Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium

Abstract

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd²⁺ in this study. The transformed yeast strains showed much higher resistance ability to Cd²⁺ compared with the control. Strikingly, their Cd²⁺ accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd²⁺ under a wide range of pH levels, from 3 to 7, without disturbing the Cu²⁺ and Hg²⁺. Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd²⁺-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

Introduction

Heavy metal ions are released into the natural environment by a variety of industrial processes with the fast development of modern industry. Moreover, their strong toxicity, non-degradable nature, and food-chain bioaccumulation have become a global environmental concern [8,20]. Taking too much heavy metal ions can lead to health hazards to human beings [5,29]. Many studies focusing on ion exchange, reverse osmosis, chemical precipitation, activated carbon adsorption, electrocoagulation, electrodialysis, ultrafiltration, and solvent extraction on the removal of heavy metal ions have been carried out [3,14]. However, conventional physical and chemical methods for heavy metal ion removal have limitations, such as high costs, incomplete removal, secondary pollution, and the lack of metal specificity [1,6,7]. Many researchers have paid more attention to the microbial biomass-based biosorption methods because they are cheap, effective, and environmentally friendly, and can work well at relatively low concentrations [3,12,15-18,21,26].

Cd2+, one type of toxic but useful heavy metal ion [22], has been widely used in many industrial processes, all of which can lead to the release of it into the environment by many different ways. Human beings often take Cd2+ directly or indirectly from food because of its food-chain bioaccumulation property [2]. In order to reduce the effect of cadmium on human health, many researchers have carried out studies on removing Cd2+ from the environment. K. Kuroda and Ueda [17,18] successfully displayed yeast metallothionein (YMT), a tandemly fused YMT and hexa-His to adsorb Cd2+. In another study, a short metal-binding peptide was displayed on the yeast surface for selective preconcentration of ultra-trace Cd2+ by Yang et al. [31], which was very useful for detection of this heavy metal ion by graphite furnace atomic absorption spectrometry (GFAAS). Many other exogenous proteins were also displayed on the cell surface of microbe to adsorb heavy metal ions [13,27]. To purify Cd2+-contaminated water by biological property inherent with microorganisms, especially with cell-surface engineered microbes, might be a potential method to this problem [11,26].

Solanum nigrum is a plant that exhibits high Cd2+ affinity ability due to its metallothionein (SMT), which is a cysteine-rich protein [10]. SMT genes encode Solanum nigrum metallothionein, which can be classified as SMT1 (subtypes a, b, c), SMT2 (subtypes a, b, c, d, e), and SMT3 (subtypes a, b, c). Among them, SMT2a, SMT2c, SMT2d, and SMT2e play an important role in heavy metal detoxification [10,25]. However, SMTs have not been displayed on the cell surface of Saccharomyces cerevisiae yet. In order to enhance the adsorption and resistance properties of Saccharomyces cerevisiae to cadmium and remove cadmium from polluted water, we displayed SMTs on it by the cell surface engineering approach and investigated the property of the cell-surface-engineered Saccharomyces cerevisiae in this study.

 

Materials and Methods

Strains and Plasmids

The host used for recombinant DNA manipulation in this study was Escherichia coli DH5α [F-, endA1, hsdR17 (rk-mk+), supE44, thi-1, λ-, recA1, gyrA96, ΔlacU169(φ80lacZΔM15)]. The yeast strains and plasmids used in this study were Saccharomyces cerevisiae CEN.PK113-5D and pYES2, pMD18-T vector. E. coli was grown on Luria-Bertani (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride) plates containing 1.5% agar and 100 μg/ml ampicillin to choose a positive colony and then amplified it in LB medium. The yeast strains bearing recombinant plasmids were selected on SC Minimal Medium and amplified in SC Minimal Medium lacking of uracil at 30℃.

Plasmids Construction

pYES2 was used as the original plasmid in this study. First, the inducer promoter GAL1 in pYES2 was replaced by the constitutive expression promoter triose-phosphate isomerase (Tpi) with SpeI and EcoRI. Second, the yeast secretion signal peptide alpha factor gene with MfeI, SpeI, and NotI was inserted after Tpi into the plasmids. Finally, the 3’ half sequence of the yeast agglutinin gene (AG) with EcoRI, MluI, and NotI was linked with the alpha factor gene in this plasmid. We named the modified plasmid as pYES2-Tpi-α-AG, whose structure is shown in Fig.1A . The primers used in this study are summarized in Table 1.

Fig. 1.Plasmid constructed for display of SMT on yeast cell surface (A) and colony PCR of transformants (B). M: DNA Marker Ladder 500; 1, 2, 3, and 4: genes SMT2a, SMT2c, SMT2d, and SMT2e obtained from transformants.

Table 1.Restriction sites are underlined and italicized.

Solanum nigrum metallothionein RNA was extracted following the standard method, and then its cDNA was obtained by reverse transcription. The SMT genes were amplified by polymerase chain reaction (PCR). The SMT genes analyzed in this study included SMT2a (EU760481.1), SMT2c (EU760483.1), SMT2d (EU760484.1), and SMT2e (EU760485.1). The primers used to amplify the SMT genes and construct the constitutive expression plasmid are shown in Table 1.

The amplified products were transformed into E. coli DH5α with the T-vector pMD-18 and sequenced by the dideoxynucleotide chain termination method [23]. The successful plasmids were extracted from E. coli DH5α, digested with EcoRI and MluI, and then inserted into the modified pYES2, which was also digested with EcoRI and MluI.

The resulting plasmid was named pYES2-Tpi-α-SMT-AG. It was sequenced to confirm that the SMT gene had inserted into the correct site of the plasmid. The successful integration plasmid for display of SMT (pYES2-Tpi-α-SMT-AG) and the integration plasmid (pYES2-Tpi-α-AG) with its SMT-encoding sequence deleted were transferred into Saccharomyces cerevisiae CEN.PK113-5D similarly.

Transformation of S. cerevisiae

The constructed plasmids were transformed to the S. cerevisiae cells with the method of electrotransformation. The transformants were isolated by incubation at 30℃ for 48-72 h on selective SC medium plates.

Cd2+ Adsorption by SMT-Displaying Yeast Cells

Prior to adsorption, the surface-engineered yeasts were precultivated and cultivated up to the stationary growth phase in SD medium containing 0.5% (w/v) casamino acids without uracil. The cells were harvested by centrifugation and then washed with 50 mM HEPES (pH 6.5). After washing, it was incubated for 2 h at 30℃ and 180 rpm in 50 mM HEPES (pH 6.5), which contained 150 μM CdCl2. It was centrifuged at 600 ×g after adsorption, and washed again with 50 mM HEPES to remove Cd2+ on the cell surface but not bound to it. Finally, the resulting concentration of Cd2+ in the rest supernatant was measured directly by flame atomic absorption spectrophotometry (FAAS), and the cell pellet’s dry weight was measured after lyophilizing for 24 h. We used the maximum adsorption capacity of cadmium as the last result and six parallel experiments were done for each type of yeast. The data are shown in Table 2.

Table 2.aYeast containing pYES2-Tpi-α-AG bYeast containing pYES2-Tpi-α-SMT2a-AG cYeast containing pYES2-Tpi-α-SMT2c-AG dYeast containing pYES2-Tpi-α-SMT2d-AG eYeast containing pYES2-Tpi-α-SMT2e-AG

Cd2+ Adsorption Under Different pH Values

The effect of pH on the adsorption ability was determined through Cd2+-binding experiment in 50 mM HEPES at pH levels between 3 and 7.

Effects of Cu2+ and Hg2+ on Bioadsorption for Cd2+

Influence of Cu2+ and Hg2+ on the adsorption of Cd2+ was investigated through co-existence of Cu2+ and Cd2+, Hg2+, and Cd2+, respectively. The pH of the Cd2+-containing solution was 6.5.

Adsorption of Ultra-Trace Cd2+ by SMT-Displaying Yeast Cells

The method used was similar to that described in the “Cd2+ adsorption by SMT-displaying yeast cells” section, with the difference being that the concentration of Cd2+ was adjusted to 100 μg/l. After adsorption by yeasts, the Cd2+ in the water was extracted by diphenylthiocarbazone-ethanol and ionic liquids. The concentration of the concentrated Cd2+ was measured by FAAS.

Growth in Cd2+-Containing Medium

Transformants were harvested from SC medium at the stationary phase and then diluted with the SC medium containing 0.5 mM CdCl2. The final culture broth at 600 nm was 0.2. After that, cells were cultivated at 30℃ and 160 rpm and cell growth was measured by absorbance of the culture broth at 600 nm.

The EMBL accession numbers of the SMT genes analyzed in this study are EU760481.1 (SMT2a), EU760483.1 (SMT2c), EU760484.1 (SMT2d), and EU760485.1 (SMT2e).

 

Results

Vector Construction, Transformation, and Positive Transformants Confirmation

The multicopy plasmid pYES2-Tpi-α-SMT-AG was constructed for the display of SMT. As a control plasmid, the modified multicopy plasmid pYES2-Tpi-α-AG lacking SMT was also constructed. The main components of cell surface displayed plasmid are as follows: Tpi promoter, yeast secretion signal alpha factor, SMT-encoding gene, and 3’ half sequence of anchoring protein-encoding gene AG. The SMT gene was located between the alpha factor and AG. The construction methods were shown in the Materials and Methods section. The structure of the plasmid is shown in Fig. 1A.

These plasmids constructed above were introduced into S. cerevisiae CEN.PK113-5D with the method of electrotransformation. The positive transformants were selected from SC plates and cultivated in SC at 30℃ and 160rpm for 48 h. The colony PCR was conducted to confirm that the transformants were correct. The results are shown in Fig. 1B.

Adsorption of Cd2+ by Surface-Engineered Yeast

The adsorption of heavy metal-containing water (150 μM CdCl2) by five types of yeast in this study was examined to determine the effect of SMT display. SMT-displaying cells with pYES2-T-a-SMT-AG showed higher adsorption ability at the cell surface than cells harboring the control plasmid pYES2-T-a-AG and the wild types (Table 2). This enhancement of adsorption ability at the cell surface indicated that the displayed SMT was functional as a chelator of heavy metal ions and contributed to the improvement of cell adsorption ability. As was shown in Table 2, the cells displaying SMT2a had the highest adsorption ability. However, there was no significant difference between the four types of displayed yeasts on the adsorption ability. All of the cell-surface- engineered yeasts could effectively adsorb Cd2+ and be applied for the biosorption and bioremediation of Cd2+-contaminated water.

Tolerance to Cd2+ by Cells Displaying SMT

The adsorption of heavy metal ions at the cell surface may serve to protect cells from the ions and contribute to tolerance to them. To investigate the protective effect of the displayed SMT against Cd2+ toxicity, surface-engineered yeasts grown to the stationary phase were inoculated into synthetic liquid medium supplemented with 0.5 mM CdCl2 (Fig. 2). SMT-displaying cells with pYES2-T-a-SMT-AG exhibited a dramatically enhanced Cd2+ tolerance in comparison with cells only harboring pYES2-T-a-AG. This result suggests that all of the SMTs displayed on the cell surface have an important effect on growth in Cd2+-containing medium and enhance cellular tolerance to Cd2+.

Fig. 2.Yeast growth in cadmium-containing medium. Yeast: () containing pYES2-Tpi-α-AG; () containing pYES2-Tpi-α-SMT2a-AG; () containing pYES2-Tpi-α-SMT2c-AG; () containing pYES2-Tpi-α-SMT2d-AG; () containing pYES2-Tpi-α-SMT2e-AG.

Adsorption of Cd2+ under Different pH Values by Surface-Engineered Yeast

The pH influence experiment suggested that the transformants can effectively adsorb Cd2+ at pH values from 3 to 7. The effect of pH on the adsorption ability of cell-surface-engineered yeast was lower than the control. The highest adsorption ability appeared at the pH levels about 6. The result is shown in Fig. 3. This result indicated that the transformants can be used to purify water with a wide range of pH that is polluted by Cd2+. For water with pH level of about 6, this type of yeast can display their highest adsorption ability. Therefore, this kind of yeast might be very useful in treating Cd2+-polluted water.

Fig. 3.the pH influence on the adsorption ability of different four kinds of displayed yeasts. yeast: (▧) containing pYES2-Tpi-α-AG; (▤) containing pYES2-Tpi-α-SMT2a-AG; (▥) containing pYES2-Tpi-α-SMT2c-AG; (▨) containing pYES2-Tpi-α-SMT2d-AG; (■) containing pYES2-Tpi-α-SMT2e-AG.

Adsorption of Cd2+ with the Co-Existing Heavy Metal Ions Cu2+ and Hg2+

Figs. 4 and 5 demonstrate that the affinity of surface-exposed SMT for cadmium exceeded the competing heavy metals ions, such as Cu2+ and Hg2+. In both cases, although high concentrations of Cu2+ and Hg2+ seriously inhibited Cd2+ adsorption, the Cd2+ uptake capacity of surface-engineered cells only decreased by 31.65%, 32.02%, 33.50%, 30.26%, and 36.13%, 32.21%, 35.54%, 33.66% as the Cu2+ and Hg2+ concentration increased to 25 mg/l, respectively. In contrast, the control decreased by 74.51% and 62.63%, respectively, when the Cu2+ and Hg2+ concentration increased to 25 mg/l.

Fig. 4.The effect of co-existing Cu2+ on the adsorption of Cd2+ by different strains of Saccharomyces cerevisiae. yeast: (▧) containing pYES2-Tpi-α-AG; (▤) containing pYES2-Tpi-α-SMT2a-AG; (▥) containing pYES2-Tpi-α-SMT2c-AG; (▨) containing pYES2-Tpi-α-SMT2d-AG; (■) containing pYES2-Tpi-α-SMT2e-AG.

Fig. 5.The effect of co-existing Hg2+ on the adsorption of Cd2+ by different strains of Saccharomyces cerevisiae. yeast: (▧) containing pYES2-Tpi-α-AG; (▤) containing pYES2-Tpi-α-SMT2a-AG; (▥) containing pYES2-Tpi-α-SMT2c-AG; (▨) containing pYES2-Tpi-α-SMT2d-AG; (■) containing pYES2-Tpi-α-SMT2e-AG.

We can infer that the surface-engineered yeasts can adsorb Cd2+ effectively when the polluted water contains Cu2+ and Hg2+ at the same time. This is very useful to purify polluted water in the environment that is polluted by several types of heavy metal ions simultaneously.

Adsorption of Ultra-Trace Cd2+ by Surface-Engineered Yeast

The adsorption ability of surface-engineered yeast was significant higher than that of the control. As can be seen from Table 3, the surface-engineered yeast could effectively adsorb ultra-trace Cd2+. This is a valuable characteristic of the surface-engineered yeast for it can purify Cd2+-polluted water much more thoroughly than the control.

Table 3.Original Cd2+ concentration: 100 ug/l; yeast cells concentration: 10 g/l.

 

Discussion

Compared with intracellular adsorption, adsorption at the cell surface has many advantages, for the following reasons: first, it is easy to recover and recycle adsorbed heavy metal ions from the surface-engineered yeasts without disintegrating them [3], which is very important in considering how to treat the heavy metal ions after their removal from wastewater. In contrast, one has to disintegrate the cells when one wants to recover adsorbed heavy metal ions from intracellular accumulated sources or plant bioremediation, which is inconvenient and costly [30]. Second, it is economically advantageous to use surfaceengineered yeast cells because they can be repeatedly used as bioadsorbents, as the cells are not severely damaged during the recovery process [4]. Therefore, the system can be easily used for adsorption, recovery, and recycling of heavy metal ions.

Four types of SMT genes were cloned from Solanum nigrum, which has the ability of hyperaccumulation to Cd2+ [10], in this study. Accordingly, four types of cell surface display plasmids were constructed with pYES2 and SMT genes. The constitutive expression plasmid constructed from pYES2 was very important for this study, as the high adsorption ability property of engineered cells largely relied on the successful construction of this plasmid.

Four types of engineered cell surface display yeasts were constructed based on the successful construction of the cell surface display plasmid described above. Colony PCR and adsorption test demonstrated that SMTs were successfully expressed and displayed on the cell surface of Saccharomyces cerevisiae. Successful display of SMTs that have the ability to adsorb Cd2+ on the yeast cell surface resulted in enhanced adsorption and recovery of heavy metal ion Cd2+ at the cell surface without cellular disintegration. This result demonstrated that SMTs displayed on the cell surface were active and successfully served as a chelator of heavy metal ion Cd2+. As can be seen from Table 2, display of SMT on the yeast cell surface is effective for the adsorption of Cd2+ and enhances the yeasts’ adsorption ability and produces valuable bioadsorbents. The adsorption ability of the engineered yeasts was almost twice that of the control, which illustrated that SMTs can effectively adsorb Cd2+ from contaminated water. As can be seen from Table 2, cells that displayed SMT2a have the highest adsorption ability. However, the adsorption ability among the engineered yeasts has no significant difference, which is consistent with the difference between their structures [22].

The resistance to Cd2+ by displayed SMT yeast results from the enhanced adsorption of toxic heavy metal ions on their surface (Fig. 2), which is similar to the resistance to Cd2+ by YMT and hexa-His-displaying yeast cells [17,18] Binding heavy metal ions at the cell surface is probably a mechanism of resistance like intracellular sequestration and efflux pumping [9,24,28]. The cell division of nonengineered yeasts was inhibited by 80 μM Cd2+ [16] whereas the engineered ones lived in the 0.5 mM Cd2+-containing medium (Fig. 2). It is considered that the surfaceengineered yeast resistance to Cd2+ was by reducing the opportunity of metal permeation into the cytosol through adsorption and binding them at the cell surface. Therefore, it is feasible that the trapping of heavy metal ions on the cell surface is a functional phenomenon, serving as a mechanism for tolerating the presence of toxic heavy metal ions. As can be seen from Fig. 2, the inhibitory effect of Cd2+ on cell division is thought to result in a longer proliferation period in the Cd2+-containing medium. However, the clarification of the mechanism and the reason for the long lag period require further investigations.

Compared with control yeast cells, the engineered yeasts are influenced less by pH variations and can effectively adsorb Cd2+ at wide pH levels, as shown in Fig. 3, which is very useful for their use in different contaminated waters. The SMT may be functional and their construction was probably very stable under a wide range of pH. However, the detail of this specific mechanism and the reason for this stable property still need to be investigated.

Several works on yeast surface display of metal-binding proteins/peptides have been carried out [16-18]. Although these surface-engineered cells showed high affinity to a wide range of heavy metals, they lacked selectivity to certain heavy metals. The display of cadmium-binding protein on the yeast surface obviously changes its binding property to the metal of interest. In this particular case, the improvement on binding selectivity was one of the main focus. Figs. 4 and 5 demonstrate that co-existing heavy metal ions Cu2+ and Hg2+ did not affect the adsorption ability for Cd2+ of engineered yeast significantly. These cells exhibit preference for Cd2+ adsorption rather than for Cu2+ and Hg2+. These data suggest that SMTs displayed on S. cerevisiae could selectively adsorb Cd2+ from mixed heavy metal ions contaminated water.

When the surface-engineered yeasts to were exposed ultra-trace heavy metal ions, they exhibited higher adsorption ability than that of the control. The main reason for this difference probably is that the cadmium-binding protein displayed on the cell surface and could detect the Cd2+ more easily. The controls do not have cell surface displayed protein, so they have much more difficulty in detecting ultra-trace Cd2+.

Microorganism–metal interactions have been reported and classified into active and passive mechanisms [11]. Living cells have been reported to preferentially adsorb Cd2+ by energy-dependent metabolism, whereas non-living ones adsorb heavy-metal ions by metabolism-independent surface binding method [19]. Accordingly, both the living and non-living cells constructed in this study can be used as effective bioadsorbents of Cd2+.

The potential ability of surface-engineered yeasts displaying proteins as bioadsorbents is largely due to the properties of the displayed protein. Therefore, in order to get engineered cells with high adsorption ability, we should focus future research on selecting proteins with high metal-binding capacity.