Screening of Saccharomyces cerevisiae and Torulaspora delbrueckii strains in relation to their effect on malolactic fermentation.

The use of Torulaspora delbrueckii in the alcoholic fermentation (AF) of grape must is increasingly studied and used in the wine industry. In addition to the organoleptic improvement of wines, the synergy of this yeast species with the lactic acid bacterium Oenococcus oeni is an interesting field of study. In this work, 60 strain combinations were compared: 3 strains of Saccharomyces cerevisiae (Sc) and 4 strains of Torulaspora delbrueckii (Td) in sequential AF, and four strains of O. oeni (Oo) in malolactic fermentation (MLF). The objective was to describe the positive or negative relationships of these strains with the aim of finding the combination that ensures better MLF performance. In addition, a new synthetic grape must has been developed that allows the success of AF and subsequent MLF. Under these conditions, the Sc-K1 strain would be unsuitable for carrying out MLF unless there is prior inoculation with Td-Prelude, Td-Viniferm or Td-Zymaflore always with the Oo-VP41 combination. However, from all the trials performed, it appears that the combinations of sequential AF with Td-Prelude and Sc-QA23 or Sc-CLOS, followed by MLF with Oo-VP41, reflected a positive effect of T. delbrueckii compared to inoculation of Sc alone, such as a reduction in L-malic consumption time. In conclusion, the obtained results highlight the relevance of strain selection and yeast-LAB strain compatibility in wine fermentations. The study also reveals the positive effect on MLF of some T. delbrueckii strains.

Transcriptomic and proteomic analysis of Oenococcus oeni PSU-1 response to ethanol shock.

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

New insights into the toxicity mechanism of octanoic and decanoic acids on Saccharomyces cerevisiae.

Octanoic (C8) and decanoic (C10) acids are produced in hypoxic conditions by the yeast Saccharomyces cerevisiae as by-products of its metabolism and are considered fermentation inhibitors in the presence of ethanol at acidic pH. This study aims to broaden our understanding of the physiological limits between toxicity and ester production in yeast cells. To this end, the non-inhibitory concentration (NIC) and maximum inhibitory concentration (MIC) values were first established for C8 and C10 at physiological pH (5.8) without ethanol. The results showed that when these acids were added to culture medium at these values, they tended to accumulate in different cellular fractions of the yeast. While C8 was almost entirely located in the cell wall fraction, C10 was found in the endocellular fraction. Cell fatty acid detoxification was also different; while the esterification of fatty acids was more efficient in the case of C10, the peroxisome was activated regardless of which fatty acid was added. Furthermore, the study of the Pdr12 and Tpo1 transporters that evolved during the detoxification process revealed that C8 was mostly expelled by the Pdr12 carrier, which was related to higher ß-oxidative damage in the presence of endocellular C10. C10 is more toxic at lower concentrations than C8. Although they are produced by yeast, the resulting intracellular medium-chain fatty acids (MCFAs) caused a level of toxicity which promoted cell death. However, MCFAs are involved in the production of beverage flavours.

ATG18 and FAB1 are involved in dehydration stress tolerance in Saccharomyces cerevisiae.

Recently, different dehydration-based technologies have been evaluated for the purpose of cell and tissue preservation. Although some early results have been promising, they have not satisfied the requirements for large-scale applications. The long experience of using quantitative trait loci (QTLs) with the yeast Saccharomyces cerevisiae has proven to be a good model organism for studying the link between complex phenotypes and DNA variations. Here, we use QTL analysis as a tool for identifying the specific yeast traits involved in dehydration stress tolerance. Three hybrids obtained from stable haploids and sequenced in the Saccharomyces Genome Resequencing Project showed intermediate dehydration tolerance in most cases. The dehydration resistance trait of 96 segregants from each hybrid was quantified. A smooth, continuous distribution of the anhydrobiosis tolerance trait was found, suggesting that this trait is determined by multiple QTLs. Therefore, we carried out a QTL analysis to identify the determinants of this dehydration tolerance trait at the genomic level. Among the genes identified after reciprocal hemizygosity assays, RSM22, ATG18 and DBR1 had not been referenced in previous studies. We report new phenotypes for these genes using a previously validated test. Finally, our data illustrates the power of this approach in the investigation of the complex cell dehydration phenotype.

Metabolomic charactetization of yeast cells after dehydration stress

In this study, we analyzed the metabolite features of the yeasts Saccharomyces cerevisiae, Naumovia castellii, and Saccharomyces mikatae. The three species are closely related genetically but differ in their tolerance of desiccation stress. Specifically, we determined whether certain metabolites correlated with cell viability after stress imposition. The metabolomic profiles of these strains were compared before cell desiccation and after cell rehydration. In S. mikatae, the presence of lysine or glutamine during rehydration led to a 20% increase in survival whereas during dehydration the levels of both amino acids in this yeast were drastically reduced (AU)

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Membrane lipid variability in Saccharomyces cerevisiae wine strains rehydrated in the presence of metabolic activators.

Slight variations in lipid composition of wine yeast membranes can alter some essential functions including selective nutrient transport and ion permeability. The absence of oxygen during alcoholic fermentation inhibits fatty acid desaturation and sterol biosynthesis, thereby reducing the stress resistance of yeast cells. In this work, membrane lipids in two commercial active dry yeast strains rehydrated in the presence of three activators (ergosterol, tetrahydrofolic acid, and manganese) were studied. Each was assayed at three different concentrations. The effect of these activators on the phospholipid, neutral lipid, and fatty acid contents in cell membranes was assessed. Also, cell viability and fermentation kinetics were determined. Ergosterol was found to shorten the lag phase and improve cell viability and membrane lipid composition; tetrahydrofolic acid raised neutral lipid levels; and manganese(II) increased cell viability and modified phospholipid composition and linoleic acid concentration. All activators interacted with yeasts in a strain-dependent way.

Metabolomic charactetization of yeast cells after dehydration stress.

In this study, we analyzed the metabolite features of the yeasts Saccharomyces cerevisiae, Naumovia castellii, and Saccharomyces mikatae. The three species are closely related genetically but differ in their tolerance of desiccation stress. Specifically, we determined whether certain metabolites correlated with cell viability after stress imposition. The metabolomics profiles of these strains were compared before cell desiccation and after cell rehydration. In S. mikatae, the presence of lysine or glutamine during rehydration led to a 20% increase in survival whereas during dehydration the levels of both amino acids in this yeast were drastically reduced.

Genetic improvement of Saccharomyces cerevisiae wine strains for enhancing cell viability after desiccation stress.

In the last few decades spontaneous grape must fermentations have been replaced by inoculated fermentation with Saccharomyces cerevisiae strains as active dry yeast (ADY). Among the essential genes previously characterized to overcome the cell-drying/rehydration process, six belong to the group of very hydrophilic proteins known as hydrophilins. Among them, only SIP18 has shown early transcriptional response during dehydration stress. In fact, the overexpression in S. cerevisiae of gene SIP18 increases cell viability after the dehydration process. The purpose of this study was to characterize dehydration stress tolerance of three wild and one commercial S. cerevisiae strains of wine origin. The four strains were submitted to transformation by insertion of the gene SIP18. Selected transformants were submitted to the cell-drying-rehydration process and yeast viability was evaluated by both viable cell count and flow cytometry. The antioxidant capacity of SIP18p was illustrated by ROS accumulation reduction after H2 O2 attack. Growth data as cellular duplication times and lag times were calculated to estimate cell vitality after the cell rehydration process. The overexpressing SIP18 strains showed significantly longer time of lag phase despite less time needed to stop the leakage of intracellular compounds during the rehydration process. Subsequently, the transformants were tested in inoculated grape must fermentation at laboratory scale in comparison to untransformed strains. Chemical analyses of the resultant wines indicated that no significant change for the content of secondary compounds was detected. The obtained data showed that the transformation enhances the viability of ADY without affecting fermentation efficiency and metabolic behaviour.

The STF2p hydrophilin from Saccharomyces cerevisiae is required for dehydration stress tolerance.

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein.

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different Saccharomyces cerevisiae wine yeast strains.

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K (m) for both sugars. Hxk1 has a threefold higher V (max) with fructose than with glucose, whereas Hxk2 has only a slightly higher V (max) with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303-1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K (m) might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.

Effect of the cellulose-binding domain on the catalytic activity of a beta-glucosidase from Saccharomycopsis fibuligera.

Enzyme engineering was performed to link the beta-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera beta-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 ( P )) and terminator (PGK1 ( T )) and yeast mating pheromone alpha-factor secretion signal (MFalpha1 ( S )). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2-4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae.

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1(P)()) and terminator (PGK1(T)()) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Sigma1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Sigma1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway.

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h(-1), with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter(-1), 10 g of glucose plus 10 g of xylose liter(-1) or, for S. cerevisiae TMB 3400, 20 g of xylose liter(-1). S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate(-1) on glucose and 0.43 g of biomass g of consumed carbohydrate(-1) on xylose. A K(s) value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.

Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter.

The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YlRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YlRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YlRim101p and to override its effects.