酿酒酵母吸附重金属离子的研究进展

重金属污染成为当今最重要的环境问题之一。生物吸附法是处理大体积低浓度重金属废水的一种理想方法,近年来有关的研究报道不断增多,但尚未实现工业化应用。酿酒酵母(Saccharomyces cerevisiae)不仅是具有实用潜力的生物吸附剂,也是研究重金属生物吸附机理的良好材料。结合自己的研究成果,总结了酿酒酵母作为生物吸附材料的优点、研究中的表现形式和吸附性能,重点讨论了酿酒酵母生物吸附机理,介绍了等温吸附平衡模型和动力学模型在酵母生物吸附中的应用情况。最后提出生物吸附进一步的研究方向。     

Biosorption of heavy metals by Saccharomyces cerevisiae: a review

Heavy metal pollution has become one of the most serious environmental problems today. Biosorption, using biomaterials such as bacteria, fungi, yeast and algae, is regarded as a cost-effective biotechnology for the treatment of high volume and low concentration complex wastewaters containing heavy metal(s) in the order of 1 to 100 mg/L. Among the promising biosorbents for heavy metal removal which have been researched during the past decades, Saccharomyces cerevisiae has received increasing attention due to the unique nature in spite of its mediocre capacity for metal uptake compared with other fungi. S. cerevisiae is widely used in food and beverage production, is easily cultivated using cheap media, is also a by-product in large quantity as a waste of the fermentation industry, and is easily manipulated at molecular level. The state of the art in the field of biosorption of heavy metals by S. cerevisiae not only in China, but also worldwide, is reviewed in this paper, based on a substantial number of relevant references published recently on the background of biosorption achievements and development. Characteristics of S. cerevisiae in heavy metal biosorption are extensively discussed. The yeast can be studied in various forms for different purposes. Metal-binding capacity for various heavy metals by S. cerevisiae under different conditions is compared. Lead and uranium, for instances, could be removed from dilute solutions more effectively in comparison with other metals. The yeast biosorption largely depends on parameters such as pH, the ratio of the initial metal ion and initial biomass concentration, culture conditions, presence of various ligands and competitive metal ions in solution and to a limited extent on temperature. An assessment of the isotherm equilibrium model, as well as kinetics was performed. The mechanisms of biosorption are understood only to a limited extent. Elucidation of the mechanism of metal uptake is a real challenge in the field of biosorption. Various mechanism assumptions of metal uptake by S. cerevisiae are summarized.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

A cytosolic domain of the yeast Zrt1 zinc transporter is required for its post-translational inactivation in response to zinc and cadmium

Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells. Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition. Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals. We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis. During the transition from zinc-limiting to zinc-replete growth conditions (i.e. zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole. To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane. This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking. Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells.     

絮凝基因内衔接重复序列与酵母菌絮凝特性多样性及遗传稳定性

存在于酵母菌细胞表面的絮凝蛋白与邻近细胞表面寡聚甘露糖链相互作用,从而使细胞相互聚集形成细胞团的生理过程称为酵母菌絮凝。编码絮凝蛋白的基因中存在大量衔接重复序列,这些重复序列的变化不但使酵母菌呈现出絮凝特性的多样性,而且由重复序列驱动的基因内或基因间重组使酵母菌的絮凝特性具有非常明显的遗传不稳定性。文中综述了基因内重复序列对酵母菌絮凝特性和遗传稳定性的影响,将为基于序列调控策略改进酵母菌絮凝特性及遗传稳定性奠定理论基础,为絮凝特性在发酵工业或环境修复过程中的可控应用提供新的解决策略。     

Biosorption of cadmium and lead ions by ethanol treated waste baker's yeast biomass

The biosorption of cadmium and lead ions from artificial aqueous solutions using waste baker's yeast biomass was investigated. The yeast cells were treated with caustic, ethanol and heat for increasing their biosorption capacity and the highest metal uptake values (15.63 and 17.49 mg g(-1) for Cd(2+) and Pb(2+), respectively) were obtained by ethanol treated yeast cells. The effect of initial metal concentration and pH on biosorption by ethanol treated yeast was studied. The Langmuir model and Freundlich equation were applied to the experimental data and the Langmuir model was found to be in better correlation with the experimental data. The maximum metal uptake values (qmax, mg g(-1)) were found as 31.75 and 60.24 for Cd(2+) and Pb(2+), respectively. Competitive biosorption experiments were performed with Cd(2+) and Pb(2+) together with Cu(2+) and the competitive biosorption capacities of the yeast biomass for all metal ions were found to be lower than in non-competitive conditions.     

The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis

In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.     

Different life strategies in genetic backgrounds of the Saccharomyces cerevisiae yeast cells

Changes in the natural environment require an organism to make constant adaptations enabling efficient use of environmental resources and ensuring its success in competition with other organisms. Such adaptations are expressed through various life strategies, largely determined by the rate of consumption and use of available resources, affecting the life-history traits and the related trade-offs. Allocation of available resources must take into consideration the costs of cell maintenance as well as reproduction. Given that carbon metabolism plays a crucial role in resource allocation, yeast living in different ecological niches show various life-history traits. There are a lot of data about life-history strategies in yeast living in various ecological niches; however, the question is whether different life strategies will be noted for yeast strains growing under strictly controlled conditions. Our studies based on three laboratory yeast strains representing different genetic backgrounds show that each of these strains has specified life strategies which are mainly determined by the glucose uptake rate and its intracellular usage. These results suggest that specific life strategies and related differences in the physiological and metabolic parameters of the cell are the key aspects that may explain various features of cells from different yeast strains, either industrial or laboratory.     

Does the accumulation of trace metals in crustaceans affect their ecology—the amphipod example?

Crustaceans, like all aquatic invertebrates, take up and accumulate metals from a wide range of sources and the trace metal concentrations within their tissues and bodies show great variability. Trace metal uptake in crustaceans occurs from the water and food, either of which may be affected by the physico-chemical properties of the sediment. Accumulated metal concentrations in amphipods are contrasted with those of other crustaceans and examples are given to show how external and internal factors affect bioaccumulation. One of the major pathways for the uptake of trace metals is from solution directly through permeable surfaces including the gills. Changes in salinity and oxygen tension can modify the uptake characteristics from solution particularly in the case of interstitial water within sediments. Infaunal amphipods have direct contact with the sediment and the bioavailabilities of trace metals depend on the strength of the metal binding which is determined by a combination of properties including grain size, organic content, the presence of metals such as lead and iron as well as other ambient environmental conditions. Metal concentrations within amphipod bodies reflect the bioavailabilities of trace metals in their habitat. Body size is one of the major factors contributing to individual variability in trace metal concentrations within species. For some amphipod species, there are differences in trace metal accumulation with gender, breeding and developmental stage. In amphipods, accumulated body metal concentrations are the best biomarkers for environmental metal availabilities. Metal accumulation affects the ecology of crustaceans as a consequence of the energy costs associated with excreting and/or detoxifying the incoming metals. If the costs are significant, then this may result in reduced growth and reproduction. The effects of accumulated metals on communities have yet to be determined. Accumulated metals in crustacean prey species may be transferred along the food chain, but biomagnification in fish appears unlikely. One of the main ecological challenges is the need to link molecular biomarkers with ecologically relevant life history characteristics including growth, survival, reproduction and recruitment.     

Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Leucine-Binding Component and Regulation of Leucine Uptake

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.     

Cyclin Cln3p links G1 progression to hyphal and pseudohyphal development in Candida albicans

G1 cyclins coordinate environmental conditions with growth and differentiation in many organisms. In the pathogen Candida albicans, differentiation of hyphae is induced by environmental cues but in a cell cycle-independent manner. Intriguingly, repressing the G1 cyclin Cln3p under yeast growth conditions caused yeast cells to arrest in G1, increase in size, and then develop into hyphae and pseudohyphae, which subsequently resumed the cell cycle. Differentiation was dependent on Efg1p, Cph1p, and Ras1p, but absence of Ras1p was also synthetically lethal with repression of CLN3. In contrast, repressing CLN3 in environment-induced hyphae did not inhibit growth or the cell cycle, suggesting that yeast and hyphal cell cycles may be regulated differently. Therefore, absence of a G1 cyclin can activate developmental pathways in C. albicans and uncouple differentiation from the normal environmental controls. The data suggest that the G1 phase of the cell cycle may therefore play a critical role in regulating hyphal and pseudohyphal development in C. albicans.     

Metal biosorption in lignocellulosic biofuel biorefinery effluent: an initial step towards sustainability of water resources

Biosorption of metals by microorganisms is a promising technology to remove accumulated non-process elements in highly recycled biorefinery process water. Removal of these elements would enable greater water reuse and reduce the environmental impact of effluent discharge. A model lignocellulosic ethanol biorefinery wastewater was created based on pulp mill effluent. This generated a wastewater with an environmentally realistic high loading of dissolved natural organic matter (900?mg/l), a potentially important factor influencing metal biosorption. Analysis of feedstock and pulp mill effluent indicated that Mn and Zn are likely to be problematic in highly recycled lignocellulosic ethanol biorefinery process water. Therefore, the growth of several bacteria and fungi from existing collections, and some isolated from pulp mill effluent were tested in the model wastewater spiked with Mn and Zn (0.2?mM). Wastewater isolates grew the best in the wastewater. Metal uptake varied by species and was much greater for Zn than Mn. A bacterium, Novosphingobium nitrogenifigens Y88(T), removed the most metal per unit biomass, 35 and 17?mg?Mn/g. No other organism tested decreased the Mn concentration. A yeast, Candida tropicalis, produced the most biomass and removed the most total metal (38?% of Zn), while uptake per unit biomass was 24?mg?Zn/g. These results indicate that microorganisms can remove significant amounts of metals in wastewater with high concentrations of dissolved natural organic matter. Metal sorption by autochthonous microorganisms in an anaerobic bioreactor may be able to extend water reuse and therefore lower the water consumption of future biorefineries.     

Cyclic accumulation of zinc by Candida utilis during growth in batch culture

Intracellular accumulation of zinc by Candida utilis NRRL-Y-7634 was mediated by an energy-and temperature-dependent, highly specific process exhibiting saturation kinetics. In zinc-supplemented medium, uptake occured only during the lad and late-exponential phases; this type of transport did not occur with zinc in bacteria nor with iron in either yeast or bacteria. Cells of C. utilis did not possess a zinc-efflux system; they could reduce their level of intracellular zinc only by dilution of the metal into daughter cells. Zinc-deficient organisms accumulated 12 times more zinc than did cells of the same culture age grown in zinc-supplemented medium. The varied, but experimentally reproducible levels of intracellular zinc that occured in response to the physiological and environmental parameters had no detectable effects on respiration, rate of growth, total cell yield, or cell viability. Neither the mechanism underlying the cyclic accumulation of sinc nor the function of such behaviour are understood.     

Effect of mercury and organomercurials on cellular glucose utilization: a study using resting mercury-resistant yeast cells

AIMS: Mercury compounds are highly toxic to all types of living cells. Isolated yeast strains of Rhodotorula rubra showed high and low resistance pattern towards mercury and organomercurial compounds. To investigate the basis of differential sensitivity of these two types of strains, glucose utilization was measured in the presence of mercury compounds. METHODS AND RESULTS: Glucose utilization process remained unaffected in resting cells of highly Hg(2+)-resistant strain in the presence of HgCl(2) but not in the presence of phenylmercuric acetate and thimerosal. However, HgCl(2) significantly affected glucose utilization in the case of low-resistant cells. The Hg-retaining ability of the cell wall of highly Hg(2+)-resistant yeast strain was greater than that of the weakly Hg(2+)-resistant strain. The spheroplast-bound Hg(2+) was also significantly less in the highly Hg(2+)-resistant strain than in the weakly Hg(2+)-resistant strain. CONCLUSIONS: Glucose uptake machinery was not affected in the presence of toxic metal ions in the case of high-resistant strains. But in the case of low Hg(2+)-resistant strain, glucose transport system may be affected either by inactivation of sensor proteins containing -SH group associated with glucose uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell wall of mercury-resistant yeast cells may play an important role in heavy metal bioremediation process.     

Cell aggregations in yeasts and their applications

Yeasts can display four types of cellular aggregation: sexual, flocculation, biofilm formation, and filamentous growth. These cell aggregations arise, in some yeast strains, as a response to environmental or physiological changes. Sexual aggregation is part of the yeast mating process, representing the first step of meiotic recombination. The flocculation phenomenon is a calcium-dependent asexual reversible cellular aggregation that allows the yeast to withstand adverse conditions. Biofilm formation consists of multicellular aggregates that adhere to solid surfaces and are embedded in a protein matrix; this gives the yeast strain either the ability to colonize new environments or to survive harsh environmental conditions. Finally, the filamentous growth is the ability of some yeast strains to grow in filament forms. Filamentous growth can be attained by two different means, with the formation of either hyphae or pseudohyphae. Both hyphae and pseudohyphae arise when the yeast strain is under nutrient starvation conditions and they represent a means for the microbial strain to spread over a wide area to survey for food sources, without increasing its biomass. Additionally, this filamentous growth is also responsible for the invasive growth of some yeast.     

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.     

Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells

The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6–H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.