Flocculation in industrial strains of Saccharomyces cerevisiae: role of cell wall polysaccharides and lectin‐like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin‐like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae , representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin‐like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin‐like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New‐Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin‐like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast‐based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

THE STRUCTURE,SYNTHESIS AND FUNCTIONS OF THE YEAST CELL WALL—A REVIEW

Glucan, mannan, chitin, protein, phosphate and lipid are found in the walls of all brewing yeasts so far examined although the contents and composition of each varies, as does the content of wall in the cell. Both glucan and mannan have branched structures and are linked to protein. Chitin is probably also bound to protein and phosphate to mannan. Some of the enzymic activities present in the wall are associated with the mannan-protein fraction. This fraction is located on the outer surface of the wall and appears to play a part in the appearance of flocculence in brewing yeast during fermentation.     

Reserved carbohydrates of different Saccharomyces cerevisiae strains

Three Saccharomyces strains were compared with commercial pressed yeast using the following criteria: rate of growth, crude protein, percentage of trehalose and glycogen, mannan plus glucan. Results proved that Sacch. cerevisiae 14 had better characteristic aspects than the other tested organisms. Its biomass reached 567 mg dried cells/100 ml culture, with the specific growth rate of 0.026. As a function of 120 h of incubation, Sacch. cerevisiae 14 showed the highest trehalose and glycogen, mannan plus glucan content being 21.1 mg/ 100 mg dried cells. On the other hand, the highest growth densities were obtained at 81 mg of O₂/(600 ml culture. min). Thus, the highest trehalose and glycogen, mannan plus glucan contents were obtained at 81 mg O₂/(600 ml medium. min) and 189 mg O₂/(600 ml medium. min)     

Galactose-inducible Expression Systems in Candida maltosa using Promoters of Newly-isolated GAL1 and GAL10 Genes

The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa. The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts. The expression of both genes was strongly induced by galactose and repressed by glucose in the medium. Galactose-inducible expression vectors in C. maltosa were constructed on low- and high-copy number plasmids using the promoter regions of both genes. With these vectors and the β-galactosidase gene from Kluyveromyces lactis as a reporter, galactose-inducible expression was confirmed. Homologous overexpression of members of the cytochrome P-450 gene family in C. maltosa was also successful by using a high-copy-number vector under the control of these promoters. © 1997 by John Wiley & Sons, Ltd.     

Binding of heterocyclic amines by yeast cells and their fractions

The binding of mutagenic pyrolysis products to cells of 50 yeast strains and their cell fractions was investigated. Cells of all yeast strains effectively bound 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Cell walls (CW), and cell wall glucan and mannan (5 mg in each case) showed the highest binding of Trp-P-1 (50 μg ml^?1); glucan adsorbed virtually all of the Trp-P-1. Cytoplasm also showed some binding but was much less effective. Glucans also bound well with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo 4,5-quinoxaline (MeIQX) much more than CW, but 2-amino-5-phenylpyridine (Phe-P-1) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MelQ) were not effectively bound. The quantity of IQ, MeIQ, Phe-P-1 and MeIQX bound was dependent on the strain of yeast. The mutagenic pyrolysis products bound to cells were effectively extracted by aqueous methanol, ammonia (50 g litre^?1) and alcohol, but not by water. The binding was pH dependent and inhibited by metal salts. When yeast cells were heated to 100° for 15 min, the binding of Trp-P-1 decreased by about 30% but Saccharomyces cerevisiae 50 heated to 100° did not differ much from untreated cells in its binding ability.     

Comparative digestibility of carbohydrates of microbial products and their metabolisable energy values in chicks and rats

The digestibility of the carbohydrate components and the metabolisable energy (ME) values of two yeasts grown on hydrocarbon (BPG and BPL yeasts) and a filamentous fungus grown on carbohydrate waste (RHM fungus) have been investigated in rats and chicks. The microbial products were included at three different levels. Mannan, and chicks, and glycogen have been shown to be the major constituent polysaccharides of these microbial products. Glycogen in the three samples tested was found to be completely digested by both animals. Mannan, β-glucan and chitin were less digestible and their utilisation varied according to the level of inclusion in the diet. The difference in the ability of the animals to digest the cell wall carbohydrates or to metabolise the gross energy from the test ingredients was significant (P 0.001). Rats digested up to 68.1 % chitin, 47.4% β-glucan and 41.8% mannan but the highest value obtained for chicks was 12.9% for mannan. Chitin had the lowest digestibility in chicks varying between 0.7 and 2.9%, whilst values for β-glucan were between 3.3 and 11.7%. Uncorrected ME values for BPG yeast, BPL yeast and RHM fungus, respectively, were 13 617, 13 190 and 12 194 kJ kg^?1 dry matter for chicks and 16 380, 15 903 and 14 107 kJ kg^?1 DM for rats. Correction of these values to zero nitrogen retention lowered the values by about 18% in chicks and 8 % in rats. Adjusting the values to 30 and 50% nitrogen retention for rats and chicks, respectively, resulted in values which were very close to uncorrected values.     

RESERVE CARBOHYDRATE METABOLISM AND CELL SURVIVAL IN AEROBICALLY STARVING BAKER'S YEAST

Changes in the level of reserve carbohydrates, cell number, viability and medium pH values were observed in aerobically starving baker's yeast. The starvation was carried out at 35°C in the physiological saline solution using two different initial pH values (4·4 or 5·5) of the medium. In addition, the formation of ethanol and acetate as well as actual and endogenous respirations were measured at a pH of 4·4. The results revealed that the initial pH value of the medium affected the pattern of glycogen and trehalose degradation and consequently, also caused the loss of viability and cell lysis. Changes in the quantity of metabolic products and respiration activity are discussed in connection with the metabolism of the starving cells and the effect of environmental conditions.     

A Study of Saccharomyces cerevisiae Cell Wall Glucans

Much research has been carried out over the years examining cell wall glucans from Saccharomyces cerevisiae and this study further examines aspects of the binding of (1r?4)‐α‐D‐glucan in the yeast cell wall, using a number of isolation techniques as well as monoclonal antibodies able to recognize a mixed (1r?4)‐α‐D‐glucan/(1r?6)‐β‐D‐glucan. Extraction of purified glucan, from S. cerevisiae cell wall, with 0.1N HCl, at 80°C for 6 h, released into the solution (1r?4)‐α‐D‐glucan and (1r?6)‐β‐D‐glucan as the major polysaccharides, along with an insoluble pellet highly enriched in (1r?3)‐β‐D‐glucan. The released (1r?4)‐α‐D‐glucan was composed of a high molecular size >100 kDa fraction (7.2% w/w) and a medium 5–50 kDa polysaccharide (10.2% w/w), with the (1r?4)‐α‐D‐glucan covalently bound to the (1r?6)‐β‐D‐glucan. The average molar ratio of the α:β glucan was 47: 53 in this mixed polysaccharide. The structure of this polysaccharide was different from the structure of plant starch or animal glycogen as monoclonal antibodies specific to yeast (1r?4)‐α‐D‐glucan/(1r?6)‐β‐D‐glucan did not recognize the plant starch or animal glycogen standards.     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.     

YEAST GLUCANASE AND MANNANASE

Synchronously-dividing cultures of brewer's yeast show cyclical changes in activities of mannanase, glucanase and laminarinase, suggesting that these enzymes may have a function in the budding process. The same enzymes are present in autolysates of brewer's yeast; the laminarinase completely hydrolyses laminarin to monosaccharide but the others yield products larger than tetrasaccharides from the native mannan and glucan.     

A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae

A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.     

STUDY OF THE PHENOMENON OF AGGLOMERATION IN THE YEAST SACCHAROMYCES CEREVISIAE

Agglomeration or “grittiness” is detrimental to bakers' yeast quality. Gritty yeast only partially resuspends when mixed in water, most of it remaining as macroscopic cell aggregates. A macroscopic sedimentation test was developed for measuring agglomeration intensity. Expression of the gritty phenotype was investigated in two strains (N176 and GB1) of Saccharomyces cerevisiae grown on a 14-liter scale by varying fermentation conditions of agitation and aeration. Results show that yeast agglomeration is different from yeast flocculation, and is determined by both strain genetic background and environmental factors. The gritty phenotype was expressed in the strain prone to agglomeration (N176) when dissolved oxygen was limiting in the fermenter. Gritty cells had a lower phosphorus and lipid concentration and a higher protein concentration at the surface of the cell, and a higher amount of whole cell and cell wall proteins and calcium than non-gritty cells. Some proteins were also extracted from gritty cells with sodium hydroxide or mercaptoethanol, that were not present in non-gritty cells. Agglomeration did not result in major differences in the structure or composition of the structural cell wall mannoprotein (CWMP). A model for agglomeration is proposed: proteins (cognors) activated by Ca²⁺ (cofactors) to increase their binding capacity bind the mannans (cognons) of adjoining cells; binding is facilitated by the lower phosphorus and lipid concentration at the surface of gritty cells.     

Adsorption of Zearalenone by beta-D-glucans in the Saccharomyces cerevisiae cell wall

Cell walls of yeasts and bacteria are able to complex with mycotoxins and limit their bioavailability in the digestive tract when these yeasts and bacteria are given as feed additives to animals. To identify the component(s) of the yeast cell wall and the chemical interaction(s) involved in complex formation with zearalenone, four strains of Saccharomyces cerevisiae differing in their cell wall glucan and mannan content were tested. Laboratory strains wt292, fks1, and mnn9 were compared with industrial S. cerevisiae strain sc1026. The complex-forming capacity of the yeast cell walls was determined in vitro by modelling the plots of amount of toxin bound versus amount of toxin added using Hill's model. A cooperative relationship between toxin and adsorbent was shown, and a correlation between the amount of beta-D-glucans in cell walls and complex-forming efficacy was revealed (R2 = 0.889). Cell walls of strains wt292 and mnn9, which have higher levels of beta-D-glucans, were able to complex larger amounts of zearalenone, with higher association constants and higher affinity rates than those of the fks1 and sc1026 strains. The high chitin content in strains mnn9 and fks1 increased the alkali insolubility of beta-D-glucans from isolated cell walls and decreased the flexibility of these cell walls, which restricted access of zearalenone to the chemical sites of the beta-D-glucans involved in complex formation. The strains with high chitin content thus had a lower complex-forming capacity than expected based on their beta-D-glucans content. Cooperativity and the three-dimensional structure of beta-D-glucans indicate that weak noncovalent bonds are involved in the complex-forming mechanisms associated with zearalenone. The chemical interactions between beta-D-glucans and zearalenone are therefore more of an adsorption type than a binding type.     

A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.     

Yeast flocculation: receptor definition by mnn mutants and concanavalin A.

Yeast flocculation involves the binding of surface lectins on flocculent yeasts, to carbohydrate receptors present as constituents of yeast cell walls. Receptors were investigated by coflocculation of flocculent strains of Saccharomyces cerevisiae, of both Flo 1 and NewFlo phenotypes, to known mnn mutants which vary in the wall mannan structure. Strong coflocculation was found with mnn1, mnn4, mnn9 and control strains, while very little coflocculation was found with mnn2 and mnn5 strains. In contrast, aggregation of these mutants by concanavalin A, a lectin with similar sugar inhibition to NewFlo phenotype flocculation, showed strong aggregation of mnn1, mnn4 and mnn5 strains and poor aggregation of mnn2 and mnn9 strains. The mmn mutant data suggested that flocculation receptors were the outer-chain mannan side-branches, two or three mannose residues in length, confirming an earlier theory based on sugar inhibition data. The similarities and differences between flocculation and concanavalin A aggregation are discussed.     

A Lallzyme MMX‐based rapid method for fission yeast protoplast preparation

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi. Copyright © 2013 John Wiley & Sons, Ltd.     

Polyamines and cell wall organization in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.     

An Enzymatic Method for the Determination of Yeast Cell Wall Glucan in Foods

Processed cell wall β-glucans prepared from strains of baker's or brewer's yeast can be used as a thickening agent in aqueous food systems and they provide a fat-like mouthfeel. This article describes an analytical procedure for the determination of the glucan content added to a variety of food products. The method is based on measurement of reducing sugars produced when the food sample, after appropriate pretreatment, is treated with a commercial microbial β-glucanase (Zymolyase) that is specific for the β-linkages present in the glucan polysaccharide of the added yeast cell walls.     

The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae

The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases. Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase. When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained. Mutants with truncated mannan side-chains in their wall proteins also had very porous walls. The importance of the mannan side-chains for wall porosity was also seen during sexual induction. Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity. Disulphide bridges also affect cell wall porosity. They were predominantly found in the glucanase-soluble wall proteins. Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.     

Growth of a tropical marine yeast Yarrowia lipolytica NCIM 3589 on bromoalkanes: relevance of cell size and cell surface properties

Yarrowia lipolytica 3589, a tropical marine yeast, grew aerobically on a broad range of bromoalkanes varying in carbon chain length and differing in degree and position of bromide group. Amongst the bromoalkanes studied, viz. 2‐bromopropane (2‐BP), 1‐bromobutane (1‐BB), 1,5‐dibromopentane (1,5‐DBP) and 1‐bromodecane (1‐BD), the best utilized was 1‐BD, with a maximal growth rate (μ_max) of 0.055 h^?1 and an affinity ratio (μ_max/K_s) of 0.022. Utilization of these bromoalkanes as growth substrates was associated with a concomitant release of bromide (8202.9 µm ) and cell mass (36 × 10⁹ cells/ml), occurring maximally on 1‐BD. Adherence of yeast cells to these hydrophobic bromoalkanes was observed microscopically, with an increase in cell size and surface hydrophobicity. The maximal cell diameter was for 1‐BD (4.66 µm), resulting in an increase in the calculated cell surface area (68.19 µm²) and sedimentation velocity (1.31 µm/s). Cell surface hydrophobicity values by microbial adhesion to solvents (MATS) analysis for yeasts grown on bromoalkanes and glucose were significantly high, i.e. >80%. Similarly, water contact angles also indicate that the cell surface of yeast cells grown in glucose possess a relatively more hydrophilic cell surface (θ = 49.1°), whereas cells grown in 1‐BD possess a more hydrophobic cell surface (θ = 90.7°). No significant change in emulsification activity or surface tension was detected in the cell‐free supernatant. Thus adherence to the bromoalkane droplets by an increase in cell size and surface hydrophobicity leading to debromination of the substrate might be the strategy employed in bromoalkane utilization and growth by Y. lipolytica 3589. Copyright © 2011 John Wiley & Sons, Ltd.