts during the fermentation of grape must under aerated conditions. Transcriptional changes in K. lactis are mainly driven by oxygen limitation, iron requirement, and oxidative stress. Oxidative stress appears as a consequence of the hypoxic conditions achieved by K. lactis once oxygen supply is no longer sufficient to sustain fully respiratory metabolism. This species copes with low oxygen and iron availability by repressing iron consuming pathways and activating iron transport mechanisms. Most of the physiological and transcriptomic features of K. lactis in aerobic wine fermentation are not shared with the Crabtree-positive yeast S. cerevisiae.