Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Use of Saccharomyces cerevisiae for Cu2+ removal from solution: the advantages of using a flocculent strain

A flocculent strain of Saccharomyces cerevisiae S646-1B accumulated more Cu²⁺ (81 nmol mg^–1 dry wt) than the isogenic (except for the marker genes ade1 and trp1 and the gene FLO1) non-flocculent strain S646-8D (30 nmol mg^–1 dry wt), in the first 10 min of contact of the cells with Cu²⁺. Additionally, this strain flocculated in solutions of 0.2 mM Cu²⁺, Ni²⁺, Zn²⁺ and Cd²⁺. The potential of using flocculent strains in the bioremediation of heavy metals contaminated waste waters is discussed.     

Metabolic state and cell cycle as determinants of facilitated uptake of genetic information by yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The transformation efficiency of yeast cells during exponential growth might be characterised as undulatory. The aim of the study was to investigate the reason for the fluctuation in transformation efficiency of yeast Saccharomyces cerevisiae p63-DC5 cells during exponential growth. The heightened response to exogenous DNA was observed with the growing yeast culture when budded cells were predominant. To confirm this phenomenon we carried out synchronization of yeast cells with 10 mM hydroxyurea. Results showed that synchronous yeast cells in the S-phase of cell cycle have enhanced transformation efficiency. Furthermore, S. cerevisiae p63-DC5 cells in the S-phase were successfully transformed with plasmid pl13 in the absence of lithium acetate. We indicated that the permeability of yeast cells in the S-phase to tetraphenylphosphonium (TPP) cations was significantly higher than in asynchronous culture. The results of our study showed that the fluctuation in transformation efficiency was strictly dependent on the metabolic state of yeast cells and the capacity of the yeast cells to become competent was related to the S-phase of cell cycle.     

Biosorption of heavy metals by Saccharomyces cerevisiae

Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4–5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast ( 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine-needle-like crystals both on the inside and outside of the S. cerevisiae cells.     

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H⁺ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The K_M-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The V_MAX but not the K_M-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.     

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.     

Co-Flocculation of Yeast Species,a New Mechanism to Govern Population Dynamics in Microbial Ecosystems

Flocculation has primarily been studied as an important technological property of Saccharomyces cerevisiae yeast strains in fermentation processes such as brewing and winemaking. These studies have led to the identification of a group of closely related genes, referred to as the FLO gene family, which controls the flocculation phenotype. All naturally occurring S. cerevisiae strains assessed thus far possess at least four independent copies of structurally similar FLO genes, namely FLO1, FLO5, FLO9 and FLO10. The genes appear to differ primarily by the degree of flocculation induced by their expression. However, the reason for the existence of a large family of very similar genes, all involved in the same phenotype, has remained unclear. In natural ecosystems, and in wine production, S. cerevisiae growth together and competes with a large number of other Saccharomyces and many more non-Saccharomyces yeast species. Our data show that many strains of such wine-related non-Saccharomyces species, some of which have recently attracted significant biotechnological interest as they contribute positively to fermentation and wine character, were able to flocculate efficiently. The data also show that both flocculent and non-flocculent S. cerevisiae strains formed mixed species flocs (a process hereafter referred to as co-flocculation) with some of these non-Saccharomyces yeasts. This ability of yeast strains to impact flocculation behaviour of other species in mixed inocula has not been described previously. Further investigation into the genetic regulation of co-flocculation revealed that different FLO genes impact differently on such adhesion phenotypes, favouring adhesion with some species while excluding other species from such mixed flocs. The data therefore strongly suggest that FLO genes govern the selective association of S. cerevisiae with specific species of non-Saccharomyces yeasts, and may therefore be drivers of ecosystem organisational patterns. Our data provide, for the first time, insights into the role of the FLO gene family beyond intraspecies cellular association, and suggest a wider evolutionary role for the FLO genes. Such a role would explain the evolutionary persistence of a large multigene family of genes with apparently similar function.     

<Emphasis Type="Italic">Starmerella bombicola</Emphasis> influences the metabolism of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

Background  

The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

The release of sex-specific substances responsible for sexual agglutination from haploid cells of Saccharomyces cerevisiae

Cell surface substances responsible for sexual cell agglutination were successfully released in a large quantity from heterothallic haploid cells of Saccharomyces cerevisiae by a newly established autoclaving method. The conditions for this releasing phenomenon were examined. The sexual agglutination substances were solubilized most efficiently when the cells, suspended in a 30 mM Tris-HCl, pH 7.0, 5 mM EDTA solution, were autoclaved at a pressure of 1 kg/cm² at 120 °C for 3 min. The substances were specifically adsorbed by the cell surface of the opposite mating type, resulting in the masking of agglutinability of the cells of the opposite mating type. The substances were not released from the surface of cells which lacked sexual cell agglutination. The evidence suggesting the formation of a molecular complex between a- and α-agglutination substances in vitro is also presented. The above procedure is applicable to the solubilization of surfage agglutination substances from various strains of S. cerevisiae.     

Effects of Pichia kluyveri killer toxin on sensitive cells

The killer toxin produced by Pichia kluyveri 1002 kills yeast strains of the genera Candida, Saccharomyces and Torulopsis, including several S. cerevisiae killer strains.Binding of a lethal amount of the toxin to cells of S. cerevisiae SCF 1717 occurs rapidly after toxin addition. After treatment with the toxin for 10 min sensitive cells partially recovered when incubated under conditions that favor protein synthesis. Only after a lag time of 50–90 min sensitive cells changed physiologically. Killing of sensitive cells was characterized by leakage of potassium and adenosine 5-triphosphate, decrease of intracellular pH, and inhibition of the active uptake of amino acids. These effects coincided with cell shrinkage and varied with incubation conditions.Uptake of the amino acid leucine in sensitive cells involved two apparently distinct transport systems (K_m1=0.04mm; K_m2=0.46mm). The toxin showed different effects on these transport systems.     

Enzyme identification and development of a whole-cell biotransformation for asymmetric reduction of o-chloroacetophenone

Chiral 1‐(o‐chlorophenyl)‐ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o‐chloroacetophenone is a preferred method of production but well investigated chemo‐ and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o‐chloroacetophenone with useful specificity (k_cat/K_m = 340 M⁻¹ s⁻¹) and perfect S‐stereoselectivity, we developed whole‐cell catalysts from Escherichia coli and Saccharomyces cerevisiae co‐expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o‐Chloroacetophenone was unexpectedly “toxic,” and catalyst half‐life times of only 20 min (E. coli) and 30 min (S. cerevisiae) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o‐chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD⁺ (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S‐1‐(o‐chlorophenyl)‐ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity. Biotechnol. Bioeng. 2011; 108:797–803. © 2010 Wiley Periodicals, Inc.     

Use of the somatic fusion method to introduce the flocculation property into Saccharomyces diastaticus

Summary Spheroplasts of a petite mutant of the amylolitic Saccharomyces diastaticus 1376 yeast strain were successfully fused with spheroplasts of a flocculent and respiratory competent Saccharomyces cerevisiae 1161 yeast strain.Flocculent and non-flocculent stable recombinants were recovered after regeneration of the cell walls all of which formed halos around their colonies in media containing starch/dextrin as carbon source. The sporulation ability varied in some of the fusion products and the possible influence of genetic instability is discussed.     

Microarray karyotyping of maltose‐fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization

Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed‐field gel electrophoresis blots, we analysed the copy number and localization of several maltose‐related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Reduction of Aluminum Toxicity by 2-Isopropylmalic Acid in the Budding Yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae secretes 2-isopropylmalic acid (2-iPMA), an intermediate in leucine biosynthesis. Because 2-iPMA binds Al(III) in the culture medium, it is thought to reduce toxicity by Al(III). The effects of 2-iPMA and malic acid (MA) on Al toxicity were investigated in a medium with a low pH and low concentrations of phosphates and magnesium. The reduction in the growth of S. cerevisiae observed in the presence of 100 μM Al(III) ions was relieved more by the addition of 1.0 mM 2-iPMA than by 1.0 mM MA, indicating that 2-iPMA possesses superior Al(III)-ion detoxification ability. Investigations using the wild type and the Δleu4 and Δleu9 mutant strains indicated that secretion of a sufficient level of 2-iPMA was required to enhance the Al tolerance. It is thought that 2-iPMA secreted from the yeast cells chelates Al ions and prevents them from entering the cells, resulting in Al tolerance. Suzuki and Tamura contributed equally to this work.     

Characterization of Antimicrobial Activity of the Lysosomes Isolated from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The antimicrobial activity of lysosomes, a cell organelle, against a range of test microorganisms was examined in this study. The lysosomes isolated from Saccharomyces cerevisiae showed antimicrobial activity to Escherichia coli that positively correlated with the pH of the phosphate buffer as a dissolving solvent. The lysosomes from S. cerevisiae exhibited optimal activity at a concentration of 40%, at pH 4.0 of phosphate buffer, and at broad range temperature, except of over 50°C. It was also found that the lysosomes have antimicrobial activity against seven different microorganisms including E. coli. In addition, S. cerevisiae were exposed by a treatment with H₂O₂ and lysosomes were isolated from H₂O₂ exposed S. cerevisiae. We found that fluorescent intensities of each isolated lysosomes were increased depending on the increment of treated H₂O₂ concentration, and the lysosomes from 20 mM H₂O₂ treated S. cerevisiae showed higher antimicrobial activity than those from normal S. cerevisiae. Therefore, it suggests that lysosomes isolated from S. cerevisiae can be used as an antimicrobial agent. In addition, lysosomes activated by H₂O₂ enhanced its antimicrobial activity.     

Enrichment of a continuous culture of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with the yeast <Emphasis Type="Italic">Issatchenkia orientalis</Emphasis> in the production of ethanol at increasing temperatures

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47°C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42°C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40°C than at 30°C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.     

Anti-hypercalcemic effect of orally administered recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing salmon calcitonin on hypercalcemic rats

Oral delivery of salmon calcitonin (sCT) to rats via a recombinant Saccharomyces cerevisiae was assessed. A synthetic sCT gene was cloned and expressed in S. cerevisiae yAGA2-sCT. Recombinant salmon calcitonin (rsCT) expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by 3 × 10⁻⁶ M rsCT. Oral administration of 5 g lyophilized yAGA2-sCT/kg to hypercalcemic rats decreased serum calcium from 2.8 ± 0.02–2.7 ± 0.02 mM.