Impact of fluorides on the removal of heavy metals from an electroplating effluent using a flocculent brewer’s yeast strain of Saccharomyces cerevisiae

Abstract

Besides several toxic heavy metals, electroplating effluents can have in solution different cations and anions, which may influence heavy metals removal by the biomass. Among them, fluorides are commonly used in the electroplating industries and thus can be found in the respective wastewaters. In the present work, the effect of the presence of fluorides in the efficiency of chromium(III), copper(II) and nickel(II) removal, from an effluent, by heat-inactivated cells of a brewing flocculent strain of Saccharomyces cerevisiae was evaluated. The presence of fluorides severely decreased (>60%) the removal of chromium(III) by yeast biomass. This effect impaired the effective treatment of the effluent according to the US Environmental Protection Agency and the Portuguese law; conversely, a higher removal of copper(II) and nickel(II) was observed. This behaviour can be understood by metal speciation. In the presence of fluorides, chromium(III) was mainly complexed, becoming unavailable for yeast accumulation; this effect decreased the efficiency of chromium(III) removal. Thus, in the presence of fluorides, less chromium(III) is associated with biomass and consequently more yeast binding sites remain available for the uptake of other metals present in solution. This fact explains the increase of copper(II) and nickel(II) removal in the presence of fluorides.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

Properties of β-glucan synthetase from Saccharomyces cerevisiae

Properties of -glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12mm. Addition of Mg⁺⁺ or Mn⁺⁺ stimulated its activity by 60% and 21% respectively. High concentrations of EDTA and hydroxyquinoline were inhibitory. Glucan synthetase was fully active in cell-free extracts. Small concentrations of trypsin or subtilopeptidase A from Bacillus subtilis, caused only a slight increase in glucosyl transferase activity, but larger concentrations destroyed -glucan synthetase. Acid proteases were neither stimulatory nordestructive. Thus it seemsunlikelythat -glucan synthetase exists in a zymogen form. Glucan synthetase was unstable. It was inactivated more rapidly at 28 C than at 0 C. The presence of substrate, -glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect -glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or -glucans. The synthesis of -glucans was competitively inhibited by UDP (K_i=0.45mm). Glucono--lactone, a known inhibitor of -glucosidases was a strong non-competitive inhibitor of -glucan synthetase.This work was supported by grants PNCB 00071 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

Ethanol production from xylo-oligosaccharides by xylose-fermenting Saccharomyces cerevisiae expressing β-xylosidase

Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.     

Ethanol Production from Xylo-oligosaccharides by Xylose-Fermenting Saccharomyces cerevisiae Expressing β-Xylosidase

Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.     

Anticlastogenic effect of β-glucan, extracted from Saccharomyces cerevisiae, on cultured cells exposed to ultraviolet radiation

β-glucan is an important polysaccharide due to its medicinal properties of stimulating the immune system and preventing chronic diseases such as cancer. The aim of the present study was to determine the anticlastogenic effect of β-glucan in cells exposed to ultraviolet radiation (UV). Chromosome aberration assay was performed in drug-metabolizing cells (HTC) and non drug-metabolizing cells (CHO-K1 and repair-deficient CHO-xrs5), using different treatment protocols. Continuous treatment (UV + β-glucan) was not effective in reducing the DNA damage only in CHO-xrs5 cells. However, the pre-treatment protocol (β-glucan before UV exposition) was effective in reducing DNA damage only in CHO-K1 cells. In post-treatment (β-glucan after UV exposition) did not show significative anticlastogenic effects, although there was a tendency toward prevention. The data suggest that β-glucan has more than one action mechanism, being capable of exerting desmutagenic as well as bio-antimutagenic action. The findings also suggest that the presence of the xenobiotic metabolizing system can reduce the chemopreventive capacity of β-glucan. Therefore, these results indicate that β-glucan from Saccharomyces cerevisiae can be used in the prevention and/or reduction of DNA damage.     

What are “heavy metals” in Plant Sciences?

Plants do not have the ability to sense physical properties of metals, e.g. specific weight. The term “heavy metal” was defined mainly by the specific weight of metals. The definition was often connected with the expectation that the substance should be toxic. This definition is not acceptable and also inconsistent in use as already stressed in literature. However, in Plant Sciences, the term is so widely used that it is hardly possible to eliminate it. We suggest instead defining the term in a more unequivocal way. This should be done on the basis of the periodic system of elements. Here, we suggest introducing the following three subgroups forming the group of heavy metals for use in Plant Sciences. 1st subgroup: all transition elements except La and Ac (Transition metals). 2nd subgroup: rare earth elements, subdivided in the series of lanthanides and the series of actinides including La and Ac themselves (Rare earth metals). 3rd subgroup: a heterogenous group p-elements including the metal Bi, the amphoterous oxides forming elements Al, Ga, In, Tl, Sn, Pb, Sb and Po, and the metalloids Ge, As and Te. We suggest using the term “lead-group” for this 3rd subgroup of heavy metals as in Toxicology and Environmental Sciences, Pb is the most prominent representative of this group.     

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20^F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Endonuclease α from Saccharomyces cerevisiae shows increased activity on ultraviolet irradiated native DNA

Molecular Genetics and Genomics - Endonuclease α isolated from the nucleus of the yeast Saccharomyces cerevisiae is a DNA endonuclease which has been shown to act preferentially on denatured...     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Production and purification of recombinant human α2C2 adrenergic receptor using Saccharomyces cerevisiae

The objective is to generate milligram quantities of recombinant human ₂C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.     

Xylitol production using recombinant Saccharomyces cerevisiae containing multiple xylose reductase genes at chromosomal δ-sequences

Xylitol production from xylose was studied using recombinant Saccharomyces cerevisiae 2805 containing xylose reductase genes (XYL1) of Pichia stipitis at chromosomal δ-sequences. S. cerevisiae 2805-39-40, which contains about 40 copies of the XYL1 gene on the chromosome, was obtained by a sequential transformation using a dominant selection marker neo^r and an auxotrophic marker URA3. The multiple XYL1 genes were stably maintained on the chromosome even after 21 and 10 days in the non-selective sequential batch and chemostat cultures, respectively, whereas S. cerevisiae 2805:pVTXR, which harbors the episomal plasmid pVTXR having the XYL1 gene, showed mitotic plasmid instability and more than 95% of the cells lost the plasmid under the same culture conditions. In the first batch (3 days) of the sequential batch culture, volumetric xylitol productivity was 0.18 g l⁻¹ h⁻¹ for S. cerevisiae 2805-39-40, as compared to 0.21 g l⁻¹ h⁻¹ for S. cerevisiae 2805:pVTXR. However, the xylitol productivity of the latter started to decrease rapidly in the third batch and dropped to 0.04 g l⁻¹ h⁻¹ in the seventh batch, whereas the former maintained the stable xylitol productivity at 0.18 g l⁻¹ h⁻¹ through the entire sequential batch culture. The xylitol production level in the chemostat culture was about 8 g l⁻¹ for S. cerevisiae 2805-39-40, as compared to 2.0 g l⁻¹ for S. cerevisiae 2805:pVTXR after 10 days of cultures even though the xylitol production level of the latter was higher than that of the former for the first 5 days. The results of this experiment indicate that S. cerevisiae containing the multiple XYL1 genes on the chromosome is much more efficient for the xylitol production in the long-term non-selective culture than S. cerevisiae harboring the episomal plasmid containing the XYL1 gene.     

Saccharomyces cerevisiae: a potential host for carboxylic acid production from lignocellulosic feedstock?

Carboxylic acids are important bulk chemicals that can be used as building blocks for the production of polymers, as acidulants, preservatives and flavour compound or as precursors for the synthesis of pharmaceuticals. Today, their production mainly takes place through catalytic processing of petroleum-based precursors. An appealing alternative would be to produce these compounds from renewable resources, using tailor-made microorganisms. Saccharomyces cerevisiae has already demonstrated its value for bioethanol production from renewable resources. In this review, we discuss Saccharomyces cerevisiae engineering potential, current strategies for carboxylic acid production as well as the specific challenges linked to the use of lignocellulosic biomass as carbon source.