A method for improving the ability of escherichia coli to adsorb cadmium ions

Abstract

The application discloses a method for improving the cadmium ion adsorption capacity of Escherichia coli, and belongs to the technical field of genetic engineering. E. coli BL21 to Cd 2+ , and the unit point mutation of T16C, K19C and S45C to CeMT2 can significantly improve the Cd 2+ binding capacity of CeMT2, and the cell Cd 2+ concentration of the T16C, K19C and S45C mutants is increased by 1.32 times, 1.50 times and 1.41 times, respectively. E. coli The recombinant expression of CeMT2 (K19C) and LmCadC in BL21 can obviously improve the cadmium ion adsorption capacity of Escherichia coli, and the maximum adsorption capacity reaches 8.5 mg / g.

Description

Technical Field

[0001] This invention relates to a method for improving the ability of Escherichia coli to adsorb cadmium ions, belonging to the field of genetic engineering technology. Background Technology

[0002] Cadmium (Cd) is a typical highly toxic heavy metal pollutant with wide sources, strong mobility, and difficulty in natural degradation. It easily accumulates through the food chain and causes long-term damage to the kidneys and bones in humans. Microbial remediation has attracted attention due to its environmental friendliness and potential for removing low concentrations, among which Escherichia coli (E. coli) is a suitable method. Escherichia coli With its advantages of easy cultivation, rapid growth, and mature genetic manipulation system, it has become a common engineered bacterium for constructing Cd removal strains.

[0003] Currently, heavy metal binding proteins and related regulatory systems are being used to enhance E. coli's resistance to Cd. 2+ The adsorption and enrichment capabilities of [the substance] have become an effective means of solving Cd pollution. E. coli Heterologous expression of mammalian metallothioneins (MTs) in BL21 increased cellular resistance to Cd. 2+ The binding ability of this protein was observed, and E. coli expressing this protein showed significant tolerance and removal ability in high-concentration Cd environments (Odawara et al., 1995). E. coli Heterologous expression of CadC and CadA in BL21 cells increased cell growth rate and Cd levels in Cd-contaminated environments. 2+ Adsorption capacity (Kang et al., 2007). Further research showed that CadC overexpression not only activates CadA efflux function but also increases cellular adsorption to Cd. 2+ The emission of Cd reduces the accumulation of Cd in cells, thereby improving the cell's tolerance to Cd and its detoxification capacity (Singh et al., 2020).

[0004] Single heavy metal binding protein gene expression has drawbacks such as limited adsorption capacity, cytotoxicity, and poor stability, making it difficult to cope with complex polluted environments. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a method for improving the ability of Escherichia coli to adsorb cadmium ions. This method improves the adsorption capacity of cadmium ions by multi-gene synergistic expression and conformational modification of metal-binding proteins. E. coli For Cd 2+ Adsorption capacity, and reduce expression burden and stress effects, thereby achieving E. coli The goal of achieving stable and efficient Cd pollution remediation performance is to address the problems caused by defects in the single heavy metal binding protein gene system.

[0006] The first technical solution provided by this invention is a metallothionein 2 mutant, wherein the mutant is a mutation of the metallothionein 2 parent with the amino acid sequence shown in SEQ ID NO.4, involving at least one of the following mutations: (1) The threonine T at position 16 is mutated to cysteine ​​C; (2) The lysine K at position 19 is mutated to cysteine ​​C; (3) The serine S at position 45 is mutated to cysteine ​​C.

[0007] In some embodiments, the mutant is a mutation performed on the metallothionein 2 parent with the amino acid sequence shown in SEQ ID NO.4, as follows: (a) The threonine T at position 16 is mutated to cysteine ​​C; (b) The lysine K at position 19 is mutated to cysteine ​​C; (c) The serine S at position 45 is mutated to cysteine ​​C; (d) The threonine T at position 16 was mutated to cysteine ​​C, and the lysine K at position 19 was mutated to cysteine ​​C.

[0008] The present invention provides a second technical solution, which is a gene encoding the mutant described in the first technical solution.

[0009] The present invention provides a third technical solution, which is a recombinant vector carrying the gene described in the second technical solution.

[0010] In some embodiments, the recombinant vector is a plasmid pET28a or a plasmid pET30a as the expression vector.

[0011] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0012] In some embodiments, the recombinant cells are Escherichia coli as host cells.

[0013] The fifth technical solution provided by this invention is a method for improving the ability of Escherichia coli to adsorb cadmium ions, wherein the method involves overexpressing at least one of the following proteins in Escherichia coli: (1) Caenorhabditis elegans The source of metallothionein 2 (CeMT2) or the mutant described in the first technical solution; (2) Listeria monocytogenes Transcriptional regulatory factors of origin (LmCadC); (3) Staphylococcus aureus P-type ATPase (SaCadA).

[0014] In some embodiments, the amino acid sequences of CeMT2, LmCadC, and SaCadA are shown in SEQ ID NO. 4-6, respectively.

[0015] The six technical solutions provided by this invention are the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant plasmid described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, in the application of improving the ability of Escherichia coli to adsorb cadmium ions.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention can effectively enhance the expression of CeMT2, LmCadC, and SaCadA. E. coli BL21 vs Cd 2+ The adsorption / removal capacity of CeMT2 was improved, and unit point mutations at T16C, K19C, and S45C were performed on CeMT2, which significantly improved the Cd removal capacity of CeMT2. 2+ Binding capacity, cellular Cd binding capacity of T16C, K19C and S45C mutants 2+ The concentrations increased significantly, by 1.32-fold, 1.50-fold, and 1.41-fold, respectively. CeMT2(K19C) and LmCadC were used together in... E. coli Recombinant expression in BL21 significantly enhances the adsorption of cadmium ions by Escherichia coli, reaching a maximum adsorption capacity of 8.5 mg / g. Attached Figure Description

[0017] Figure 1 For different reorganizations E. Coli BL21 strain under 400 μM Cd 2+ Differences in adsorption capacity.

[0018] Figure 2 Introducing mutants of CeMT2(Wt) and different Cys in E. coli Cd in BL21 2+ Differences in adsorption capacity (***, p<0.001).

[0019] Figure 3 CeMT2 single-site and combinatorial mutation recombination E. coli BL21 vs Cd 2+ Differences in adsorption capacity (***, p<0.001).

[0020] Figure 4 Recombinant expression of single genes and dual plasmid combinations E. coli BL21 vs. Cd 2+ The differences in adsorption capacity (*, p<0.05; ***, p<0.001). Detailed Implementation

[0021] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0022] Test method: Reorganization E. coli BL21's Cd 2+ Adsorption capacity determination Select pET30a-CeMT2, pET30a-LmCadC, and pET30a-SaCadA for recombination E. coli Single colonies of strain BL21 were cultured overnight at 37°C and 220 rpm in TB liquid medium.

[0023] The recombinant cells cultured overnight were inoculated at a rate of 1% (v / v). E. coli BL21 was inoculated into TB liquid medium and cultured at 37°C and 220 rpm until OD reached. 600 The value is 0.6, and Cd is added. 2+ The solution makes Cd in the culture medium 2+ The bacterial culture was incubated at a concentration of 400 μM for 12 h. The culture was then centrifuged at 4000 rpm for 15 min, and the supernatant was filtered through a 0.22 filter membrane to determine its Cd content. 2+ The concentration of the bacterial precipitate was washed with sterile water, dried in an oven to constant weight, and then weighed. The recombinant concentration was calculated using formula (1). E. coli BL21's Cd 2+ Adsorption capacity ( q Each experiment was performed in three biological replicates.

[0024]

[0025] in C 0 and C e represents the initial and equilibrium Cd², respectively. + concentration, V The volume of the solution. m This is the dry weight of the bacterial cells.

[0026] Materials used in the examples: 1. Host strain: Commercial strain E. coli BL21 E. coli JM109 samples were all preserved in the laboratory. Expression vectors: commercial plasmids pET28a and pET30a were preserved in the laboratory.

[0027] 2. The genes involved in the following examples are shown in Table 1.

[0028] Table 1. Sources and gene sequences of heavy metal binding proteins

[0029] Example 1: Expression of Recombinant Heavy Metal Binding Protein E. coli Construction of BL21 Primers were designed based on the gene sequences of each protein (SEQ ID NO. 1~3) (Table 2), and PCR amplification was performed to obtain the target genes. The PCR products were detected by 1% agarose gel electrophoresis, then purified by enzyme digestion. The prokaryotic expression vectors pET30a-CeMT2, pET30a-LmCadC, and pET30a-SaCadA were constructed using a one-step cloning kit and transformed into these vectors. E. coli JM109 competent cells were used, and single clones were selected for sequencing after PCR verification. Plasmids with correct sequencing results were then transformed into... E. coli BL21 construction for expression of recombinant heavy metal binding protein E. coli BL21.

[0030] Table 2 Primer sequences for heavy metal binding proteins

[0031] Compared with the empty vector control group (pET-30a), the recombinant vector expressing heavy metal-related proteins showed improvement. E. coli BL21 vs. Cd² + The adsorption capacity of all showed a significant increase. Figure 1 The recombinant strains pET30a-CeMT2, pET30a-LmCadC, and pET30a-SaCadA showed better resistance to Cd compared to the empty vector control group. 2+ The adsorption capacity increased by 3.85 times, 2.13 times, and 4.48 times, respectively. The pET30a-SaCadA recombinant strain exhibited the highest Cd² adsorption capacity. + Adsorption capacity (5.23 mg / g). The above results indicate that the expression of CeMT2, LmCadC, and SaCadA can effectively enhance… E. coli BL21 vs Cd² + The adsorption / removal capacity of the samples was improved by SaCadA and CeMT2.

[0032] Example 2 Mutant Screening 1. Construction of single point mutant strains and Cd 2+ Adsorption capacity determination Based on the CeMT2 gene sequence, mutation primers were designed (Table 3), and PCR amplification was performed to obtain the single point mutation gene. After PCR product detection by 1% agarose gel electrophoresis, enzyme digestion and purification were performed. Prokaryotic expression vectors pET30a-CeMT2(WT), pET30a-CeMT2(Q10C), pET30a-CeMT2(T16C), pET30a-CeMT2(K19C), pET30a-CeMT2(D25C), pET30a-CeMT2(A37C), pET30a-CeMT2(S45C), pET30a-CeMT2(G49C), and pET30a-CeMT2(Q60C) were constructed using an end smoothing / phosphorylation / ligation kit. E. coli JM109 competent cells were used, and single clones were selected for sequencing after PCR verification. Plasmids with correct sequencing results were then transformed into... E. coli BL21 Recombination at Different Mutation Sites E. coli BL21 strain.

[0033] Table 3. Primer Design for CeMT2 Unit Point Mutation

[0034] Select pET30a-CeMT2(WT), pET30a-CeMT2(Q10C), pET30a-CeMT2(T16C), pET30a-CeMT2(K19C), pET30a-CeMT2(D25C), pET30a-CeMT2(S45C), pET30a-CeMT2(G49C), and pET30a-CeMT2(Q60C) for recombination. E. coli Single colonies of strain BL21 were cultured overnight at 37°C and 220 rpm in TB liquid medium.

[0035] To evaluate the introduction of additional Cys pairs in CeMT2 for Cd 2+ To investigate the effect of binding capacity, multiple CeMT2 single-point mutants were constructed, and their intracellular Cd content under the same conditions was compared. 2+ content( Figure 2 Compared to wild-type CeMT2 (WT), some mutants exhibited significant Cd... 2+ Enrichment was enhanced. Specifically, the T16C, K19C, and S45C mutants showed increased Cd concentrations in their cells. 2+ The concentrations increased significantly by 1.32-fold, 1.50-fold, and 1.41-fold, with K19C being the highest among all mutants (7.15 mg / g). In contrast, while other sites such as Q10C and Q606C showed some upward trends, they were not significantly different from wild-type CeMT2 (WT).

[0036] Overall, the introduction of Cys into T16C, K19C, and S45C significantly improves the Cd content of CeMT2. 2+ It has the ability to be combined, but its effect is limited or even has a negative impact in other positions.

[0037] 2. Construction of multi-site mutant strains and Cd 2+ Adsorption capacity determination Based on unit point mutation recombination E. coli BL21's Cd 2+ Based on the adsorption capacity results, sites that enhance adsorption capacity were selected for multi-site mutation. The multi-site mutation primers are shown in Table 4. Using T-M2 / 3-F and T-M2 / 3-R as mutation primers, PCR amplification was performed with pET30a-CeMT2(T16C) and pET30a-CeMT2(S45C) as templates to obtain T16C / K19C double-site mutation CeMT2 and T16C / K19C / S45C multi-site CeMT2. Using pET30a-CeMT2(S45C) as a template, PCR amplification was performed with T-M2 / 6-F, T-M2 / 6-R, T-M3 / 6-F, and T-M3 / 6-R to obtain T16C / S45C double-site mutation and K19C / S45C double-site mutation CeMT2.

[0038] After PCR products were detected by 1% agarose gel electrophoresis, they were purified by enzyme digestion. Prokaryotic expression vectors, pET30a-CeMT2(T16C / K19C), pET30a-CeMT2(T16C / S45C), pET30a-CeMT2(K19C / S45C), and pET30a-CeMT2(T16C / K19C / S45C), were constructed using an end-smoothing / phosphorylation / ligation kit. These vectors were then transformed with... E. coli JM109 competent cells were used, and single clones were selected for sequencing after PCR verification. Plasmids with correct sequencing results were then transformed into... E. coli BL21 Recombination at Different Mutation Sites E. coli BL21 strain.

[0039] Table 4. Primer Design for CeMT2 Multisite Mutations

[0040] Select pET30a-CeMT2(T16C / K19C), pET30a-CeMT2(T16C / S45C), pET30a-CeMT2(K19C / S45C), and pET30a-CeMT2(T16C / K19C / S45C) for recombination. E. coli Single colonies of strain BL21 were cultured overnight at 37°C and 220 rpm in TB liquid medium.

[0041] The results showed that ( Figure 3 Compared to single-point mutations, two-site or three-site combination mutations did not significantly increase Cd. 2+ Adsorption capacity. Among them, Cd with the T16C / K19C dual-site mutation... 2+ The adsorption capacity was comparable to that of the corresponding single-site mutations (T16C or K19C), while the adsorption capacity of the other mutation combinations decreased significantly. Notably, the K19C / S45C double-site mutation in Cd... 2+ The adsorption capacity is not only lower than that of the corresponding single-point mutation, but also significantly lower than that of wild-type CeMT2.

[0042] In conclusion, multi-site combination mutations of T16C, K19C, and S45C did not improve CeMT2 response to Cd. 2+ The adsorption performance of Cd² was improved by single-point mutations T16C, K19C, and S45C. + The binding ability is significant, with the K19C mutation showing the most pronounced effect.

[0043] Example 3: Construction and transformation of dual plasmids Primers were designed based on the gene sequences of LmCadC and SaCadA (Table 5), and PCR amplification was performed to obtain the target genes. The PCR products were detected by 1% agarose gel electrophoresis, then purified by enzyme digestion. The prokaryotic expression vectors pET28a-LmCadC and pET28a-SaCadA were constructed using a one-step cloning kit and transformed into these vectors. E. coli JM109 competent cells were selected for single-clone sequencing after PCR verification.

[0044] The constructed pET28a-LmCadC and pET28a-SaCadA were combined with pET30a-CeMT2(K19C) and pET30a-LmCadC with pET28a-SaCadA, respectively, and then converted to... E. coli In BL21, positive clones carrying both plasmids were obtained by screening on selective medium containing kanamycin and ampicillin.

[0045] Table 5 Primer Design for the Dual Plasmid System

[0046] Pick those carrying two plasmids E. coliSingle colonies of the recombinant strains BL21 (pET30a-CeMT2(K19C)+pET28a-LmCadC), (pET30a-CeMT2(K19C)+pET28a-SaCadA), and (pET30a-LmCadC+pET28a-SaCadA) were cultured overnight at 37°C and 220 rpm in TB liquid medium.

[0047] Dual plasmid co-expression for recombination E. coli BL21 Cd 2+ The effect of adsorption capacity, such as Figure 4 As shown, different gene combinations exhibited significantly different effects on Cd. The CeMT2(K19C)+LmCadC and CeMT2(K19C)+SaCadA dual-protein expression strains showed better Cd expression compared to single-protein expression. 2+ The adsorption capacity was significantly improved, with CeMT2(K19C)+LmCadC reaching the maximum adsorption capacity (8.5 mg / g), indicating that multi-gene synergistic expression can improve adsorption performance. In contrast, the LmCadC+SaCadA dual plasmid combination for Cd... 2+ The adsorption capacity was lower than that of the combination containing CeMT2 (K19C), but still significantly higher than that of LmCadC or SaCadA expressed alone.

[0048] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A metallothionein 2 mutant, characterized in that, The mutant is a mutation of the metallothionein 2 parent with the amino acid sequence shown in SEQ ID NO.4, involving at least one of the following mutations: (1) threonine T at position 16 is mutated to cysteine ​​C; (2) lysine K at position 19 is mutated to cysteine ​​C; (3) serine S at position 45 is mutated to cysteine ​​C.

2. The mutant according to claim 1, characterized in that, The mutant is a mutation of the metallothionein 2 parent with the amino acid sequence shown in SEQ ID NO.4, in which: (a) threonine T at position 16 is mutated to cysteine ​​C; (b) lysine K at position 19 is mutated to cysteine ​​C; (c) serine S at position 45 is mutated to cysteine ​​C; (d) threonine T at position 16 is mutated to cysteine ​​C, and lysine K at position 19 is mutated to cysteine ​​C.

3. The gene encoding the mutant according to any one of claims 1 to 2.

4. A recombinant vector carrying the gene of claim 3.

5. The recombinant vector of claim 4, wherein, The recombinant vector is expressed using plasmid pET28a or plasmid pET30a.

6. Expressing the mutant of any one of claims 1 to 2, or containing the gene of claim 3, or transforming a recombinant cell containing the recombinant plasmid of any one of claims 4 to 5.

7. The recombinant cell of claim 6, wherein, The recombinant cells used Escherichia coli as the host cells.

8. A method for improving the ability of Escherichia coli to adsorb cadmium ions, characterized in that, The method involves overexpressing at least one of the following proteins in Escherichia coli: (1) Caenorhabditis elegans The source is metallothionein 2 CeMT2 or the mutant described in any one of claims 1 to 2; (2) Listeria monocytogenes LmCadC, a transcriptional regulatory factor from which this originates; (3) Staphylococcus aureus P-type ATPase SaCadA.

9. The method according to claim 8, characterized in that, The amino acid sequences of CeMT2, LmCadC, and SaCadA are shown in SEQ ID NO.4~6, respectively.

10. The use of the mutant according to any one of claims 1 to 2, or the gene according to claim 3, or the recombinant plasmid according to any one of claims 4 to 5, or the recombinant cell according to any one of claims 6 to 7, or the method according to any one of claims 8 to 9 in improving the ability of Escherichia coli to adsorb cadmium ions.

Images