Mannoprotein Release Research Articles

Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells.

A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast.

Pulsed electric fields accelerate release of mannoproteins from Saccharomyces cerevisiae during aging on the lees of Chardonnay wine

The potential of PEF for triggering autolysis of Saccharomyces cerevisiae and accelerating the release of mannoproteins during aging on the lees of Chardonnay wine was evaluated.Release of mannoproteins in Chardonnay wine increased drastically in samples containing PEF-treated (5 and 10 kV/cm, 75 μs) yeasts. No mannoprotein release was observed in the first seven days of aging on the lees in wine containing untreated yeast; however, after the same time interval, the concentration of those compounds increased by 40 and 60% in wines containing yeast treated by PEF at 5 and 10 kV/cm, respectively. After 30 days of incubation, the mannoprotein concentration in wines containing yeast treated under the most intense PEF conditions reached the maximum value. Control cells, on the other hand, required six months to reach that maximum level.Chromatic characteristics, total polyphenol index, total volatile acidity, pH, ethanol, and CIELAB parameters of the wine were not affected during aging on the lees with untreated and PEF-treated yeast. On the other hand, the capability of the mannoproteins released from yeast treated by PEF for decreasing wine turbidity, foaming, and interacting with tannins was similar to that of those released from untreated yeast; the differences observed were a consequence of the varying concentration of mannoproteins.The result obtained demonstrates that PEF permits the acceleration of the aging-on-lees step while avoiding or reducing the problems associated with it. To achieve this effect, intense treatment is not required. Therefore, wineries could process lees by using the most economical PEF devices on the market.

Hyperproduction of β-Glucanase Exg1 Promotes the Bioconversion of Mogrosides in Saccharomyces cerevisiae Mutants Defective in Mannoprotein Deposition.

Bacteria and fungi can secrete extracellular enzymes to convert macromolecules into smaller units. Hyperproduction of extracellular enzymes is often associated with alterations in cell wall structure in fungi. Recently, we identified that Saccharomyces cerevisiae kre6Δ mutants can efficiently convert mogroside V into mogroside III E, which has antidiabetic properties. However, the underlying efficient bioconversion mechanism is unclear. In the present study, the mogroside (MG) bioconversion properties of several cell wall structure defective mutants were analyzed. We also compared the cell walls of these mutants by transmission electron microscopy, a zymolyase sensitivity test, and a mannoprotein release assay. We found zymolyase-sensitive mutants (including kre1Δ, las21Δ, gas1Δ, and kre6Δ), with defects in mannoprotein deposition, exhibit efficient MG conversion and excessive leakage of Exg1; such defects were not observed in wild-type cells, or mutants with abnormal levels of glucans in the cell wall. Thus, yeast mutants defective in mannoprotein deposition may be employed to convert glycosylated bioactive compounds.

Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae×S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters).

Effect of Winery Yeast Lees on Touriga Nacional Red Wine Color and Tannin Evolution

Red wine aging on lees is a winemaking practice used to achieve more rounded and less astringent wines. In two different trials, external yeast lees were added to a red wine and their effects on wine color and tannin evolution during aging were studied. Results indicated that the addition of yeast lees did not affect color stabilization during the studied period. Color compounds and condensed tannins were rapidly adsorbed to the yeast lees at the beginning of the experiment. There was a retarding effect on proanthocyanidin polymerization reaction by the addition of yeast lees, leading to the maintenance of low and medium molecular weight tannins in solution. Two different interactions were observed: first, proanthocyanidin adsorption by the yeast lees, primarily ones with the highest polymerization degree, and second, the retarding of proanthocyanidin polymerization, likely by the mannoproteins released by yeast lees. The age of the yeast lees was a factor in mannoprotein release and its effect in wine.

Flocculation and transcriptional adaptation to fermentation conditions in a recombinant wine yeast strain defective for KNR4/SMI1

KNR4 defective recombinant wine yeast strains were previously shown to oversecrete mannoproteins during alcoholic fermentation and, depending on the genetic background, to contribute to protein stability of white wines. We have tried to get a deeper insight into the consequences of KNR4 deletion in a wine yeast strain, from both a biological and an enological standpoint, and to understand the mechanisms leading to improved mannoprotein release. In fermentation experiments, followed by aging on lees, and compared to the parent strain, the recombinant strain shows increased release of mannoproteins during the fermentation but little increase during aging. Mannoprotein release by the recombinant strain takes place mainly during the fermentation step. In contrast, autolysis of the recombinant strain keeps going after aging for 78 days. In addition, the recombinant strain is moderately flocculent, which would be interesting for the production of sparkling wines. This might be related to changes in the expression of Flo1p-regulated genes. The new biological processes affected by KNR4 deletion in wine yeasts, as revealed by this transcriptomic study are flocculation, adaptation to anaerobiosis, oxidative stress response, and ethanol tolerance, as well as FKS1 overexpression; but no overexpression was detected for genes coding for major structural mannoproteins of the cell wall.

A <em>Saccharomyces cerevisiae</em> wine yeast strain overproducing mannoproteins selected through classical genetic methods

<p style="text-align: justify;"><strong>Aims</strong>: Developing, by classical genetic methods, new wine yeast strains showing improved release of mannoproteins during wine fermentation, as well as suitable selection procedures for this purpose. These strains would be useful to improve quality characters associated to wine mannoprotein content.</p><p style="text-align: justify;"><strong>Methods and results</strong>: UV mutagenesis was used for genetic improvement of the industrial wine yeast strain ADY1. Cell wall-related phenotypes were used as primary selection criteria; an additional screening procedure was developed based on the detection of the released mannoproteins by hybridization with peroxidase-labeled Concanavalin A. Mannoprotein overproduction was assessed in laboratory media as well as in grapevine juice. One mutant strain, renamed HPS, was selected using these criteria. HPS showed increased mannoprotein release in different culture media, including natural must. Moreover, white wines fermented with this improved strain were less susceptible to protein haze than equivalent wines fermented with the original ADY1 strain. Red wines fermented with the mutant strain were also polysaccharide-enriched as compared to the original one.</p><p style="text-align: justify;"><strong>Conclusion</strong>: No clear correlation between a specific cell wall-related phenotype, or a combination of them, and improved release of polysaccharides by yeast random mutants could be established, and not all strains identified by in vitro assays as mannoprotein overproducing mutants were found positive for mannoprotein release in industrial conditions. Nevertheless, UV mutagenesis, combined with Concanavalin A detection, seems to be a viable way to improve mannoprotein release by industrial wine yeast strains.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This study is one of the few recent reports on genetic improvement of wine yeast strains by non-recombinant genetic tools. It shows that mannoprotein release can be genetically improved and, for the first time, describes a successful selection procedure for such a complex character. These strains are potentially useful for the improvement of mannoprotein-related characters of white and red wines.</p>

Three Different Targets for the Genetic Modification of Wine Yeast Strains Resulting in Improved Effectiveness of Bentonite Fining

Bentonite fining is used in the clarification of white wines to prevent protein haze. This treatment results in the loss of a significant portion of the wine itself, as well as aroma compounds important for the quality of white wines. Among other interesting effects on wine quality, yeast cell wall mannoproteins have been shown to stabilize wine against protein haze. A previous work showed that wine yeast strains engineered by deletion of KNR4 release increased amounts of mannoproteins and produce wines showing attenuated responses in protein haze tests. This paper describes the technological properties of several new recombinant wine yeast strains, deleted for genes involved in cell-wall biogenesis, as well as the regulatory gene KNR4. Stabilization of wines produced by three of the six recombinant strains analyzed required 20-40% less bentonite than those made with their nonrecombinant counterparts. The availability of multiple targets for genetically improving yeast mannoprotein release, as shown in this work, is relevant not only for genetic engineering of wine yeast but especially for the feasibility of genetically improving this character by classical methods of strain development such as random mutagenesis or sexual hybridization.

A RecombinantSaccharomyces cerevisiaeStrain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.

Mannoproteins from the cell wall ofKluyveromyces lactis

Wall mannoproteins from Kluyveromyces lactis have been solubilised by treatment of cell walls with sodium dodecyl sulphate (SDS) or zymolyase. While the former reagent liberates a large number of molecular species, zymolyase preferentially releases a high-molecular-weight material that is sensitive to endo-β-N-acetylglucosaminidase H, and a 29-kDa molecule that reacts with the antiserum raised against a similar species from walls of Saccharomyces cerevisiae. In contrast with observations on isolated walls of S. cerevisiae, dithiothreitol pretreatment of K. lactis walls does not enhance the effect of zymolyase upon mannoprotein release. However, the action of thiol agents is still necessary to obtain protoplasts by zymolyase digestion from K. lactis whole cells.