Opportunistic Strains of Saccharomyces cerevisiae: A Potential Risk Sold in Food Products

In recent decades, fungal infections have emerged as an important health problem associated with more people who present deficiencies in the immune system, such as HIV or transplanted patients. Saccharomyces cerevisiae is one of the emerging fungal pathogens with a unique characteristic: its presence in many food products. S. cerevisiae has an impeccably good food safety record compared to other microorganisms like virus, bacteria and some filamentous fungi. However, humans unknowingly and inadvertently ingest large viable populations of S. cerevisiae (home-brewed beer or dietary supplements that contain yeast). In the last few years, researchers have studied the nature of S. cerevisiae strains and the molecular mechanisms related to infections. Here we review the last advance made in this emerging pathogen and we discuss the implication of using this species in food products.RP-T was supported by CICYT grants (ref. AGL2012-39937-CO2-01 and -02) from the Spanish Ministry of Education and Science and FEDER. This work was supported by grant PROMETEO (project PROMETEO/2009/019) from the Generalitat Valenciana.Peer reviewedPeer Reviewe

Increased mannoprotein content in wines produced by Saccharomyces kudriavzevii × Saccharomyces cerevisiae hybrids

Several wine quality aspects are influenced by yeast mannoproteins on account of aroma compounds retention, lactic-acid bacterial growth stimulation, protection against protein haze and astringency reduction. Thus selecting a yeast strain that produces high levels of mannoproteins is important for the winemaking industry. In this work, we observed increased levels of mannoproteins in S. cerevisiae × S. kudriavzevii hybrids, compared to the S. cerevisiae strain, in wine fermentations. Furthermore, the expression of a key gene related to mannoproteins biosynthesis, PMT1, increased in the S. cerevisiae × S. kudriavzevii hybrid. We showed that artificially constructed S. cerevisiae × S. kudriavzevii hybrids also increased the levels of mannoproteins. This work demonstrates that either natural or artificial S. cerevisiae × S. kudriavzevii hybrids present mannoprotein overproducing capacity under winemaking conditions, a desirable physiological feature for this industry. These results suggest that genome interaction in hybrids generates a physiological environment that enhances the release of mannoproteins.R. Pérez-Torrado was supported by the JAEDOC postdoctoral program. L. Pérez-Través was supported by an I3P fellowship from the CSIC. This work was supported by CICYT grants (ref. AGL2012-39937-CO2-01 and AGL2012-39937-CO2-02 and AGL2015-67504-C3-1-R) from the Spanish Ministry of Education and Science and FEDER, and by grant PROMETEO (Project PROMETEO/2009/019) from the Generalitat Valenciana.Peer reviewe

Ethanol Effects Involve Non-canonical Unfolded Protein Response Activation in Yeast Cells

The unfolded protein response (UPR) is a conserved intracellular signaling pathway that controls transcription of endoplasmic reticulum (ER) homeostasis related genes. Ethanol stress has been recently described as an activator of the UPR response in yeast Saccharomyces cerevisiae, but very little is known about the causes of this activation. Although some authors ensure that the UPR is triggered by the unfolded proteins generated by ethanol in the cell, there are studies which demonstrate that protein denaturation occurs at higher ethanol concentrations than those used to trigger the UPR. Here, we studied UPR after ethanol stress by three different approaches and we concluded that unfolded proteins do not accumulate in the ER under. We also ruled out inositol depletion as an alternative mechanism to activate the UPR under ethanol stress discarding that ethanol effects on the cell decreased inositol levels by different methods. All these data suggest that ethanol, at relatively low concentrations, does not cause unfolded proteins in the yeasts and UPR activation is likely due to other unknown mechanism related with a restructuring of ER membrane due to the effect of ethanol.EN was supported by a FPU grant from the Ministerio de Educación y Ciencia (ref. AP2009-0787). This work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and FEDER and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ.Peer reviewe

Trx2p-dependent regulation of Saccharomyces cerevisiae oxidative stress response by the Skn7p transcription factor under respiring conditions

The whole genome analysis has demonstrated that wine yeasts undergo changes in promoter regions and variations in gene copy number, which make them different to lab strains and help them better adapt to stressful conditions during winemaking, where oxidative stress plays a critical role. Since cytoplasmic thioredoxin II, a small protein with thiol-disulphide oxidoreductase activity, has been seen to perform important functions under biomass propagation conditions of wine yeasts, we studied the involvement of Trx2p in the molecular regulation of the oxidative stress transcriptional response on these strains. In this study, we analyzed the expression levels of several oxidative stress-related genes regulated by either Yap1p or the co-operation between Yap1p and Skn7p. The results revealed a lowered expression for all the tested Skn7p dependent genes in a Trx2p-deficient strain and that Trx2p is essential for the oxidative stress response during respiratory metabolism in wine yeast. Additionally, activity of Yap1p and Skn7p dependent promoters by β-galactosidase assays clearly demonstrated that Skn7p-dependent promoter activation is affected by TRX2 gene deficiency. Finally we showed that deleting the TRX2 gene causes Skn7p hyperphosphorylation under oxidative stress conditions. We propose Trx2p to be a new positive efector in the regulation of the Skn7p transcription factor that controls phosphorylation events and, therefore, modulates the oxidative stress response in yeast. © 2013 Gómez-Pastor et al.This work has been supported by grants AGL 2008-00060 and AGL2011-24353 from the Spanish Ministry of Education and Science (MEC) to EM. EG was a predoctoral fellow of the FPI program from the MEC (Spanish Ministry of Education). RG-P was a predoctoral fellow of the I3P program from the CSIC (Spanish National Research Council). RP-T was a postdoctoral fellow of the JAEDOC program from the CSIC.Peer Reviewe

Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress

Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.BO was supported by CAPES the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Brazilian Ministry of Education). This work has been supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government, FEDER, and Generalitat Valenciana PROMETEOII/2014/042 to AQ, GA CR 15-03708S from the Czech National Foundation to HS, and by the European Commission FP7: Marie Curie Initial Training Network CORNUCOPIA no. 264717 to AQ and HS.Peer reviewe

Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast

Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there is a room for ethanol tolerance improvement by enhancing UPR response.EN was supported by a FPU grant from the Ministerio de Educación, Cultura y Deporte (ref. AP2009-0787). RP was supported by a JAEDOC postdoctoral program (IATA-CSIC), co-funded by FSE. This work has been supported by grants AGL2012-39937-C02-01 from the Spanish Government, FEDER and Generalitat Valenciana PROMETEOII/2014/042 to AQ. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased Vmax of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD+/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter

Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

<p>Abstract</p> <p>Background</p> <p>Wine <it>Saccharomyces cerevisiae </it>strains, adapted to anaerobic must fermentations, suffer oxidative stress when they are grown under aerobic conditions for biomass propagation in the industrial process of active dry yeast production. Oxidative metabolism of sugars favors high biomass yields but also causes increased oxidation damage of cell components. The overexpression of the <it>TRX2 </it>gene, coding for a thioredoxin, enhances oxidative stress resistance in a wine yeast strain model. The thioredoxin and also the glutathione/glutaredoxin system constitute the most important defense against oxidation. Trx2p is also involved in the regulation of Yap1p-driven transcriptional response against some reactive oxygen species.</p> <p>Results</p> <p>Laboratory scale simulations of the industrial active dry biomass production process demonstrate that <it>TRX2 </it>overexpression increases the wine yeast final biomass yield and also its fermentative capacity both after the batch and fed-batch phases. Microvinifications carried out with the modified strain show a fast start phenotype derived from its enhanced fermentative capacity and also increased content of beneficial aroma compounds. The modified strain displays an increased transcriptional response of Yap1p regulated genes and other oxidative stress related genes. Activities of antioxidant enzymes like Sod1p, Sod2p and catalase are also enhanced. Consequently, diminished oxidation of lipids and proteins is observed in the modified strain, which can explain the improved performance of the thioredoxin overexpressing strain.</p> <p>Conclusions</p> <p>We report several beneficial effects of overexpressing the thioredoxin gene <it>TRX2 </it>in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in <it>TRX2 </it>overexpressing strain can be of special interest for several industrial applications.</p

Correction: reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

AbstractFollowing publication of this work [Gomez-Pastor et al, Microbial Cell Factories 2010, 9:9] we have noticed a production error in the article. Figure 1 in the original version showed incorrect results, with graphs having been duplicated in error from another figure. The correct results for Figure 1 are shown below. Legend to Figure 1 Improved performance of TTRX2 strain in biomass production process. (A) Biomass produced (continuous line) and oxygen saturation (discontinuous line) along bench-top trials of biomass propagation for T73 (black diamond), TTRX2 (white square) and TGSH1 (white triangle) strains by measuring OD600 from diluted samples. Average of three independent experiments and standard deviations are shown. (B) Fermentative capacity of yeast biomass collected at the end of the batch and fed-batch stages of growth in bench-top trials of ADY production. Biomass from wild-type T73 (black bars) and TTRX2 (white bars) were dehydrated until 8% moisture before performing the analysis. Data were normalized to the fermentative capacity of the batch sample from T73 strain. Average of three independent experiments and standard deviations are shown. Significantly different values compared to the control (p < 0.001) were marked by asterisk. (C) Sugar consumption profiles during microvinification experiments using natural Bobal must for T73 (closed symbols) and TTRX2 (open symbols) strains. The start of must fermentation was followed in detail during the first 6 hours for both strains T73 (closed symbol) and TTRX2 (open symbol). Averages were obtained from two independent experiments with three technical replicates for each one.Peer Reviewe