Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Reaction kinetics of -glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of -glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of -glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.     

Regio- and enantioselective reduction of methyleneketoesters mediated by Saccharomyces cerevisiae

Methyleneketoesters were readily prepared in high yields by performing a direct -methylenation of the corresponding ketoesters using a previously described protocol. Reactions of ethyl 2-methylene-3-oxo-3-arylpropanoates 2a–c catalyzed by S. cerevisiae were performed with good conversions to give reductions of the CC, CO or both, depending on the reaction conditions and on the substitution of the aryl moiety. Reaction of 3-methylene-2-oxo-4-phenylbutyrate 2d was carried out with free yeast cells and with yeast cells immobilized with calcium alginate, in which the major products resulted from CC and CO bond reduction.

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The use of immobilized Saccharomyces cerevisiae on radiation crosslinked acrylamide–maleic acid hydrogel carriers for production of ethyl alcohol

Radiation crosslinked acrylamide/maleic acid (AAm/MA) copolymers were prepared by γ-irradiation. They were used in experiments on swelling, diffusion, and immobilization of yeast cells (Saccharomyces cerevisiae) for the production of ethyl alcohol. AAm/MA hydrogels containing different amount of MA, irradiated at different doses, were used for swelling and diffusion studies. The parameters of swelling, diffusional exponents, network constants, diffusion coefficients and percent porosity of the hydrogel/penetrant systems were calculated and evaluated. Yeast cells were immobilized on to the hydrogels by adsorption during multiplication, and ethyl alcohol production by the hydrogels was investigated. Swelling of AAm/MA increased with increase in MA content. Ethyl alcohol production also increased with increasing MA in the hydrogels but decreased with an increase of irradiation dose.     

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Elevation of glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant Saccharomyces cerevisiae

To increase the NAD(P)H-dependent xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis, the activity of glucose 6-phosphate dehydrogenase (G6PDH) encoded by the ZWF1 gene was amplified to increase the metabolic flux toward the pentose phosphate pathway and NADPH regeneration. Compared with the control strain, the specific G6PDH activity was enhanced approximately 6.0-fold by overexpression of the ZWF1 gene. Amplification in the G6PDH activity clearly improved the NAD(P)H-dependent xylitol production in the recombinant S. cerevisiae strain. With the aid of an elevated G6PDH level, maximum xylitol concentration of 86 g/l was achieved with productivity of 2.0 g/l h in the glucose-limited fed-batch cultivation, corresponding to 25% improvement in volumetric xylitol productivity compared with the recombinant S. cerevisiae strain containing the xylose reductase gene only.     

Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator $\text{m-}\text{chlorocarbonylcyanide}$ phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.     

Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na⁺/P_i cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their P_i uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na⁺ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na⁺ was omitted, this activity decreased significantly, reaching values close to those exhibited by the Δpho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of P_i limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na⁺/P_i cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent K_m of 0.5 μM and a pH optimum of 9.5, and is highly specific for Na⁺; activation of transport is maximal at a Na⁺ concentration of 25 mM. Received: 2 November 1997 / Accepted: 20 February 1998     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p

STE50 is required to sustain pheromone-induced signal transduction in S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G_α, G_β, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth. Received: 20 February 1998 / Accepted: 17 March 1998     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p

The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease. Received: 7 May 1998 / Accepted: 16 July 1998     

Critical analysis of application of generalized distance function for optimization of important variables for esterase synthesis by Saccharomyces cerevisiae

The generalized distance function approach is employed to obtain a suitable near optimal conditions of variables. The optimal values of variables (medium constituents, microbiological parameters, and process parameters) have been evaluated separately using single responses (either specific esterase activity or cell mass) as per central-composite-design and multi-responses following generalized distance function approach. The optimal conditions (medium composition (g l⁻¹): dextrose, 13.43; peptone, 7.285; yeast extract, 2.55; and malt extract, 1.695; microbiological parameters: slant age, 39.9 h; inoculum age, 9.6 h; and number of cells, 1.49 × 10⁸ numbers ml⁻¹; process conditions: temperature, 29.9 °C; and pH, 6.2) obtained by generalized distance approach can be considered as a ‘near optimal’ solution of interactive multi-response systems of intracellular esterase synthesis by Saccharomyces cerevisiae.