Deletion of BCY1 from the Saccharomyces cerevisiae Genome Is Semidominant and Induces Autolytic Phenotypes Suitable for Improvement of Sparkling Wines

Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3′ end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines.     

Construction of a recombinant autolytic wine yeast strain overexpressing the csc1‐1 allele

During the aging step of sparkling wines and wines aged on lees, yeast cells kept in contact with the wine finally die and undergo autolysis, releasing cellular compounds with a positive effect on the wine quality. In view of the interest of autolysis for wine properties, biotechnologists have tried to improve autolytic yield during winemaking. In this work we used genetic engineering techniques to construct an autolytic industrial strain by expressing the csc1‐1 allele from the RDN1 locus. The expression of this mutant allele, that causes a “constitutive in autophagy phenotype,” resulted in accelerated autolysis of the recombinant strain. Although autophagic phenotype due to csc1‐1 expression has been reported to require the mutant allele in multicopy, autolytic acceleration was achieved by expressing only one or two copies of the gene under the control of the constitutive promotor pTDH3. The acceleration of autolysis together with the unaltered fermentative capacity, strongly supported the overexpression of csc1‐1 allele as a strategy to obtain wines with aged‐like properties in a shortened time. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Chiral analysis of amino acids from conventional and transgenic yeasts

Autolysis of Saccharomyces cerevisiae yeast is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work, a genetically modified yeast (LS11) is compared to its isogenic wild type strain (BY4741) after autolysis. Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) is used to identify and quantify the main D- and L-amino acids from both strains after accelerated autolysis. The procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC-LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 30 mM SDS, 20 mM beta-CD at pH 10.0. The D- and L-forms of Arg, Asn, Ala, Glu and Asp, corresponding to the major amino acids found in these samples, are separated in less than 30 min with efficiencies up to 800,000 plates/m and high sensitivity (i.e., LODs as low as 40 nM were obtained for D-Arg for a signal to noise ratio of three). From these results it is corroborated that the genetic modification brings a faster autolysis of the yeast, releasing a higher amount of L-amino acids to the medium in a short time. Interestingly, the pattern of release of D-amino acids was also different between the transgenic and the conventional yeast strains.     

Proteins influencing foam formation in wine and beer: the role of yeast

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa--characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins.     

Enological functions of parietal yeast mannoproteins

Parietal yeast mannoproteins play a very important role in the overall vinification process. Their production and release, both during winemaking and aging on lees, depends on the specific yeast strain and the nutritional conditions. The following enological functions of parietal yeast mannoproteins have been described: (a) adsorption of ochratoxin A; (b) combination with phenolic compounds; (c) increased growth of malolactic bacteria; (d) inhibition of tartrate salt crystallization; (e) interaction with flor wines; (f) prevention of haze; (g) reinforcement of aromatic components; (h) wine enrichment during aging on fine lees; (i) yeast flocculation and autolysis in sparkling wines. Further discoveries related to their enological functions are foreseeable. Yeast-derived mannoproteins may well induce chemical, sensorial and health benefits, thus greatly improving wine quality.     

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.     

Bulk sparkling wine production by external encapsulated yeast bioreactor

Summary A new method for bulk sparkling wine production is proposed. The use of an external bioreactor with high immobilized yeast loading for second fermentation was studied. The new process is much faster than the traditional one and the sparkling wines obtained are perfectly clear without showing quality faults.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002     

Preliminary assessment on the use of immobilized yeast cells in sodium alginate for sparkling wine processes

Summary Use of alginate-immobilized yeasts in the production of sparkling wine using the champenois method was investigated. The results indicate that there are no variations in the principal chemical-physical characteristics between sparkling wines obtained through immobilized yeast and traditional sparkling wines.     

Cross regulation between Candida albicans catalytic and regulatory subunits of protein kinase A

In the pathogen Candida albicans protein kinase A (PKA) catalytic subunit is encoded by two genes TPK1 and TPK2 and the regulatory subunit by one gene, BCY1. PKA mediates several cellular processes such as cell cycle regulation and the yeast to hyphae transition, a key factor for C. albicans virulence. The catalytic isoforms Tpk1p and Tpk2p share redundant functions in vegetative growth and hyphal development, though they differentially regulate glycogen metabolism, the stress response pathway and pseudohyphal formation. In Saccharomyces cerevisiae it was earlier reported that BCY1 overexpression not only increased the amount of TPK3 mRNA but also its catalytic activity. In C. albicans a significant decrease in Bcy1p expression levels was already observed in tpk2Δ null strains. In this work we showed that the upregulation in Bcy1p expression was observed in a set of strains having a TPK1 or TPK2 allele reintegrated in its own locus, as well as in strains expressing the TPKs under the control of the constitutive ACT1 promoter. To confirm the cross regulation event between Bcy1p and Tpkp expression we generated a mutant strain with the lowest PKA activity carrying one TPK1 and a unique BCY1 allele with the aim to obtain two derived strains in which BCY1 or TPK1 were placed under their own promoters inserted in the RPS10 neutral locus. We found that placing one copy of BCY1 upregulated the levels of Tpk1p and its catalytic activity; while TPK1 insertion led to an increase in BCY1 mRNA, Bcy1p and in a high cAMP binding activity. Our results suggest that C. albicans cells were able to compensate for the increased levels of either Tpk1p or Tpk2p subunits with a corresponding elevation of Bcy1 protein levels and vice versa, implying a tightly regulated mechanism to balance holoenzyme formation.     

The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

The Yeast Deletion Collection: A Decade of Functional Genomics

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general.     

Evaluation of Fermentation Efficiency of Yeast Strains and their Effect on Quality of Young Wines

The yeast has important role in fermentation of wine grapes and wine quality. The fermentation of wine grapes affect by efficiency of particular yeast strain, sugar content, pH, available temperature, etc. To evaluate the efficiency of yeast strains (Premier Cuvee, RS-1, RS-2, RS-3 and natural), present study was conducted on two wine grape varieties viz.; Sauvignon Blanc (White) and Cabernet Sauvignon (Red). Efficiency of yeast strains was evaluated in terms of conversion rate of sugar into alcohol. As per recorded data, strain RS-3 (Pichia kudriavzevii) was found more efficient than other strains in fermentation of Cabernet Sauvignon with efficiency of 84.4 per cent but in case of Sauvignon Blanc, the commercial culture Premier Cuevee was found superior over RS-3. The quality parameters of young wines of both the varieties were also affected by the used strains. Considering the efficiency and impact on various parameters of wines, local strain, i.e., RS-3 was found at par with commercial culture (Premier Cuvee). The RS-3 strain has potential to produce quality wines. However, studies on effects of RS-3 strain on some specific quality parameters of wines like varietal aroma compounds, flavours etc. are needed.     

Characterization of yakju brewed from glutinous rice and wild-type yeast strains isolated from nuruks

Korean traditional rice wines yakju and takju are generally brewed with nuruk as the source of the saccharogenic enzymes by natural fermentation. To improve the quality of Korean rice wine, the microorganisms in the nuruk need to be studied. The objective of this research was to improve the quality of Korean wine with the wild-type yeast strains isolated from the fermentation starter, nuruk. Only strain YA-6 showed high activity in 20% ethanol. Precipitation of Y89-5-3 was similar to that of very flocculent yeast (?80%) at 75.95%. Using 18S rRNA sequencing, all 10 strains were identified as Saccharomyces cerevisiae. Volatile compounds present in yakju were analyzed by gas chromatography-mass selective detector. The principal component analysis (PCA) of the volatile compounds grouped long-chain esters on the right side of the first principal component, PC1; these compounds were found in yakju that was made with strains YA-6, Y89-5-3, Y89-5- 2, Y90-9, and Y89-1-1. On the other side of PC1 were short-chain esters; these compounds were found in wines that were brewed with strains Y183-2, Y268-3, Y54-3, Y98-4, and Y88-4. Overall, the results indicated that using different wild-type yeast strains in the fermentation process significantly affects the chemical characteristics of the glutinous rice wine.     

Flor yeast strains from culture collection: Genetic diversity and physiological and biochemical properties

Sixteen flor yeast strains from the Magarach Collection of the Microorganisms for Winemaking (Yalta, Crimea), which are used for production of sherry, were analyzed for morphophysiological, cultural, and biochemical properties. Long-term storage did not affect their viability or the preservation of major properties, such as their flor- and aldehyde-forming abilities, and the ability to produce wines with typical sherry properties. Significant variation in the strains was observed mainly in the aldehyde-forming and flor-forming abilities and flor properties. Interdelta typing was shown to be the most informative technique to study the genetic diversity of flor yeast strains. Certain correlations between genetic polymorphisms and the enological properties of the strains were observed. The presence of a 24-bp long deletion in the ITS1 spacer of the ribosomal gene cluster, a typical feature of Spanish flor yeast strains, is correlated with a high level of production of aldehydes and acetales, efficient flor formation, and the ability to produce high quality sherry. The presence of a specific deletion in the promoter of the FLO11 gene appeared to be less informative, since the aldehyde and acetal production and flor formation abilities of such strains were variable. The studies of intraspecies genetic polymorphism by various molecular markers have revealed a high degree of phylogenetic closeness of some yeast flor strains from different geographic regions.     

Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Yeast strain effect on the concentration of major volatile compounds and sensory profile of wines from Vitis vinifera var. Treixadura

The fermentative ability of five Saccharomyces cerevisiae strains and their influence on the aroma and sensory properties of wine from Treixadura were evaluated to determine the most suitable yeast that produces a high quality wine from this grapevine variety. The results indicated that all strains, except T2, were able to lead the vinification process and have good fermentative powers. The chemical composition of wines obtained with resident cellar yeasts consisted of a significant amount of glycerol, a compound that contributes to the structure and smoothness in taste of the wine. In addition, these strains were good producers of acetates, ethyl esters and fatty acids, compounds that positively influence wine aroma. Compounds with a direct contribution to the aroma of Treixadura wines (Odour Activity Value >1) included 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isoamyl and ethyl acetate and the ethyl esters, ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. Sensory analysis supported the fact that cellar strains produced more fruity and floral wines than commercial or spontaneous fermentations. We conclude that resident cellar yeasts enhanced sensory quality of wines from Treixadura.