Introduction

Mercury (Hg) is a highly toxic heavy metal to human health, and the Agency for Toxic Substances and Disease Registry had ranked it the third in the priority list of hazardous substances [1, 2]. Hg was released to environment through many industrial processes and could be accumulated by plants or animals which might be used for foods by humans [3, 4]. In recent years, most of the Hg in the environment was released by natural processes and human activities including oceanic emission, biomass burning, mining, metal manufacturing and fossil fuel burning, which resulted in environmental pollution and potential danger to human beings [5,6,7,8]. A large portion of Hg existed in the environment was Hg2+, and it can influence human health seriously [8,9,10].

The impacts of Hg contamination on environment and human health impelled researchers to find effective ways for treating this toxic metal. The remediation techniques for Hg contamination mainly include physical treatment, chemical remediation and bioremediation [11, 12]. Conventional physico-chemistry methods used to remove Hg from contaminated environment were often inadequate and lack of selectivity to reduce Hg2+; they were usually unfriendly to environment and expensive [13,14,15].

Bioremediation technology had been wildly applied for treating Hg contamination due to its cheap, green and environmentally friendly characters [16,17,18]. Microbial-based remediation technology is a critical important part of bioremediation because of the certain properties of microorganisms [12, 19]. A lot of microbes had been examined for bioremediation of Hg [20, 21]. The mer (mercury resistance) operon system in bacteria could transfer Hg2+ into the cell where it was reduced to Hg0 and volatilized from the bacterial cell to the atmosphere at last [22, 23]. Therefore, the bacteria-owned mer operon system had been attempted to be used to reduce Hg2+ contamination [24,25,26]. But this microbial volatilization system is often inhibited by high concentration of heavy metal ions [15]. In order to enhance the tolerance and adsorption ability for Hg of the microorganisms, researchers were trying to modify these microbes. Kiyono and Pan-hou [27] constructed an engineered Escherichia coli expressing Hg transport system and organomercury lyase for accumulation and transformation of Hg. Engineered bacterium expressing Hg transport system and metallothionein was constructed for biosorption of Hg2+ by Deng and Jia [28]. However, the Hg2+ adsorbed by these engineered bacteria was usually difficult to recover because it was in the cytoplasm or volatilized to the atmosphere [29, 30]. These problems might be solved by displaying the target proteins that have high affinity to Hg2+ on the cell surface. MerR is a metalloregulatory protein in the mer operon system which contained in many bacteria [18, 22, 31] and it can selectively adsorb Hg2+ [32]. Escherichia coli’s Hg2+ adsorption ability was enhanced by displaying MerR on its cell surface [33]. The cell surface-engineered microorganisms might be a potential effective way for treating Hg pollutions.

The Saccharomyces cerevisiae is one of the most suitable microorganisms for the development of cell surface display system, and many heterologous proteins had been displayed on it [34,35,36]. We hypothesized that S. cerevisiae displaying MerR might adsorb Hg2+ effectively and not result in secondary pollution. It might be a potential method to purify Hg2+-polluted water. In this study, we displayed MerR on the cell surface of S. cerevisiae with an α-agglutinin-based display system to enhance its adsorption ability for Hg2+. The properties of the engineered yeast showed that it could adsorb Hg2+ effectively and could be used to purify Hg-polluted water.

Materials and Methods

Strains, Media and Plasmids

Escherichia coli DH5α [F −, endA1, hsdR17 (r − k m + k ), supE44, thi-1, λ−, recA1, gyrA96, ΔlacU169(φ 80lacZΔM15)] was used as the host for recombinant DNA in this study. Saccharomyces cerevisiae CEN.PK113-5D (named C5D) was used as the host strain for genetic engineering. The Luria–Bertani (LB) medium plate used for selecting positive E. coli was made of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1% (w/v) sodium chloride, 1.5% (w/v) agar, 100 μg/mL ampicillin, 40 μg/mL 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) and 1 mM isopropyl–d-thiogalactopyranoside (IPTG). The Luria–Bertani (LB) medium used for amplifying E. coli was made of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1% (w/v) sodium chloride and 100 μg/mL ampicillin. The recombinant yeast strain was chosen on the SC minimal medium containing 0.67% (w/v) yeast nitrogen base (YNB), 2% (w/v) glucose, 0.01% (w/v) (adenine, arginine, cysteine, leuine, lysine, threonine, tryptophan), 0.005% (w/v) (aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine) and 2% (w/v) agar. The plasmids used in this study were pYES2 and pMD18-T.

Plasmids Construction

The original plasmid used in this study was pYES2. Firstly, we used the constitutive expression promoter triose-phosphateisomerase promoter (Tpi) to replace the inducible promoter GAL1 in pYES2. Secondly, we inserted the yeast secretion signal peptide gene after Tpi. Finally, the alpha factor gene was connected with the 3′ half sequence of yeast agglutinin gene (AG) [37]. The structure of the constructed plasmids is shown in Fig. 1, and it was named as pYES2-Tpi-α-AG.

Fig. 1
figure 1

Structure of the plasmid pYES2-Tpi-α-AG

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The merR gene sequence (CP019338.1:1310586-1311020) was downloaded from NCBI GeneBank database, and the sequence was optimized by JCat (http://www.jcat.de/). The EcoR I and Mlu I sites were added to the sequence after adaptation and then synthesized by Sangon Biotech company (shanghai). At last, the sequence was digested by EcoR I and Mlu I and then inserted between the alpha factor gene and the 3′ half sequence of yeast agglutinin gene (AG) in the modified pYES2 which was also digested by EcoR I and Mlu I. The obtained cell surface display plasmid was named as pYES2-Tpi-α-merR-AG (Fig. 2), and the merR gene was confirmed in the right site through sequencing with the Sanger dideoxynucleotide chain termination method [38]. The successful integration plasmid for displaying MerR (pYES2-Tpi-α-merR-AG) and the integration plasmid (pYES2-Tpi-α-AG) without MerR-encoding sequence were transferred into the S. cerevisiae similarly.

Fig. 2
figure 2

Structure of the plasmid pYES2-Tpi-α-merR-AG

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Transformation of Saccharomyces cerevisiae

The electrotransformation method [39] was used to transform the aforementioned integration plasmids to the S. cerevisiae competent cells. The competent S. cerevisiae cells were obtained through dealing with lithium acetate (LiAc) and dithiothreitol (DTT) [40]. The transformants were cultured on the selective SC medium plate and cultivated at 30 °C until the colony appeared. The yeast strains were named C5D-C and C5D-MerR, respectively.

Colony PCR

The colony PCR was conducted based on the transformants according to the following procedures: 94 °C 5 min, 94 °C 30 s, 58 °C 40 s, 72 °C 45 s and 72 °C 10 min. The primers used were merRF (5′ GAATTCATGGAAAACAACT3′) and merRR (5′ACGCGTCTGTGGTGGTGGT3′).

Hg2+ Adsorption by MerR-Displaying Yeast Cells

Firstly, the yeasts were grown up to stationery growth phase before adsorption experiment. The yeast cells were harvested with the method of centrifugation and then washed with 50 mM HEPES (pH 7.0). The cells were put in 20 mg/L HgCl2 solution after washing and incubated for 2 h. After adsorption, the yeast cells were harvested and washed again. The weight of the yeast cells was calculated after lyophilizing for 24 h. The final concentration of Hg2+ in the rest supernatant was measured directly with the method of hydride generation atomic absorption spectrometry. We did six parallel experiments for each type of yeast strain.

Hg2+ Adsorption Under Different pH

The Hg2+ solution with pH levels between 4 and 8 was prepared before adsorption experiment. The yeasts cells were obtained through the method described in the “Hg2+ adsorption by MerR-displaying yeast cells” section. The adsorption experiment procedure was the same as the “Hg2+ adsorption by MerR-displaying yeast cells” section. The Hg2+ concentration was measured by the hydride generation atomic absorption spectrometry method. Six parallel experiments were conducted for each type of yeast strain.

Effect of Cu2+ and Cd2+ on the Adsorption for Hg2+

The solution (pH 7.0) with coexistence of Cu2+, Cd2+ and Hg2+ was prepared before adsorption experiment. The yeast cells were collected, and the adsorption experiments were conducted according to the methods described in the “Hg2+ adsorption by MerR-displaying yeast cells” section. Six parallel experiments were conducted for each type of yeast strain.

The Ultra-Trace Adsorption Ability of Hg2+ by MerR-Displaying Yeast Cells

The yeast cells were harvested, and the adsorption experiment was conducted in 100 μg/L Hg2+ solution (pH 7.0). The rest method is similar to the “Hg2+ adsorption by MerR-displaying yeast cells” section. The Hg2+ concentration was measured by hydride generation atomic absorption spectrometry method. Six parallel experiments were conducted for each type of yeast strain.

Growth in Hg2+-Containing Medium

The yeast strains were harvested at stationary phase and diluted by the SC medium (the final culture broth at 600 nm was 0.2) whose Hg2+ concentration was 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 mg/L, respectively. Then, they were cultivated at 30 °C and 160 rpm in shaking incubator. The cell growth condition was measured through the light absorbance value of the culture broth at 600 nm by NanoDrop 2000C Spectrophotometer. The experiment was conducted in triplicate.

Results

Plasmid Construction and Transformant Confirmation

The plasmid pYES2-Tpi-α-merR-AG constructed above was used for displaying MerR. The control plasmid lacking of merR was pYES2-Tpi-α-AG. The two types of plasmids were confirmed by sequencing, and the Tpi promoter, yeast secretion signal alpha factor, 3′ half sequence of anchoring protein-encoding gene AG and MerR-encoding gene were all in the right site of the plasmid (Figs. 1, 2).

The plasmids pYES2-Tpi-α-merR-AG and pYES2-Tpi-α-AG were transformed to S. cerevisiae through electrotransformation, respectively. The positive colony was reconfirmed by PCR (Fig. 3). The enlargement culturing of correct positive colony was carried out in SC medium and can be used for the next experiment.

Fig. 3
figure 3

Colony PCR of transformed yeast strain C5D-MerR

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Adsorption ability of Hg2+ by Different Yeast Strains

The adsorption ability for Hg2+ by the three yeast strains (C5D, C5D-C and C5D-MerR) was tested in order to verify the effect of MerR display on the cell surface. The result showed that the display of MerR on the cell surface enhanced its adsorption ability for Hg2+ (Table 1). The enhancement of this ability indirectly indicted that the MerR was successfully displayed on the cell surface of S. cerevisiae, and the MerR protein was also functional as an Hg2+ chelator. The engineered S. cerevisiae that displaying MerR could adsorb Hg2+ effectively and could be used to purify Hg2+-polluted water.

Table 1 Adsorption of Hg2+ by different types of yeast
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Hg2+ Tolerance Ability of Different Yeast Strains

The experiment showed that the Hg2+ tolerance ability of C5D-MerR was stronger than that of C5D and C5D-C. C5D and C5D-C could not grow in solution with Hg2+ of 40 mg/L, while the engineered yeast strain C5D-MerR could grow in solution not exceed 100 mg/L of Hg2+ (Fig. 4). The stronger tolerance ability might suggest that most of the Hg2+ was combined by MerR on the cell surface and little Hg2+ flowed into the cell. This character of the engineered yeast makes it can be used in purifying polluted water with high concentration of Hg2+.

Fig. 4
figure 4

Yeast strains’ growth condition in different Hg2+ solutions. a C5D; b C5D-CT; c C5D-MerR

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The Influence of pH on the Adsorption Ability for Hg2+ by Different Yeast Strains

The pH influence experiment indicted that the C5D-MerR could effectively adsorb Hg2+ under pH levels between 4 and 8. The influence of pH on the adsorption ability of C5D-MerR was lower than that of C5D and C5D-C. The highest adsorption ability appeared when the pH was about 7 (Fig. 5). The results of the experiment suggested that the C5D-MerR could be used to purify Hg2+-polluted water with wide range of pH levels. This might be very useful for purifying Hg2+-polluted water.

Fig. 5
figure 5

pH influence on the adsorption ability for Hg2+ of different yeast strains. Error bars represented the standard deviation of the experiment

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The Adsorption Ability for Hg2+ of Different Yeast Strains with Coexistence of Cu2+ and Cd2+

The coexistence of heavy metal ions probably has effect on the cells’ adsorption ability. The result of the experiment also supported our hypothesis. When the concentration of Cu2+ and Cd2+ equals to 20 mg/L, the adsorption ability of C5D-MerR for Hg2+ decreased 5.04%, while the adsorption ability of C5D and the C5D-C decreased 62.46 and 62.62%, respectively (Fig. 6). We inferred that the C5D-MerR could selectively adsorb Hg2+ with the coexistence of Cu2+ and Cd2+. Therefore, the C5D-MerR could be used to purify water that is polluted by different kinds of heavy metal ions simultaneously.

Fig. 6
figure 6

Adsorption ability for Hg2+ of different yeast strains with coexistence of Cu2+ and Cd2+. Error bars represented the standard deviation of the experiment

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Adsorption Ability of Ultra-trace Hg2+ by Different Yeast Strains

The display of MerR exactly enhanced the adsorption ability of C5D-MerR for ultra-trace Hg2+. We found that the adsorption ability of C5D-MerR was stronger than that of C5D and C5D-C (Table 2). It demonstrated that the C5D-MerR could adsorb the Hg2+ in the water much more thoroughly than that of C5D and C5D-C did. And we could obtain pure water at accepted level after its handling.

Table 2 Adsorption of ultra-trace Hg2+ by different types of yeast
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Discussion

Cell surface display system can display the metal binding protein on the cell surface. The cell surface-displayed engineered microbe cells have many advantages when compared with other engineered microbes that have to adsorb metal ions into cells. Firstly, the cells with surface metal binding protein on its surface can adsorb metal ions onto the cell surface rather than into the cell, which can alleviate their toxicity to the cell [41, 42]. On the other hand, the recovery of the metal ions from cell surface was much easier than that from the inside of the cell. We can easily recover the metal ions from the cell surface without disintegrating the cells, while the cells must be damaged when you recover metal ions adsorbed into the cells [17]. Therefore, the recovery of metal ions adsorbed on the cell surface is convenient and economic, which is very important in the practical application for purifying polluted water and recovering metal ions from the bioadsorbents. In contrast, it is not convenient and economic to recover heavy metal ions from intracellular bioremediation adsorbents [43]. Furthermore, the cell surface-engineered adsorbents can be reused because the cells did not damage during the adsorbing and recovering processes [33, 44]. We supposed that the cell surface-engineered yeast cells could be used to adsorb, recover and recycle of metal ions.

The display of MerR on the yeast cell surface enhanced its adsorption ability for Hg2+. This result and the colony PCR experiment suggested that MerR was expressed and successfully displayed on the cell surface of S. cerevisiae. The MerR displayed on the cell surface was active and could bind Hg2+ effectively. Our results showed that the engineered C5D-MerR could adsorb almost three times Hg2+ as much as that of C5D and C5D-C (Table 1). The enhancement of the adsorption ability makes C5D-MerR a potential bioadsorbents to purify Hg2+-polluted water.

The tolerance ability for Hg2+ of C5D-MerR was much stronger than that of C5D and C5D-C. The C5D-MerR could be cultured in medium with Hg2+ under the concentration of 100 mg/L, while the C5D and C5D-C’s growth could be inhibited by Hg2+ that is excess of 30 mg/L. The resistance character to heavy metal ions of C5D-MerR also found in other cell surface-displayed engineered yeast cells [45]. The strong resistance ability of C5D-MerR for Hg2+ mainly thanks to the successful display of MerR on its surface, which can inhibit the Hg2+ permeating into its cytosol. Therefore, it is a feasible way to enhance the resistance ability of yeast cells by displaying metal binding protein on their surface. On the other hand, the cell division period was lengthened by Hg2+ in the medium when compared with the cells growth in the medium without Hg2+. However, the reason for this phenomenon is still unclear.

The pH levels influenced C5D-MerR less than that of C5D and C5D-C. The C5D-MerR has the highest adsorption ability at about pH 7, but it can effectively adsorb Hg2+ at a wide range of pH levels, which will be very useful in treating different types of water with different pH levels (Fig. 5). The mechanism for the relative stability of the C5D-MerR at a wide range of pH levels still needs much more investigation.

The coexistence of heavy metal ions Cu2+ and Cd2+ seriously affected the adsorption ability of C5D and C5D-C for Hg2+. This indicted that these two yeast strains could not selectively adsorb Hg2+ from water polluted by Hg2+, Cu2+ and Cd2+ at the same time. On the other hand, the adsorption ability of C5D-MerR was almost not influenced by the coexistence of Cu2+ and Cd2+. This result demonstrated that C5D-MerR had higher affinity and selectivity toward Hg2+ than C5D and C5D-C. The MerR displayed on the cell surface was functional and could be used as an effective binder for Hg2+. The engineered yeast strain C5D-MerR can be used to adsorb Hg2+ from mixed heavy metals ions, and this character will be very useful in the practical application.

The adsorption ability for ultra-trace Hg2+ of C5D-MerR was much stronger than that of C5D and C5D-C. We predicted that this enhancement was mainly attributed to the MerR displayed on the cell surface because it could easily detect the ultra-trace Hg2+ in the environment. The C5D and C5D-C do not have displayed protein on their surface, so they have much difficult to detect ultra-trace Hg2+ in the water.

The mechanisms of microorganism–metal interreaction were classified into active and passive types [46]. The active type adsorbs heavy metal ions by energy-dependent metabolism, while the other type is likely to adsorb heavy metal ions with surface binding mechanism without energy consuming [47]. Therefore, we inferred that the engineered C5D-MerR constructed in this study could be used as a functional bioadsorbents for Hg2+ under both living and non-living conductions.

The function of the engineered yeast mainly depends on the character of the protein displayed on the cell surface. Therefore, finding more proteins that have high affinity, capacity and selectivity for certain heavy metal ions should be the future research focus.