Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.
The biological control of flocculation interactions by factors related to growth under different conditions of aeration was documented with a new assay for flocculence. The degree of flocculence expressed in a genetically defined Saccharomyces cerevisiae strain (FLO1/FLO1 ade1/ade1) remained constant during aerobic growth but varied with aeration. Flocculence was repressed in anaerobically growing cells but was induced in stationary cells or cells returned to aerobic growth. Repression was correlated with the selective inactivation of cell surface lectin-like components. The changes in flocculence were accompanied by changes in 16 extractable proteins separated by electrophoresis; however, a clear correlation between specific protein bands and flocculence could not be established. The study clearly demonstrated that the phenotypic expression of FLO1 could be reproducibly manipulated for experimental purposes by aeration alone.
Flocculation is an attractive property for Saccaromyces cerevisiae, which plays important roles in fermentation industry and environmental remediation. The process of flocculation is mediated by a family of cell surface flocculins. As one member of flocculins, Flo1 is characterized by four families of repeats (designated as repeat units A, B, C and D) in the central domain. It is generally accepted that variation of repeat unit A in length in Flo1 influences the degree of flocculation or specificity for sugar recognization. However, no reports were observed for other repeat units. Here, we compared the flocculation ability and its sensitivity to environmental factors between yeast strain YSF1 carrying the intact FLO1 gene and yeast strains carrying the derived forms of FLO1 with partial or complete deletion of repeats in unit C. No obvious differences in flocculation ability and specificity of carbohydrate recognition were observed among these yeast strains, which indicates the truncated flocculins can stride across the cell wall and cluster the N-terminal domain on the surface of yeast cells as the intact Flo1 thereby improving intercellular binding. However, yeast strains with the truncated flocculins required more mannose to inhibit completely the flocculation, displayed broad tolerance of flocculation to pH fluctuation, and the fewer the repeats in unit C, the stronger adaptability of flocculation to pH change, which was not relevant to the position of deletion. This suggests that more stable active conformation is obtained for flocculin by deletion the repeat unit C in the central domain of Flo1, which was validated further by the higher hydrophobicity on the surface of cells of YSF1c with complete deletion of unit C under neutral and alkaline conditions and the stabilization of GFP conformation by fusion with flocculin with complete deletion of unit C in the central domain.
In many industrial fermentation processes, the Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation, a high suspended yeast count is required to maintain a satisfactory rate of fermentation, while at completion, efficient settling is desired to enhance product clarification and recovery. In most fermentation industries, currently used starter cultures do not satisfy this ideal, probably because nonflocculent yeast strains were selected to avoid fermentation problems. In this paper, we assess molecular strategies to optimize the flocculation behavior of S. cerevisiae. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5, and FLO11, of the haploid nonflocculent, noninvasive, and non-flor-forming S. cerevisiae FY23 strain were placed under the transcriptional control of the promoters of the ADH2 and HSP30 genes. All six promoter-gene combinations resulted in specific flocculation behaviors in terms of timing and intensity. The strategy resulted in stable expression patterns providing a platform for the direct comparison and assessment of the specific impact of the expression of individual dominant FLO genes with regard to cell wall characteristics, such as hydrophobicity, biofilm formation, and substrate adhesion properties. The data also clearly demonstrate that the flocculation behavior of yeast strains can be tightly controlled and fine-tuned to satisfy specific industrial requirements.
The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait.
Flocculation; FLO1; drug resistance; evolution; ethanol; selfish gene; Darwin; Dawkins; Hamilton; green beard gene
The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used.
Harvesting; Microalgae; Bio-flocculation
A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47°C. At 43°C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43°C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50°C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.
Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 107 CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic.
Cell aggregation in unicellular organisms, induced by either cell non-sexual adhesion to yield flocs and biofilm, or pheromone-driving sexual conjugation is of great significance in cellular stress response, medicine, and brewing industries. Most current literatures have focused on one form of cell aggregation termed flocculation and its major molecular determinants, the flocculation (FLO) family genes. Here, we implemented a map-based approach for dissecting the molecular basis of non-sexual cell aggregation in Saccharomyces cerevisiae. Genome-wide mapping has identified four major quantitative trait loci (QTL) underlying nature variation in the cell aggregation phenotype. High-resolution mapping following up with knockout and allele replacement experiments resolved the QTL into the underlying genes (AMN1, RGA1, FLO1, and FLO8) or even into the causative nucleotide. Genetic variation in the QTL genes can explain up to 46% of phenotypic variation of this trait. Of these genes, AMN1 plays the leading role, differing from the FLO family members, in regulating expression of cell clumping phenotype through inducing cell segregation defect. These findings provide novel insights into the molecular mechanism of how cell aggregation is regulated in budding yeast, and the data will be directly implicated to understand the molecular basis and evolutionary implications of cell aggregation in other fungus species.
cell aggregation; map-based cloning; QTL analysis; Saccharomyces cerevisiae
We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.
The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.
We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.
Hyphal morphogenesis in Candida albicans is regulated by multiple pathways which act by either inducing or repressing filamentation. Most notably, Tup1, Nrg1, and Rfg1 are transcriptional repressors, while Efg1, Flo8, Cph1, and Czf1 can induce filamentation. Here, we present the functional analysis of CaSFL1, which encodes the C. albicans homolog of the Saccharomyces cerevisiae SFL1 (suppressor of flocculation) gene. Deletion of CaSFL1 results in flocculation (i.e., the formation of clumps) of yeast cells, which is most pronounced in minimal medium. The flocs contained hyphae already under noninducing conditions, and filamentation could be enhanced with hypha-inducing cues at 37°C. Expression of SFL1 in a heterozygous mutant under the control of the CaMET3 promoter was shown to complement these defects and allowed switching between wild-type and mutant phenotypes. Interestingly, increased expression of SFL1 using a MET3prom-SFL1 construct prior to the induction of filamentation completely blocked germ tube formation. To localize Sfl1 in vivo, we generated a SFL1-GFP fusion. Sfl1-green fluorescent protein was found in the nucleus in both yeast cells and, to a lesser extent, hyphal cells. Using reverse transcription-PCR, we find an increased expression of ALS1, ALS3, HWP1, ECE1, and also FLO8. Our results suggest that Sfl1 functions in the repression of flocculation and filamentation and thus represents a novel negative regulator of C. albicans morphogenesis.
The inhibition of growth by octanoic or decanoic acids, two subproducts of ethanolic fermentation, was evaluated in Saccharomyces cerevisiae and Kluyveromyces marxianus in association with ethanol, the main product of fermentation. In both strains, octanoic and decanoic acids, at concentrations up to 16 and 8 mg/liter, respectively, decreased the maximum specific growth rate and the biomass yield at 30°C as an exponential function of the fatty acid concentration and increased the duration of growth latency. These toxic effects increased with a decrease in pH in the range of 5.4 to 3.0, indicating that the undissociated form is the toxic molecule. Decanoic acid was more toxic than octanoic acid. The concentrations of octanoic and decanoic acids were determined during the ethanolic fermentation (30°C) of two laboratory media (mineral and complex) by S. cerevisiae and of Jerusalem artichoke juice by K. marxianus. Based on the concentrations detected (0.7 to 23 mg/liter) and the kinetics of growth inhibition, the presence of octanoic and decanoic acids cannot be ignored in the evaluation of the overall inhibition of ethanolic fermentation.
Cell–cell and cell–surface adherence represents initial steps in forming multicellular aggregates or in establishing cell–surface interactions. The commonly used Saccharomyces cerevisiae laboratory strain S288c carries a flo8 mutation, and is only able to express the flocculin-encoding genes FLO1 and FLO11, when FLO8 is restored. We show here that the two flocculin genes exhibit differences in regulation to execute distinct functions under various environmental conditions. In contrast to the laboratory strain Σ1278b, haploids of the S288c genetic background require FLO1 for cell–cell and cell–substrate adhesion, whereas FLO11 is required for pseudohyphae formation of diploids. In contrast to FLO11, FLO1 repression requires the Sin4p mediator tail component, but is independent of the repressor Sfl1p. FLO1 regulation also differs from FLO11, because it requires neither the KSS1 MAP kinase cascade nor the pathways which lead to the transcription factors Gcn4p or Msn1p. The protein kinase A pathway and the transcription factors Flo8p and Mss11p are the major regulators for FLO1 expression. Therefore, S. cerevisiae is prepared to simultaneously express two genes of its otherwise silenced FLO reservoir resulting in an appropriate cellular surface for different environments.
Eight strains of Kluyveromyces marxianus were screened, and all of them were found to ferment the aldopentose d-xylose directly to ethanol under aerobic conditions. One of these strains, K. marxianus SUB-80-S, was grown in a medium containing 20 g of d-xylose per liter, and the following results were obtained: maximum ethanol concentration, 5.6 g/liter; ethanol yield, 0.28 g of ethanol per g of d-xylose (55% of theoretical); maximum specific growth rate, 0.12 h−1; 100% d-xylose utilization was completed in 48 h.
For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.
Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.
The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.
Mitochondrial DNA from the yeast Kluyveromyces marxianus var. lactis (K.lactis) is a circular molecule of 39 kilobase-pairs. A genetic and physical map was constructed. We found that this genome contained a large number of guanine-cytosine (GC)-rich sequence clusters, many of which are characterized by the presence of SacII restriction sites (CCGCGG). The primary sequence of the GC clusters often showed a palindromic structure. These GC clusters were present in several varieties of K.marxianus, but not in others. The presence of these clusters is a major feature that distinguishes K.lactis strains from those of K.marxianus var. marxianus (including K.fragilis).
To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.
The 5′ upstream regions of the Saccharomyces cerevisiae glucoamylase-encoding genes STA1 to -3 and of the MUC1 (or FLO11) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent. Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view. Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level. To investigate the transcriptional regulation, we compared the transcription levels conferred by the STA2 and MUC1 promoters under various growth conditions. Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects. We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression. We also identified the STA10 repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in S. cerevisiae.
The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell−cell and cell−surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.
Mss11; FLO; cellular adhesion; cell wall
A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.
Kluyveromyces marxianus possesses a useful potential to assimilate a wide variety of substrates at a high temperature, but the negative effect by coexisting glucose is critical for utilization of biomass containing various sugars. Such a negative effect on the activity of inulinase, which is the sole enzyme to hydrolyze sucrose, raffinose and inulin, has been demonstrated in K. marxianus without analysis at the gene level. To clarify the utilization capability of sucrose, raffinose and inulin and the glucose effect on inulinase in K. marxianus DMKU 3-1042, its growth and metabolite profiles on these sugars were examined with or without glucose under a static condition, in which glucose repression evidently occurs. Consumption of sucrose was not influenced by glucose or 2-deoxyglucose. On the other hand, raffinose and inulin consumption was hampered by glucose at 30°C but hardly hampered at 45°C. Unlike Saccharomyces cerevisiae, increase in glucose concentration had no effect on sucrose utilization. These sugar-specific glucose effects were consistent with the level of inulinase activity but not with that of the KmINU1 transcript, which was repressed in the presence of glucose via KmMig1p. This inconsistency may be due to sufficient activity of inulinase even when glucose is present. Our results encourage us to apply K. marxianus DMKU 3-1042 to high-temperature ethanol fermentation with biomass containing these sugars with glucose.
Kluyveromyces marxianus; inulinase; glucose repression; INU1; MIG1
We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.
Yeast flocculation has been found to be important in many biotechnological processes. It has been suggested that flocculation is promoted by decreasing electrostatic repulsion between cells. In this study, we used an unconventional rapid technique—permittivity test—for determination of the flocculation properties and surface charge values of three industrial yeast strains with well-known flocculation characteristics: Saccharomyces cerevisiae NCYC 1017 (brewery, ale), S. pastorianus NCYC 680 (brewery, lager), and Debaryomyces occidentalis LOCK 0251 (unconventional amylolytic yeast). The measurements of permittivity were compared with the results from two classical methods for determination of surface charge: Alcian blue retention and Sephadex DEAE attachment. The permittivity values for particular strains correlated directly with the results of Alcian blue retention (r = 0.9). The results also confirmed a strong negative relationship between the capacitance of yeast suspensions and their flocculation abilities. The highest permittivity was noted for the ale strain NCYC 1017, with weak flocculation abilities, and the lowest for the flocculating lager yeast NCYC 680. This paper is the first to describe the possibility of using a rapid permittivity test to evaluate the surface charge of yeast cells and their flocculation abilities. This method is of practical value in various biotechnological industries where flocculation is applied as a major method of cell separation.
Yeasts; Flocculation; Surface charge; Permittivity; Biocontrol