Publications by authors named "Agustín Aranda"

33 Publications

Wine yeast peroxiredoxin plays a role in growth, stress response and trehalose metabolism in biomass propagation.

Microorganisms 2020 Oct 6;8(10). Epub 2020 Oct 6.

Institute for Integrative Systems Biology, I2SysBio, University of Valencia-CSIC, 7, 46980 Paterna, Spain.

Peroxiredoxins are a family of peroxide-degrading enzymes for challenging oxidative stress. They receive their reducing power from redox-controlling proteins called thioredoxins, and these, in turn, from thioredoxin reductase. The main cytosolic peroxiredoxin is Tsa1, a moonlighting protein that also acts as protein chaperone a redox switch controlling some metabolic events. Gene deletion of peroxiredoxins in wine yeasts indicate that , thioredoxins and thioredoxin reductase are required for normal growth in medium with glucose and sucrose as carbon sources. gene deletion also diminishes growth in molasses, both in flasks and bioreactors. The mutation brings about an expected change in redox parameters but, interestingly, it also triggers a variety of metabolic changes. It influences trehalose accumulation, lowering it in first molasses growth stages, but increasing it at the end of batch growth, when respiratory metabolism is set up. Glycogen accumulation at the entry of the stationary phase also increases in the D mutant. The mutation reduces fermentative capacity in grape juice, but the vinification profile does not significantly change. However, acetic acid and acetaldehyde production decrease when is absent. Hence, plays a role in the regulation of metabolic reactions leading to the production of such relevant enological molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8101537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600145PMC
October 2020

Basal catalase activity and high glutathione levels influence the performance of non-Saccharomyces active dry wine yeasts.

Food Microbiol 2020 Dec 30;92:103589. Epub 2020 Jun 30.

Institute for Integrative Systems Biology (I2SysBio), Universitat de València-CSIC, C/ Catedrático José Beltrán, 2, 46980, Paterna, Valencia, Spain. Electronic address:

Non-Saccharomyces wine yeasts are useful tools for producing wines with complex aromas or low ethanol content. Their use in wine would benefit from their production as active dry yeast (ADY) starters to be used as co-inocula alongside S. cerevisiae. Oxidative stress during biomass propagation and dehydration is a key factor in determining ADY performance, as it affects yeast vitality and viability. Several studies have analysed the response of S. cerevisiae to oxidative stress under dehydration conditions, but not so many deal with non-conventional yeasts. In this work, we analysed eight non-Saccharomyces wine yeasts under biomass production conditions and studied oxidative stress parameters and lipid composition. The results revealed wide variability among species in their technological performance during ADY production. Also, for Metschnikowia pulcherrima and Starmerella bacillaris, better performance correlates with high catalase activity and glutathione levels. Our data suggest that non-Saccharomyces wine yeasts with an enhanced oxidative stress response are better suited to grow under ADY production conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fm.2020.103589DOI Listing
December 2020

Role of Nutrient Signaling Pathways During Winemaking: A Phenomics Approach.

Front Bioeng Biotechnol 2020 22;8:853. Epub 2020 Jul 22.

Institute for Integrative Systems Biology (I2SysBio), University of Valencia-CSIC, Valencia, Spain.

The ability of the yeast to adapt to the changing environment of industrial processes lies in the activation and coordination of many molecular pathways. The most relevant ones are nutrient signaling pathways because they control growth and stress response mechanisms as a result of nutrient availability or scarcity and, therefore, leave an ample margin to improve yeast biotechnological performance. A standardized grape juice fermentation assay allowed the analysis of mutants for different elements of many nutrient signaling pathways under different conditions (low/high nitrogen and different oxygenation levels) to allow genetic-environment interactions to be analyzed. The results indicate that the cAMP-dependent PKA pathway is the most relevant regardless of fermentation conditions, while mutations on TOR pathways display an effect that depends on nitrogen availability. The production of metabolites of interest, such as glycerol, acetic acid and pyruvate, is controlled in a coordinated manner by the contribution of several components of different pathways. Ras GTPase Ras2, a stimulator of cAMP production, is a key factor for achieving fermentation, and is also relevant for sensing nitrogen availability. Increasing cAMP concentrations by deleting an enzyme used for its degradation, phosphodiesterase Pde2, proved a good way to increase fermentation kinetics, and offered keys for biotechnological improvement. Surprisingly glucose repression protein kinase Snf1 and Nitrogen Catabolite Repression transcription factor Gln3 are relevant in fermentation, even in the absence of starvation. Gln3 proved essential for respiration in several genetic backgrounds, and its presence is required to achieve full glucose de-repression. Therefore, most pathways sense different types of nutrients and only their coordinated action can ensure successful wine fermentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.00853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387434PMC
July 2020

Saccharomyces cerevisiae nutrient signaling pathways show an unexpected early activation pattern during winemaking.

Microb Cell Fact 2020 Jun 6;19(1):124. Epub 2020 Jun 6.

Institute for Integrative Systems Biology, I2SysBio, University of Valencia-CSIC, Parc Cientific UV. Av. Agustín Escardino 9, Paterna, 46980, Valencia, Spain.

Background: Saccharomyces cerevisiae wine strains can develop stuck or sluggish fermentations when nutrients are scarce or suboptimal. Nutrient sensing and signaling pathways, such as PKA, TORC1 and Snf1, work coordinately to adapt growth and metabolism to the amount and balance of the different nutrients in the medium. This has been exhaustively studied in laboratory strains of S. cerevisiae and laboratory media, but much less under industrial conditions.

Results: Inhibitors of such pathways, like rapamycin or 2-deoxyglucose, failed to discriminate between commercial wine yeast strains with different nutritional requirements, but evidenced genetic variability among industrial isolates, and between laboratory and commercial strains. Most signaling pathways involve events of protein phosphorylation that can be followed as markers of their activity. The main pathway to promote growth in the presence of nitrogen, the TORC1 pathway, measured by the phosphorylation of Rps6 and Par32, proved active at the very start of fermentation, mainly on day 1, and ceased soon afterward, even before cellular growth stopped. Transcription factor Gln3, which activates genes subject to nitrogen catabolite repression, was also active for the first hours, even when ammonium and amino acids were still present in media. Snf1 kinase was activated only when glucose was exhausted under laboratory conditions, but was active from early fermentation stages. The same results were generally obtained when nitrogen was limiting, which indicates a unique pathway activation pattern in winemaking. As PKA remained active throughout fermentation, it could be the central pathway that controls others, provided sugars are present.

Conclusions: Wine fermentation is a distinct environmental situation from growth in laboratory media in molecular terms. The mechanisms involved in glucose and nitrogen repression respond differently under winemaking conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12934-020-01381-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275465PMC
June 2020

Evaluation of yeasts from Ecuadorian chicha by their performance as starters for alcoholic fermentations in the food industry.

Int J Food Microbiol 2020 Mar 26;317:108462. Epub 2019 Nov 26.

Institute for Integrative Systems Biology (I2SysBio), Universitat de València-CSIC, C/ Catedrático José Beltran, 2, 46980 Paterna, Valencia, Spain. Electronic address:

Yeasts involved in the spontaneous fermentation of traditional beverages like chicha (indigenous Andean beer) may have the potential to be used as starter cultures to improve the quality and microbiological safety of these products, but also as non-conventional alternatives to other food alcoholic fermentations. In this research, we isolated, identified and characterised yeast strains from four Ecuadorian chichas made by using four different raw materials: rice (RC), oat (OC), grape (GC) and a mixture of seven corn varieties (yamor, YC). Finally, 254 yeast isolates were obtained and identified by molecular methods. Eleven yeast genera and 16 yeast species were identified with relatively few isolates belonging to Saccharomyces cerevisiae (9.1% belonging to 6 strains) and Torulaspora delbrueckii (18.6% belonging to 2 strains). In order to select good candidates for fermentative starter production, different analyses were performed. The results of the stress response tests showed a wide variability between species and strains, and identified some yeasts displaying high stress tolerance, similarly to commercial wine strains. Amylase production was screened as being indicative of the capacity to degrade and ferment starch-rich substrates. A Cryptococcus sp. isolate showed the highest amylase activity. The growth rate and fermentative capacity in molasses medium was measured for three S. cerevisiae, T. delbrueckii and Candida sp. strains as tests for yield and performance in biomass production. Based on their excellent behaviour, three S. cerevisiae strains and one T. delbrueckii strain were selected for further analyses, including dehydration tolerance and invertase activity as additional desired traits for chicha starters. All the S. cerevisiae strains exhibited high invertase activity and one also displayed high resistance to dehydration. The yeasts selected in this study can thus be suitably used as dry starters for the microbiologically controlled production of traditional beverages, and also for other alcoholic fermentations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijfoodmicro.2019.108462DOI Listing
March 2020

Saccharomyces cerevisiae Cytosolic Thioredoxins Control Glycolysis, Lipid Metabolism, and Protein Biosynthesis under Wine-Making Conditions.

Appl Environ Microbiol 2019 04 22;85(7). Epub 2019 Mar 22.

Institute for Integrative Systems Biology, I2SysBio, University of Valencia-CSIC, Paterna, Spain

Thioredoxins are small proteins that regulate the cellular redox state, prevent oxidative damage, and play an active role in cell repair. Oxidative stress has proven to be of much relevance in biotechnological processes when the metabolism of is mainly respiratory. During wine yeast starter production, active dry yeast cytosolic thioredoxin Trx2p is a key player in protecting metabolic enzymes from being oxidized by carbonylation. Less is known about the role of redox control during grape juice fermentation. A mutant strain that lacked both cytosolic thioredoxins, Trx1p and Trx2p, was tested for grape juice fermentation. Its growth and sugar consumption were greatly impaired, which indicates the system's relevance under fermentative conditions. A proteomic analysis indicated that deletion of the genes and caused a reduction in the ribosomal proteins and factors involved in translation elongation in addition to enzymes for glycolysis and amino acid biosynthesis. A metabolomic analysis of the Δ Δ mutant showed an increase in most proteogenic amino acids, phospholipids, and sphingolipids and higher fatty acid desaturase Ole1p content. Low glycolytic activity was behind the reduced growth and fermentative capacity of the thioredoxin deletion strain. All three hexokinases were downregulated in the mutant strain, but total hexokinase activity remained, probably due to posttranslational regulation. Pyruvate kinase Cdc19p presented an early level of aggregation in the Δ Δ mutant, which may contribute to a diminished hexose metabolism and trigger regulatory mechanisms that could influence the level of glycolytic enzymes. Oxidative stress is a common hazardous condition that cells have to face in their lifetime. Oxidative damage may diminish cell vitality and viability by reducing metabolism and eventually leading to aging and ultimate death. Wine yeast also faces oxidative attack during its biotechnological uses. One of the main yeast antioxidant systems involves two small proteins called thioredoxins. When these two proteins are removed, wine yeast shows diminished growth, protein synthesis, and sugar metabolism under wine-making conditions, and amino acid and lipid metabolism are also affected. Altogether, our results indicate that proper redox regulation is a key factor for metabolic adaptations during grape juice fermentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.02953-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585497PMC
April 2019

Yeast thioredoxin reductase Trr1p controls TORC1-regulated processes.

Sci Rep 2018 11 7;8(1):16500. Epub 2018 Nov 7.

Institute for Integrative Systems Biology, I2SysBio, University of Valencia-CSIC, Valencia, Spain.

The thioredoxin system plays a predominant role in the control of cellular redox status. Thioredoxin reductase fuels the system with reducing power in the form of NADPH. The TORC1 complex promotes growth and protein synthesis when nutrients, particularly amino acids, are abundant. It also represses catabolic processes, like autophagy, which are activated during starvation. We analyzed the impact of yeast cytosolic thioredoxin reductase TRR1 deletion under different environmental conditions. It shortens chronological life span and reduces growth in grape juice fermentation. TRR1 deletion has a global impact on metabolism during fermentation. As expected, it reduces oxidative stress tolerance, but a compensatory response is triggered, with catalase and glutathione increasing. Unexpectedly, TRR1 deletion causes sensitivity to the inhibitors of the TORC1 pathway, such as rapamycin. This correlates with low Tor2p kinase levels and indicates a direct role of Trr1p in its stability. Markers of TORC1 activity, however, suggest increased TORC1 activity. The autophagy caused by nitrogen starvation is reduced in the trr1Δ mutant. Ribosomal protein Rsp6p is dephosphorylated in the presence of rapamycin. This dephosphorylation diminishes in the TRR1 deletion strain. These results show a complex network of interactions between thioredoxin reductase Trr1p and the processes controlled by TOR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-34908-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220292PMC
November 2018

Non-canonical regulation of glutathione and trehalose biosynthesis characterizes non- wine yeasts with poor performance in active dry yeast production.

Microb Cell 2018 Jan 26;5(4):184-197. Epub 2018 Jan 26.

Department of Biotechnology, Institute for Agrochemistry and Food Technology, CSIC, Valencia, Spain.

Several yeast species, belonging to and non- genera, play fundamental roles during spontaneous must grape fermentation, and recent studies have shown that mixed fermentations, co-inoculated with and non- strains, can improve wine organoleptic properties. During active dry yeast (ADY) production, antioxidant systems play an essential role in yeast survival and vitality as both biomass propagation and dehydration cause cellular oxidative stress and negatively affect technological performance. Mechanisms for adaptation and resistance to desiccation have been described for but no data are available on the physiology and oxidative stress response of non- wine yeasts and their potential impact on ADY production. In this study we analyzed the oxidative stress response in several non- yeast species by measuring the activity of reactive oxygen species (ROS) scavenging enzymes, e.g., catalase and glutathione reductase, accumulation of protective metabolites, e.g., trehalose and reduced glutathione (GSH), and lipid and protein oxidation levels. Our data suggest that non-canonical regulation of glutathione and trehalose biosynthesis could cause poor fermentative performance after ADY production, as it corroborates the corrective effect of antioxidant treatments, during biomass propagation, with both pure chemicals and food-grade argan oil.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15698/mic2018.04.624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5878686PMC
January 2018

Herbicide glufosinate inhibits yeast growth and extends longevity during wine fermentation.

Sci Rep 2017 09 29;7(1):12414. Epub 2017 Sep 29.

Institute of Agrochemistry and Food Technology (IATA-CSIC), Paterna, Valencia, Spain.

Glufosinate ammonium (GA) is a widely used herbicide that inhibits glutamine synthetase. This inhibition leads to internal amino acid starvation which, in turn, causes the activation of different nutrient sensing pathways. GA also inhibits the enzyme of the yeast Saccharomyces cerevisiae in such a way that, although it is not used as a fungicide, it may alter yeast performance in industrial processes like winemaking. We describe herein how GA indeed inhibits the yeast growth of a wine strain during the fermentation of grape juice. In turn, GA extends longevity in a variety of growth media. The biochemical analysis indicates that GA partially inhibits the nutrient sensing TORC1 pathway, which may explain these phenotypes. The GCN2 kinase mutant is hypersensitive to GA. Hence the control of translation and amino acid biosynthesis is required to also deal with the damaging effects of this pesticide. A global metabolomics analysis under winemaking conditions indicated that an increase in amino acid and in polyamines occurred. In conclusion, GA affects many different biochemical processes during winemaking, which provides us with some insights into both the effect of this herbicide on yeast physiology and into the relevance of the metabolic step for connecting nitrogen and carbon metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-12794-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622092PMC
September 2017

Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking.

FEMS Yeast Res 2017 01;17(1)

Institute of Agrochemistry and Food Technology (IATA-CSIC), Paterna, 46980 Valencia, Spain.

Grape juice fermentation is a harsh environment with many stressful conditions, and Saccharomyces cerevisiae adapts its metabolism in response to those environmental challenges. Many nutrient-sensing pathways control this feature. The Tor/Sch9p pathway promotes growth and protein synthesis when nutrients are plenty, while the transcription factor Gcn4p is required for the activation of amino acid biosynthetic pathways. We previously showed that Sch9p impact on longevity depends on the nitrogen/carbon ratio. When nitrogen is limiting, SCH9 deletion shortens chronological life span, which is the case under winemaking conditions. Its deletion also increases glycerol during fermentation, so the impact of this pathway on metabolism under winemaking conditions was studied by transcriptomic and metabolomic approaches. SCH9 deletion causes the upregulation of many amino acid biosynthesis pathways. When Gcn4p was overexpressed during winemaking, increased glycerol production was also observed. Therefore, both pathways are related in terms of glycerol production. SCH9 deletion increased the amount of the limiting enzyme in glycerol biosynthesis, glycerol-3-P dehydrogenase Gpd1p at the protein level. The impact on the metabolome of SCH9 deletion and GCN4 overexpression differed, although both showed a downregulation of glycolysis. SCH9 deletion downregulated the amount of most proteinogenic amino acids and increased the amount of lipids, such as ergosterol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/femsyr/fow106DOI Listing
January 2017

RNA binding protein Pub1p regulates glycerol production and stress tolerance by controlling Gpd1p activity during winemaking.

Appl Microbiol Biotechnol 2016 Jun 4;100(11):5017-27. Epub 2016 Feb 4.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, IATA-CSIC, Avda. Agustín Escardino, 7, 46980, Paterna, Spain.

Glycerol is a key yeast metabolite in winemaking because it contributes to improve the organoleptic properties of wine. It is also a cellular protective molecule that enhances the tolerance of yeasts to osmotic stress and promotes longevity. Thus, its production increases by genetic manipulation, which is of biotechnological and basic interest. Glycerol is produced by diverting glycolytic glyceraldehyde-3-phosphate through the action of glycerol-3-phosphate dehydrogenase (coded by genes GPD1 and GPD2). Here, we demonstrate that RNA-binding protein Pub1p regulates glycerol production by controlling Gpd1p activity. Its deletion does not alter GPD1 mRNA levels, but protein levels and enzymatic activity increase, which explains the higher intracellular glycerol concentration and greater tolerance to osmotic stress of the pub1∆ mutant. PUB1 deletion also enhances the activity of nicotinamidase, a longevity-promoting enzyme. Both enzymatic activities are partially located in peroxisomes, and we detected peroxisome formation during wine fermentation. The role of Pub1p in life span control depends on nutrient conditions and is related with the TOR pathway, and a major connection between RNA metabolism and the nutrient signaling response is established.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-016-7340-zDOI Listing
June 2016

Food-grade argan oil supplementation in molasses enhances fermentative performance and antioxidant defenses of active dry wine yeast.

AMB Express 2015 Dec 1;5(1):75. Epub 2015 Dec 1.

Departament de Bioquímica i Biologia Molecular, Universitat de València, València, Spain.

The tolerance of the yeast Saccharomyces cerevisiae to desiccation is important for the use of this microorganism in the wine industry, since active dry yeast (ADY) is routinely used as starter for must fermentations. Both biomass propagation and dehydration cause cellular oxidative stress, therefore negatively affecting yeast performance. Protective treatments against oxidative damage, such as natural antioxidants, may have important biotechnological implications. In this study we analysed the antioxidant capacity of pure chemical compounds (quercetin, ascorbic acid, caffeic acid, oleic acid, and glutathione) added to molasses during biomass propagation, and we determine several oxidative damage/response parameters (lipid peroxidation, protein carbonylation, protective metabolites and enzymatic activities) to assess their molecular effects. Supplementation with ascorbic, caffeic or oleic acids diminished the oxidative damage associated to ADY production. Based on these results, we tested supplementation of molasses with argan oil, a natural food-grade ingredient rich in these three antioxidants, and we showed that it improved both biomass yield and fermentative performance of ADY. Therefore, we propose the use of natural, food-grade antioxidant ingredients, such as argan oil, in industrial processes involving high cellular oxidative stress, such as the biotechnological production of the dry starter.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13568-015-0159-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666183PMC
December 2015

Interplay among Gcn5, Sch9 and mitochondria during chronological aging of wine yeast is dependent on growth conditions.

PLoS One 2015 6;10(2):e0117267. Epub 2015 Feb 6.

Department of Biotechnology, Institute of Agrochemistry and Food Technology, IATA-CSIC, Paterna, Spain.

Saccharomyces cerevisiae chronological life span (CLS) is determined by a wide variety of environmental and genetic factors. Nutrient limitation without malnutrition, i.e. dietary restriction, expands CLS through the control of nutrient signaling pathways, of which TOR/Sch9 has proven to be the most relevant, particularly under nitrogen deprivation. The use of prototrophic wine yeast allows a better understanding of the role of nitrogen in longevity in natural and more demanding environments, such as grape juice fermentation. We previously showed that acetyltransferase Gcn5, a member of the SAGA complex, has opposite effects on CLS under laboratory and winemaking conditions, and is detrimental under the latter. Here we demonstrate that integrity of the SAGA complex is necessary for prolonged longevity, as its dismantling by SPT20 deletion causes a drop in CLS under both laboratory and winemaking conditions. The sch9Δ mutant is long-lived in synthetic SC medium, as expected, and the combined deletion of GCN5 partially suppresses this phenotype. However it is short-lived in grape juice, likely due to its low nitrogen/carbon ratio. Therefore, unbalance of nutrients can be more relevant for life span than total amounts of them. Deletion of RTG2, which codes for a protein associated with Gcn5 and is a component of the mitochondrial retrograde signal, and which communicates mitochondrial dysfunction to the nucleus, is detrimental under laboratory, but not under winemaking conditions, where respiration seems not so relevant for longevity. Transcription factor Rgm1 was found to be a novel CLS regulator Sch9-dependently.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117267PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319768PMC
February 2016

Acetyltransferase SAS2 and sirtuin SIR2, respectively, control flocculation and biofilm formation in wine yeast.

FEMS Yeast Res 2014 Sep 26;14(6):845-57. Epub 2014 Jun 26.

Laboratorio de MicrobiologÍa Enológica, Facultad de Ciencias, Universidad de Cádiz, Puerto Real, Cádiz, Spain.

Cell-to-cell and cell-to-environment interactions of microorganisms are of substantial relevance for their biotechnological use. In the yeast Saccharomyces cerevisiae, flocculation can be an advantage to clarify final liquid products after fermentation, and biofilm formation may be relevant for the encapsulation of strains of interest. The adhesion properties of wine yeast strains can be modified by the genetic manipulation of transcriptional regulatory proteins, such as histone deacetylases, and acetylases. Sirtuin SIR2 is essential for the formation of mat structures, a kind of biofilm that requires the expression of cell-wall protein FLO11 as its deletion reduces FLO11 expression, and adhesion of cells to themselves and to agar in a commercial wine strain. Deletion of acetyltransferase GCN5 leads to a similar phenotype. A naturally flocculant wine yeast strain called P2 was characterized. Its flocculation happens only during grape juice fermentation and is due to the presence of a highly transcribed version of flocculin FLO5, linked to the presence of a δ sequence in the promoter. Deletion of acetyltransferase SAS2 enhances this phenotype and maltose fermentation even more. Therefore, the manipulation of acetylation/deacetylation machinery members is a valid way to alter the interaction of industrial yeast to their environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1567-1364.12173DOI Listing
September 2014

Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking.

Microb Cell Fact 2013 Jan 2;12. Epub 2013 Jan 2.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos-CSIC, Av, Agustín Escardino, 7, Paterna 46980, Spain.

Background: Yeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS), while glycerol extends it.

Results: Different age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS.

Conclusions: Manipulation of regulators of gene expression at both transcriptional (i.e., sirtuins) and posttranscriptional (i.e., mRNA binding protein Pub1) levels allows to modulate yeast life span during its biotechnological use. Due to links between aging and metabolism, it also influences the production profile of metabolites of industrial relevance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1475-2859-12-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583744PMC
January 2013

Two-carbon metabolites, polyphenols and vitamins influence yeast chronological life span in winemaking conditions.

Microb Cell Fact 2012 Aug 8;11:104. Epub 2012 Aug 8.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos-CSIC, Av. Agustín Escardino 7, Paterna 46980, Spain.

Background: Viability in a non dividing state is referred to as chronological life span (CLS). Most grape juice fermentation happens when Saccharomyces cerevisiae yeast cells have stopped dividing; therefore, CLS is an important factor toward winemaking success.

Results: We have studied both the physical and chemical determinants influencing yeast CLS. Low pH and heat shorten the maximum wine yeast life span, while hyperosmotic shock extends it. Ethanol plays an important negative role in aging under winemaking conditions, but additional metabolites produced by fermentative metabolism, such as acetaldehyde and acetate, have also a strong impact on longevity. Grape polyphenols quercetin and resveratrol have negative impacts on CLS under winemaking conditions, an unexpected behavior for these potential anti-oxidants. We observed that quercetin inhibits alcohol and aldehyde dehydrogenase activities, and that resveratrol performs a pro-oxidant role during grape juice fermentation. Vitamins nicotinic acid and nicotinamide are precursors of NAD+, and their addition reduces mean longevity during fermentation, suggesting a metabolic unbalance negative for CLS. Moreover, vitamin mix supplementation at the end of fermentation shortens CLS and enhances cell lysis, while amino acids increase life span.

Conclusions: Wine S. cerevisiae strains are able to sense changes in the environmental conditions and adapt their longevity to them. Yeast death is influenced by the conditions present at the end of wine fermentation, particularly by the concentration of two-carbon metabolites produced by the fermentative metabolism, such as ethanol, acetic acid and acetaldehyde, and also by the grape juice composition, particularly its vitamin content.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1475-2859-11-104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503821PMC
August 2012

Wine yeast sirtuins and Gcn5p control aging and metabolism in a natural growth medium.

Mech Ageing Dev 2012 May 2;133(5):348-58. Epub 2012 Apr 2.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos-CSIC, Av. Agustín Escardino 7, Paterna 46980, Spain.

Grape juice fermentation by wine yeast is an interesting model to understand aging under conditions closer to those in nature. Grape juice is rich in sugars and, unlike laboratory conditions, the limiting factor for yeast growth is nitrogen. We tested the effect of deleting sirtuins and several acetyltransferases to find that the role of many of these proteins during grape juice fermentation is the opposite to that under standard laboratory aging conditions using synthetic complete media. For instance, SIR2 deletion extends maximum chronological lifespan in wine yeasts grown under laboratory conditions, but shortens it in winemaking. Deletions of sirtuin HST2 and acetyltransferase GCN5 have the opposite effect to SIR2 mutation in both media. Acetic acid, a well known pro-aging compound in laboratory conditions, does not play a determinant role on aging during wine fermentation. We discovered that gcn5Δ mutant strain displays strongly increased aldehyde dehydrogenase Ald6p activity, caused by blocking of Ald6p degradation by autophagy under nitrogen limitation conditions, leading to acetic acid accumulation. We describe how nitrogen limitation and TOR inhibition extend the chronological lifespan under winemaking conditions and how the TOR-dependent control of aging partially depends on the Gcn5p function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mad.2012.03.013DOI Listing
May 2012

Phylogenetic origin and transcriptional regulation at the post-diauxic phase of SPI1, in Saccharomyces cerevisiae.

Cell Mol Biol Lett 2012 Sep 18;17(3):393-407. Epub 2012 May 18.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Spain.

The gene SPI1, of Saccharomyces cerevisiae, encodes a cell wall protein that is induced in several stress conditions, particularly in the postdiauxic and stationary phases of growth. It has a paralogue, SED1, which shows some common features in expression regulation and in the null mutant phenotype. In this work we have identified homologues in other species of yeasts and filamentous fungi, and we have also elucidated some aspects of the origin of SPI1, by duplication and diversification of SED1. In terms of regulation, we have found that the expression in the post-diauxic phase is regulated by genes related to the PKA pathway and stress response (MSN2/4, YAK1, POP2, SOK2, PHD1, and PHO84) and by genes involved in the PKC pathway (WSC2, PKC1, and MPK1).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2478/s11658-012-0017-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275683PMC
September 2012

Oxidative stress tolerance, adenylate cyclase, and autophagy are key players in the chronological life span of Saccharomyces cerevisiae during winemaking.

Appl Environ Microbiol 2012 Apr 10;78(8):2748-57. Epub 2012 Feb 10.

Departamento de Biotecnología, IATA-CSIC, Paterna, Spain.

Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene, RDL2, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.07261-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318821PMC
April 2012

The Saccharomyces cerevisiae flavodoxin-like proteins Ycp4 and Rfs1 play a role in stress response and in the regulation of genes related to metabolism.

Arch Microbiol 2011 Jul 27;193(7):515-25. Epub 2011 Mar 27.

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, Paterna, Spain.

SPI1 is a gene whose expression responds to many environmental stimuli, including entry into stationary phase. We have performed a screening to identify genes that activate SPI1 promoter when overexpressed. The phosphatidylinositol-4-phosphate 5-kinase gene MSS4 was identified as a positive activator of SPI1. Another SPI1 transcriptional regulator isolated was the flavodoxin-like gene YCP4. YCP4 and its homolog RFS1 regulate the expression of many genes during the late stages of growth. The double deletion mutant in YCP4 and its homolog RFS1 has an impact on gene expression related to metabolism by increasing the expression of genes involved in hexose transport and glycolysis, and decreasing expression of genes of amino acid metabolism pathways. Genes related to mating and response to pheromone show a decreased expression in the double mutant, while transcription of genes involved in translational elongation is increased. Deletion of these genes, together with the third member of the family, PST2, has a complex effect on the stress response. For instance, double mutant ycp4Δrfs1Δ has an increased response to oxidative stress, but a decreased tolerance to cell-damaging agent SDS. Additionally, this mutation affects chronological aging and slightly increases fermentative capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00203-011-0696-7DOI Listing
July 2011

Btn2p is involved in ethanol tolerance and biofilm formation in flor yeast.

FEMS Yeast Res 2008 Nov 12;8(7):1127-36. Epub 2008 Jun 12.

Department of Biotechnology, IATA (CSIC), Paterna, Valencia, Spain.

Flor yeasts are a particular kind of Saccharomyces cerevisiae strains involved in Sherry wine biological ageing. During this process, yeasts form a film on the wine surface and use ethanol as a carbon source, producing acetaldehyde as a by-product. Acetaldehyde induces BTN2 transcription in laboratory strains. Btn2p is involved in the control of the subcellular localization of different proteins. The BTN2 gene shows a complex expression pattern in wine yeast, increasing its expression by acetaldehyde, but repressing it by ethanol. A flor yeast strain transcribes more BTN2 than a first fermentation yeast during growth, but less under different stress conditions. BTN2 deletion decreases flor yeast resistance to high ethanol concentrations. Surprisingly, this effect is suppressed by the addition of high amounts of amino acids to the growth medium, indicating that the role of Btn2p protein in amino acid transport is important for ethanol resistance. Btn2p deletion increases the fermentative capacity of flor yeast and its overexpression prevents its growth on nonfermentable carbon sources. BTN2 deletion also affects the biofilm formation ability of flor yeast, and it increases its sliding motility, resulting in increased mat formation. This correlates with an increased transcription of the FLO11 gene, a gene essential for biofilm formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1567-1364.2008.00397.xDOI Listing
November 2008

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells.

Nucleic Acids Res 2007 13;35(7):2191-8. Epub 2007 Mar 13.

Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Madrid, Spain.

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkm118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874631PMC
June 2007

The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds.

Antonie Van Leeuwenhoek 2007 Jul 25;92(1):61-75. Epub 2007 Jan 25.

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Dr. Moliner, 50, 46100, Burjassot, Valencia, Spain.

Nitrogen starvation may lead to stuck and sluggish fermentations. These undesirable situations result in wines with high residual sugar, longer vinification times, and risks of microbial contamination. The typical oenological method to prevent these problems is the early addition of ammonium salts to the grape juice, although excessive levels of these compounds may lead to negative consequences for the final product. This addition reduces the overall fermentation time, regardless of the time of addition, but the effect is more significant when nitrogen is added during the yeast exponential phase. In this work we analysed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion in order to understand yeast metabolic changes that lead to the adaptation to the new conditions. These studies were carried out in a synthetic must that mimics the composition of the natural must. Furthermore, we studied how this addition affects fermentative behaviour, the levels of several yeast volatile compounds in the final product, arginase activity, and the expression of several genes involved in stress response and nitrogen metabolism during vinification. We found that the nature of the nitrogen source added during yeast late exponential growth phase introduces changes to the volatile compounds profile and to the gene expression. On the other hand, arginase activity and the expression of the stress response gene ACA1 are useful to monitor nitrogen depletion/addition during growth of the wine yeast considered under our vinification conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10482-006-9135-1DOI Listing
July 2007

A novel approach for the improvement of stress resistance in wine yeasts.

Int J Food Microbiol 2007 Feb 20;114(1):83-91. Epub 2006 Dec 20.

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Dr. Moliner, 50. E-46100, Burjassot, Valencia, Spain.

During wine production yeast cells are affected by several stress conditions that could affect their viability and fermentation efficiency. In this work we describe a novel genetic manipulation strategy designed to improve stress resistance in wine yeasts. This strategy involves modifying the expression of the transcription factor MSN2, which plays an important role in yeast stress responses. The promoter in one of the genomic copies of this gene has been replaced by the promoter of the SPI1 gene, encoding for a cell wall protein of unknown function. SPI1 is expressed at late phases of growth and is regulated by Msn2p. This modification allows self-induction of MSN2 expression. MSN2 gene transcription, Msn2p protein levels and cell viability increase under several stress conditions in the genetically modified strain. The expression of stress response genes regulated by Msn2p also increases under these situations. Cells containing this promoter change are able to carry out vinifications at 15 and 30 degrees C with higher fermentation rates during the first days of the process compared to the control strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijfoodmicro.2006.10.043DOI Listing
February 2007

Sulfur and adenine metabolisms are linked, and both modulate sulfite resistance in wine yeast.

J Agric Food Chem 2006 Aug;54(16):5839-46

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biologiques, Universitat de València, Burjassot, Spain.

Sulfite treatment is the most common way to prevent grape must spoilage in winemaking because the yeast Saccharomyces cerevisiae is particularly resistant to this chemical. In this paper we report that sulfite resistance depends on sulfur and adenine metabolism. The amount of adenine and methionine in a chemically defined growth medium modulates sulfite resistance of wine yeasts. Mutations in the adenine biosynthetic pathway or the presence of adenine in a synthetic minimal culture medium increase sulfite resistance. The presence of methionine has the opposite effect, inducing a higher sensitivity to SO(2). The concentration of methionine, adenine, and sulfite in a synthetic grape must influences the progress of fermentation and at the transcriptional level the expression of genes involved in sulfur (MET16), adenine (ADE4), and acetaldehyde (ALD6) metabolism. Sulfite alters the pattern of expression of all these genes. This fact indicates that the response to this stress is complex and involves several metabolic pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jf060851bDOI Listing
August 2006

Incidence of acute respiratory distress syndrome and its relation to age.

J Crit Care 2005 Sep;20(3):274-80

Critical Care and Emergency Department, Virgen de las Nieves University Hospital, 18013 Granada, Spain.

Purpose: The incidence of acute respiratory distress syndrome (ARDS) was previously considered to be relatively low, at less than 10 cases per 100,000 inhabitants per year, but recent reports suggest a higher incidence, especially in elderly patients. The objective was to determine the incidence and mortality of ARDS in our setting, both overall and by age group.

Materials And Methods: We conducted a prospective, observational study of patients older than 14 years, admitted to the intensive care units of all hospitals in a province of southern Spain (Granada) during a 5-month period in 2001. American-European Consensus Conference criteria for ARDS were used. Patients were divided into 5 age groups, and the hospital mortality was recorded.

Results: During the study period, 61 Granada-residing patients developed ARDS criteria. This represents an overall incidence of 23 cases per 100,000 inhabitants per year in the province. The incidence of ARDS in the age groups of 15 to 29, 30 to 44, 45 to 59, 60 to 74, and older than 74 years was 4.6, 13.6, 21.6, 51, and 73.9 cases per 100,000 inhabitants per year, respectively. The overall hospital mortality rate was 66%.

Conclusions: The incidence of ARDS is higher than reported a decade ago and is especially elevated in the elderly. The mortality remains high.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcrc.2005.05.008DOI Listing
September 2005

Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms.

Mol Cell 2004 Jul;15(2):303-13

Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Spain.

Most studies of eukaryotic gene regulation have been done looking at mature mRNA levels. Nevertheless, the steady-state mRNA level is the result of two opposing factors: transcription rate (TR) and mRNA degradation. Both can be important points to regulate gene expression. Here we show a new method that combines the use of nylon macroarrays and in vivo radioactive labeling of nascent RNA to quantify TRs, mRNA levels, and mRNA stabilities for all the S. cerevisiae genes. We found that during the shift from glucose to galactose, most genes undergo drastic changes in TR and mRNA stability. However, changes in mRNA levels are less pronounced. Some genes, such as those encoding mitochondrial proteins, are coordinately regulated in mRNA stability behaving as decay regulons. These results indicate that, although TR is the main determinant of mRNA abundance in yeast, modulation of mRNA stability is a key factor for gene regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2004.06.004DOI Listing
July 2004

Exposure of Saccharomyces cerevisiae to acetaldehyde induces sulfur amino acid metabolism and polyamine transporter genes, which depend on Met4p and Haa1p transcription factors, respectively.

Appl Environ Microbiol 2004 Apr;70(4):1913-22

Departament de Bioquímica i Biología Molecular, Facultat de Ciències Biològiques, Universitat de València, València, Spain.

Acetaldehyde is a toxic compound produced by Saccharomyces cerevisiae cells under several growth conditions. The adverse effects of this molecule are important, as significant amounts accumulate inside the cells. By means of global gene expression analyses, we have detected the effects of acetaldehyde addition in the expression of about 400 genes. Repressed genes include many genes involved in cell cycle control, cell polarity, and the mitochondrial protein biosynthesis machinery. Increased expression is displayed in many stress response genes, as well as other families of genes, such as those encoding vitamin B1 biosynthesis machinery and proteins for aryl alcohol metabolism. The induction of genes involved in sulfur metabolism is dependent on Met4p and other well-known factors involved in the transcription of MET genes under nonrepressing conditions of sulfur metabolism. Moreover, the deletion of MET4 leads to increased acetaldehyde sensitivity. TPO genes encoding polyamine transporters are also induced by acetaldehyde; in this case, the regulation is dependent on the Haa1p transcription factor. In this paper, we discuss the connections between acetaldehyde and the processes affected by this compound in yeast cells with reference to the microarray data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383134PMC
http://dx.doi.org/10.1128/aem.70.4.1913-1922.2004DOI Listing
April 2004

Response to acetaldehyde stress in the yeast Saccharomyces cerevisiae involves a strain-dependent regulation of several ALD genes and is mediated by the general stress response pathway.

Yeast 2003 Jun;20(8):747-59

Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Valencia, Spain.

One of the stress conditions that yeast may encounter is the presence of acetaldehyde. In a previous study we identified that, in response to this stress, several HSP genes are induced that are also involved in the response to other forms of stress. Aldehyde dehydrogenases (ALDH) play an important role in yeast acetaldehyde metabolism (e.g. when cells are growing in ethanol). In this work we analyse the expression of the genes encoding these enzymes (ALD) and also the corresponding enzymatic activities under several growth conditions. We investigate three kinds of yeast strains: laboratory strains, strains involved in the alcoholic fermentation stage of wine production and flor yeasts (responsible for the biological ageing of sherry wines). The latter are very important to consider because they grow in media containing high ethanol concentrations, and produce important amounts of acetaldehyde. Under several growth conditions, further addition of acetaldehyde or ethanol in flor yeasts induced the expression of some ALD genes and led to an increase in ALDH activity. This result is consistent with their need to obtain energy from ethanol during biological ageing processes. Our data also suggest that post-transcriptional and/or post-translational mechanisms are involved in regulating the activity of these enzymes. Finally, analyses indicate that the Msn2/4p and Hsf1p transcription factors are necessary for HSP26, ALD2/3 and ALD4 gene expression under acetaldehyde stress, while PKA represses the expression of these genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/yea.991DOI Listing
June 2003