Method for realizing precise green restoration of environmental heavy metal pollutants by coupling heavy metal sensing module and aerobic sulfate reduction module

Abstract

The invention discloses a method for realizing precise green restoration of environmental heavy metal pollutants by coupling a heavy metal sensing module and an aerobic sulfate reduction module. The method comprises the following steps: constructing an aerobic sulfate reduction pathway by heterologous reconstruction of cysteine feedback inhibition-resistant serine acetyltransferase and cysteine desulfylase, putting the aerobic sulfate reduction pathway under the control of a sensing module composed of heavy metal sensing protein genes, so as to construct a'sensing integration 'module, and transferring the'sensing integration' module into environmental microorganisms. The heavy metal in-situ remediation engineering strain is formed. When a specific heavy metal sensing module in the engineering strain recognizes heavy metal pollution, expression of an aerobic sulfate reduction module can be effectively started, so that heavy metal ions are converted into stable metal sulfide, and in-situ green low-carbon remediation of environmental heavy metal pollutants is realized. The genetic method disclosed by the invention is simple and efficient, saves the cost, can realize accurate perception and accurate control of environmental heavy metal pollution, and has important environmental protection application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental pollution remediation, and specifically relates to a method for coupling a heavy metal sensing module and an aerobic sulfate reduction module to achieve precise green remediation of environmental heavy metal pollutants. The method uses environmental microorganisms as chassis microorganisms and utilizes synthetic biology technology to couple heavy metal sensing pathways and aerobic sulfate reduction pathways to achieve in-situ precise remediation of environmental heavy metals. Background Art

[0002] Heavy metal pollution can lead to the destruction of soil structure, the decline of biodiversity, the inhibition of soil microbial activity, and serious impacts on the self-purification capacity of soil and the quality and yield of crops. Therefore, controlling the migration of heavy metal ions in the environment is of great significance to maintaining ecological security and environmental health.

[0003] The existing technology has adopted a variety of methods to deal with environmental heavy metal pollution, including physical methods, chemical methods and biological methods. The physical method mainly reduces the bioavailability of heavy metals by soil replacement and solidification, but the cost is high and causes secondary pollution. Chemical methods include the use of chemical reagents for precipitation, complexation, etc. Although these methods have fast processing speeds, they are often costly, difficult to sustain, and prone to secondary environmental pollution. Therefore, biological methods, especially microbial remediation methods, are considered to be a green, environmentally friendly and low-cost way to remediate heavy metal pollution due to their ability to absorb and transform heavy metals.

[0004] Sulfate-Reducing Bacteria (SRB) are a type of anaerobic bacteria that can use sulfate as an electron acceptor to perform sulfate respiration. SRB can be widely distributed in anaerobic habitats rich in organic matter and sulfate, such as soil, seawater, river water underground pipelines, oil and gas wells, flooded rice field soil, river and lake sediments, and swamp mud. Previous studies have mainly found that SRB produces S through the dissimilatory sulfate reduction pathway. 2- It can passivate heavy metals into insoluble metal sulfides, thereby reducing the environmental migration of heavy metals. However, SRB is an obligate anaerobe, and its growth conditions require a strict anaerobic environment, which greatly limits its application scenarios in actual environments. 2- It can also be produced through the assimilative sulfate reduction process to synthesize cysteine ​​and methionine, and the assimilative sulfate reduction pathway does not require strict anaerobic conditions. However, there are still the following difficulties in solving the problem of heavy metal pollution control through this aerobic assimilative sulfate reduction pathway: (1) Unlike dissimilatory sulfate reduction, the assimilative sulfate reduction pathway process is strictly inhibited by negative feedback from cysteine. Therefore, conventional bacteria almost do not produce and secrete S 2-(2) In the actual environment, the aerobic assimilation sulfate reduction pathway will indiscriminately precipitate various metal ions, thus causing potential adverse effects on the ecological environment and environmental health. Therefore, the current application of sulfate-reducing bacteria in the in situ remediation of environmental heavy metals is severely limited.

[0005] In order to achieve green and low-carbon remediation of environmental heavy metals, microorganisms are used to produce S 2- Converting heavy metals into insoluble metal sulfides is an important approach. Therefore, using synthetic biology strategies to develop new sulfate-reducing microorganisms and achieve controllable and efficient sulfate reduction under aerobic conditions will have important application value for in situ bioremediation and green management of heavy metals in my country's environment. Summary of the invention

[0006] In order to solve the bottleneck problem of the current sulfate-reducing bacteria in the remediation of heavy metal pollution in the environment, the present invention, based on the synthetic biology strategy, introduces two unique enzymes through genetic engineering technology to construct an aerobic sulfate reduction pathway: (1) serine acetyltransferase (CysE) which is insensitive to negative feedback inhibition of cysteine. 768 ), which directs sulfate reduction to generate excess cysteine; (2) cdsH, encoding cysteine ​​desulfurase (CdsH), converts excess cysteine ​​into S 2- At the same time, by introducing a heavy metal sensing module to control the aerobic sulfate reduction process, a "sensing and controlling integrated" genetic module was constructed, and it was transformed into environmental microorganisms to construct a new heavy metal remediation engineering strain. When the engineering strain senses the presence of heavy metals, it can directly use sulfate in the environment as a sulfur source and produce S through aerobic reduction pathways. 2- , converting heavy metal ions into insoluble metal sulfides, thereby achieving green and low-carbon remediation of heavy metal pollution.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for coupling a heavy metal sensing module and an aerobic sulfate reduction module to achieve precise green remediation of environmental heavy metal pollutants. Through synthetic biology strategies, the heavy metal sensing module and the aerobic sulfate reduction module are coupled to each other to construct a heavy metal "sensing and controlling integrated" module, and the module is used to transform environmental microorganisms and construct a heavy metal remediation engineering strain; the engineering strain can specifically turn on the expression of the aerobic sulfate reduction module according to the type of environmental heavy metal pollution, thereby converting heavy metal ions into stable metal sulfides, and achieving in-situ green and low-carbon remediation of environmental heavy metal pollutants. The steps are as follows:

[0009] (1) Through gene editing, CysE, which is not affected by cysteine ​​negative feedback inhibition, is 768The protein combines with the CdsH protein that can convert cysteine ​​into H2S to construct an aerobic sulfate reduction module;

[0010] (2) Different types of heavy metal sensing protein genes and their corresponding inducible promoters were inserted into recombinant plasmids to construct heavy metal sensing modules to respond to different environmental heavy metal pollutants;

[0011] (3) The constructed heavy metal sensing module and aerobic sulfate reduction module are coupled into a "sensing and controlling integrated" remediation module, and introduced into environmental microorganisms to construct a new sulfate reduction engineering strain;

[0012] (4) Using this engineered strain, S2- can be efficiently produced under aerobic conditions, thereby converting heavy metal pollutants into insoluble metal sulfides, achieving accurate perception and precise remediation of heavy metals.

[0013] The specific steps of the present invention are:

[0014] (1) The cysE gene was cloned from the E. coli genome to construct the shuttle plasmid pBF-cysE; primers were designed to mutate the cysE gene, mutating the base G at 768 bp to A, and constructing the shuttle plasmid pBF-cysE 768 ; Synthesize the cdsH gene and integrate it into the shuttle plasmid pBF-cysE 768 , construct the recombinant plasmid pBF-P lac -cdsH-cysE 768 , and removed the endogenous promoter gspA of the cdsH gene to obtain the assimilation sulfate reduction module; the recombinant plasmid was introduced into E. coli DH5α or S. oneidensis MR-1 by electroporation and conjugation transfer for functional verification.

[0015] (2) Synthesize or amplify heavy metal sensing protein genes from different sources and the corresponding inducible promoters to construct the recombinant plasmid pBF-P lac -XP X -egfp, to obtain the heavy metal sensing module; where X represents different types of heavy metal sensing protein genes, P X Represents an inducible promoter regulated by heavy metal sensing protein; the recombinant plasmid was introduced into E.coli DH5α by electroporation and conjugation transfer, and the green fluorescence signal was used to detect the specificity and sensitivity of the sensing protein.

[0016] The heavy metal sensing module constructed by the present invention can specifically respond to environmental heavy metals such as cadmium, copper, lead, arsenic and mercury.

[0017] (3) The constructed heavy metal sensing module and aerobic sulfate reduction module were coupled into an integrated sensing and control repair module by homologous recombination. The recombinant plasmid was introduced into E. coli DH5α or S. oneidensis MR-1 by electroporation and conjugation transfer, and the integrated repair module was verified. After successful verification, it was transferred to environmental microorganisms by CRISPR gene editing technology to construct a new sulfate reduction engineered strain.

[0018] (4) Using a novel sulfate-reducing engineered strain, S2- can be efficiently generated under aerobic conditions, thereby converting heavy metal pollutants into insoluble metal sulfides, and detecting the removal effect of heavy metal ions. The engineered strain constructed by the present invention can be used for low-cost remediation of heavy metals in soil, heavy metals in water, and industrial heavy metal wastewater.

[0019] The template strain E. coli MG1655 of the present invention was purchased from the American Escherichia coli Culture Collection, with the collection number being CGSC 6300.

[0020] One of the chassis microorganisms of the present invention is a Gram-negative bacterium E. coli DH5α having an aerobic assimilation sulfate reduction pathway, which was purchased from the American Type Culture Collection and has a strain number of ATCC PTA-1798.

[0021] One of the chassis microorganisms of the present invention is a Gram-negative bacterium S. oneidensis MR-1 with an aerobic assimilation sulfate reduction pathway, which was purchased from the American Type Culture Collection and has a strain number of ATCC 700550. TM .

[0022] The complete nucleotide sequence of the cysE gene of the present invention is shown in SEQ ID No: 1.

[0023] CysE of the present invention 768 The complete nucleotide sequence of the gene is shown in SEQ ID No: 2.

[0024] The complete nucleotide sequence of the cdsH gene of the present invention is shown in SEQ ID No:3.

[0025] The complete nucleotide sequence of the cadC gene of the present invention is shown in SEQ ID No:4.

[0026] The promoter P of the present invention cadC The complete nucleotide sequence is shown in SEQ ID No:5.

[0027] The complete nucleotide sequence of the cueR gene of the present invention is shown in SEQ ID No:6.

[0028] The promoter P of the present invention copA The complete nucleotide sequence is shown in SEQ ID No:7.

[0029] The complete nucleotide sequence of the pbrR gene of the present invention is shown in SEQ ID No:8.

[0030] The promoter P of the present invention pbr The complete nucleotide sequence is shown in SEQ ID No:9.

[0031] The complete nucleotide sequence of the merR gene of the present invention is shown in SEQ ID No: 10.

[0032] The promoter P of the present invention merT The complete nucleotide sequence is shown in SEQ ID No:11.

[0033] The complete nucleotide sequence of the arsR gene of the present invention is shown in SEQ ID No:12.

[0034] The promoter P of the present invention arsR The complete nucleotide sequence is shown in SEQ ID No: 13.

[0035] The present invention mutates the cysE gene by gene editing and successfully obtains CysE that is insensitive to cysteine ​​negative feedback inhibition. 768 .

[0036] The present invention obtains the recombinant plasmid pBF-P by genetic engineering method lac -cdsH-cysE 768 , and the endogenous promoter gspA of the cdsH gene was removed to express CysE in E. coli DH5α and S. oneidensis MR-1 768 and CdsH proteins, successfully releasing S through the assimilation sulfate reduction pathway under aerobic conditions. 2- .

[0037] The present invention obtains the recombinant plasmid pBF-XP by genetic engineering method X -cdsH-cysE 768 , which can respond to the induction of corresponding heavy metal ions and turn on the expression of the sulfate reduction module.

[0038] The engineered strain obtained by the invention can precipitate heavy metal ions to form stable heavy metal sulfides, and the removal rates of different types of heavy metal ions are all as high as over 70%.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) The aerobic sulfate reduction module avoids the dissimilatory sulfate reduction pathway used by conventional sulfate-reducing bacteria and instead uses a synthetic biology strategy to transform the serine acetyltransferase mutant gene cysE 768 It integrates with cysteine ​​desulfurase cdsH to convert sulfate into S via the assimilatory reduction pathway. 2- This method avoids the anaerobic environment required for dissimilatory sulfate reduction and the degradation of cysteine ​​in the aerobic sulfate assimilation pathway. 2- The severe negative feedback inhibition generated by the reaction enables it to achieve aerobic reduction of sulfate and S 2- Efficient production of for in-situ solidification of heavy metals.

[0041] (2) The “sensing and control integrated” module can select specific heavy metal sensing modules according to the category of heavy metals in the environment. When the heavy metal exceeds the sensing threshold, the sulfate reduction module begins to be expressed to perform in-situ solidification of the heavy metal. When the heavy metal is lower than the sensing threshold, the sulfate reduction module is shut down, thereby achieving accurate sensing and precise remediation of heavy metals in the environment.

[0042] (3) The constructed heavy metal remediation strain can directly utilize sulfate that is ubiquitous in the environment without the need to add additional sulfur sources; when heavy metals exceed the perception threshold, the engineered strain can convert sulfate into S 2- , thereby achieving in-situ passivation and fixation of environmental heavy metals.

[0043] (4) Ordinary sulfate-reducing bacteria are absolute anaerobes and can only perform dissimilatory sulfate reduction under absolute anaerobic conditions. Their absolute anaerobic characteristics severely limit their application in practical environments. The assimilative sulfate reduction module constructed by the present invention can achieve efficient sulfate reduction under aerobic conditions to produce S 2- , thus achieving the passivation and fixation of heavy metals. This significantly enhances the environmental adaptability of the repair strain.

[0044] (5) The sulfate reduction process of common sulfate-reducing bacteria is uncontrollable, making it difficult to achieve accurate repair of environmental heavy metals. The present invention places the sulfate reduction module under the control of the heavy metal sensing module. Only when specific heavy metals are present will the sulfate reduction process of the repair strain be turned on, thereby achieving accurate perception and precise control of heavy metal pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Represents the entire technical solution of the present invention.

[0046] Figure 2 The plasmid map constructed in the present invention is shown.

[0047] Figure 3Represents E.coli DH5α / pBF-cadC-P cadC -cdsH-cysE 768 S 2- And the ability to precipitate heavy metal cadmium.

[0048] Figure 4 Indicates E.coli DH5α / pBF-cueR-P copA -cdsH-cysE 768 S 2- And the ability to precipitate heavy metal copper.

[0049] Figure 5 S. oneidensis MR-1 / pBF-pbrR-P pbr -cdsH-cysE 768 S 2- And the ability to precipitate heavy metal lead.

[0050] Figure 6 S. oneidensis MR-1 / pBF-merR-P merT -cdsH-cysE 768 S 2- and the ability to precipitate heavy metal mercury.

[0051] Figure 7 S. oneidensis MR-1 / pBF-arsR-P arsR -cdsH-cysE 768 S 2- and the ability to precipitate heavy metal arsenic.

[0052] Figure 8 Indicates the growth of E. coli engineered strains.

[0053] Fig. 9 Represents the growth of S. oneidensis engineered strains.

[0054] Fig.10 Represents the identification of CdS precipitation by SEM and EDS.

[0055] Fig.11 Represents E.coli DH5α / pBF-cadC-P cadC -cdsH-cysE 768 Removal rate of heavy metal Cd by the strain.

[0056] Fig.12 Indicates E.coli DH5α / pBF-cueR-P copA -cdsH-cysE768 Removal rate of heavy metal Cu by strains.

[0057] Fig.13 S. oneidensis MR-1 / pBF-pbrR-P pbr -cdsH-cysE 768 Removal rate of heavy metal Pb by strains.

[0058] Fig.14 S. oneidensis MR-1 / pBF-merR-P merT -cdsH-cysE 768 Removal rate of heavy metal Hg by strains.

[0059] Fig.15 S. oneidensis MR-1 / pBF-arsR-P arsR -cdsH-cysE 768 Removal rate of heavy metal As by strains. DETAILED DESCRIPTION

[0060] The LB medium (1 L) used in each example consists of 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. The solvent is deionized water. The solid medium needs to be supplemented with 1.5% agar B.

[0061] The composition of the M9 medium (1 L) used in each example is: 1 g NH4Cl, 12 g NaCl, 11 g Tris, 2 mM MgSO4, 0.013 g CaSO4, 0.3 g KCl, 2 g MgCl2, 10 mL 40% glycerol, 2 mM sodium glycerophosphate, the solvent is deionized water, and the pH is adjusted to 7.

[0062] The mineral salt medium (1 L) used in each example consists of: HEPES 12 g, 10× major elements 100 mL, MgSO4·7H2O (1000×) 1 mL, 100× trace elements 10 mL, sodium lactate 50 mM. The solvent is deionized water, and the pH is adjusted to 7.

[0063] Example 1

[0064] In this embodiment, a "sensing-control-in-one" heavy metal remediation engineering strain that specifically responds to Cd is constructed for accurate perception and precise remediation of the environmental heavy metal Cd.

[0065] (1) Construction of shuttle plasmid pBF-cysE

[0066] Plasmid pBF-P was stored in the laboratory lac-B0035-egfp was used as a template, and primers pBF-linear-F and pBF-liner-R were used to amplify the plasmid backbone; the E. coli MG1655 genome was used as a template, and primers cysE-F and cysE-R were used to amplify the cysE gene sequence.

[0067] (2) Construction of shuttle plasmid pBF-cysE 768

[0068] The shuttle plasmid pBF-cysE was used as a template and the plasmid sequence was amplified using primers cysE-mu-F and cysE-mu-R.

[0069] (3) Construction of recombinant plasmid pBF-P lac -cdsH-cysE 768

[0070] shuttle plasmid pBF-cysE 768 Primers pBF-cysE-linear-F and pBF-cysE-linear-R were used as templates to amplify the backbone sequence of the plasmid; primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence using pUC57-cdsH plasmid as a template.

[0071] (4) Construction of recombinant plasmid pBF-P lac -cdsH-ΔgspA-cysE 768

[0072] pBF-P lac -cdsH-cysE 768 The plasmid was used as a template and the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R.

[0073] (5) Construction of recombinant plasmid pBF-cadC-P cadC -cdsH-ΔgspA-cysE 768

[0074] pBF-P lac -cdsH-ΔgspA-cysE 768 The plasmid was used as a template, and the primers pBF-cadC-linear-F and pBF-cadC-linear-R were used to amplify the backbone sequence of the plasmid; the pSB1K3-cadC-gfp plasmid was used as a template, and the primers cadC-F and cadC-R were used to amplify the cadC gene and its downstream P cadC Promoter sequence.

[0075] (6) Using shake flask fermentation, the constructed engineered strain was tested for assimilation of sulfate reduction pathway under aerobic conditions to evaluate its S production 2- and the ability to precipitate Cd(II).

[0076] The strains and plasmids used in this example are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0077] Table 1 Bacterial species and plasmids used in the present invention

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] The specific implementation steps are as follows:

[0084] (1) Expression of CysE insensitive to cysteine ​​negative feedback inhibition in E. coli DH5α 768 protein and can convert excess cysteine ​​into S 2- CdsH protein, construct S 2- strains( Figure 1 ). The specific construction steps are as follows:

[0085] ①Preserve plasmid pBF-P in the laboratory lac -B0035-egfp was used as a template, and the plasmid backbone was amplified using primers pBF-linear-F and pBF-liner-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E.coli MG1655 genome as a template, the cysE gene sequence was amplified using primers cysE-F and cysE-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0086] Table 3 Plasmid construction PCR reaction system

[0087]

[0088]

[0089] ② The above two fragments were excised, recovered and purified, and the linear vector and target fragment were assembled by Gibson. The process of Gibson assembly of linear vector and target fragment is as follows: 20μL assembly system, including Gibson reaction solution, target fragment, linear vector and ultrapure water. When performing recombination reaction, the amount of vector is generally 50-100ng, and the target fragment and vector are generally mixed in a 5:1 molar ratio. React in a PCR instrument at a constant temperature of 50℃ for 1h, then electrotransform into E.coli DH5α, and spread on a 50mg / L kanamycin resistance plate. The positive transformant E.coli DH5α / pBF-cysE ( Figure 1 ).

[0090] ③ Using the shuttle plasmid pBF-cysE as a template, primers cysE-mu-F and cysE-mu-R were used to amplify the plasmid sequence; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 1 min, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0091] ④Same as step ② above. Screen the successful integration positive transformant DH5α / pBF-cysE by colony PCR and sequencing 768 ( Figure 1 ).

[0092] ⑤ With shuttle plasmid pBF-cysE 768 As a template, primers pBF-cysE-linear-F and pBF-cysE-linear-R were used to amplify the backbone sequence of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 3min, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-cdsH plasmid as a template, primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0093] ⑥Same as step ② above. Screen the successful integration positive transformant E.coliDH5α / pBF-P by colony PCR and sequencing. lac -cdsH-cysE 768 ( Figure 1 ).

[0094] ⑦ Using the recombinant plasmid pBF-Plac-cdsH-cysE 768 As a template, the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 3 min 30 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0095] ⑧Same as step ② above. Screen the successful integration positive transformant E.coliDH5α / pBF-P by colony PCR and sequencing lac -cdsH-ΔgspA-cysE 768 ( Figure 1 ).

[0096] (2) Integrate the cadmium heavy metal sensing protein gene and its corresponding inducible promoter into pBF-P lac -cdsH-ΔgspA-cysE 768 The cdsH gene upstream of the plasmid was electroporated into E. coli DH5α to form an engineered strain that can sense cadmium ( Figure 1 ). The specific construction steps are as follows:

[0097] ① Plasmid pBF-P lac -cdsH-ΔgspA-cysE 768 As a template, primers pBF-cadC-linear-F and pBF-cadC-linear-R were used to amplify the backbone of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the pSB1K3-cadC-gfp plasmid as a template, primers cadC-F and cadC-R were used to amplify the cadC gene and P cadC The promoter sequence was amplified; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0098] ② The above DNA fragments were excised and purified, and the linear vector and target fragments were assembled and electrotransformed into E. coli DH5α as shown in step (1) ②. The positive transformants E. coli DH5α / pBF-cadC-P that were successfully integrated were screened by colony PCR and sequencing. cadC -cdsH-cysE 768 ( Figure 1 ).

[0099] (3) E. coli DH5α / pBF-cadC-P cadC -cdsH-cysE 768 S 2- And the ability to precipitate Cd(Ⅱ) is evaluated. The specific steps are as follows:

[0100] ① Store E. coli DH5α / Wide-type and E. coli DH5α / pBF-cadC-P at -80℃ cadC -cdsH-cysE 768 The strains were inoculated into 5 mL of LB medium and cultured overnight at 37°C and 200 rpm. The strains cultured overnight were expanded and inoculated into 100 mL of LB medium and cultured at 37°C and 200 rpm for 150 min-180 min. When the bacterial concentration reached about 0.6-0.8, the bacterial solution was poured into two 50 mL centrifuge tubes and centrifuged at 4°C and 5000 g for 5 min to collect the bacteria. The bacteria were resuspended in M9 medium, added to 50 mL of medium, the bacterial concentration was adjusted to about 0.6, and 50 μM CdCl2 was added. Lead acetate test paper was attached to the bottle mouth and the bottle mouth was sealed with plastic wrap to prevent the generated H2S gas from escaping. After 6 hours of shaking culture at 37°C and 200 rpm, the lead acetate test paper was successfully observed to turn black ( Figure 3 ).

[0101] ② As in step ①, the growth curve of the engineered strain was measured. The addition of 50 μM CdCl2 had no effect on the growth of the engineered strain ( Figure 8 ).

[0102] ③ As in step ①, after 24 hours of shaking culture at 37°C and 200 rpm, the bacterial solution was successfully observed to change. After centrifugation, a yellow precipitate was clearly found ( Figure 3 ).

[0103] ④Same as above step ②E.coli DH5α / pBF-cadC-P cadC -cdsH-cysE 768 After yellow particles appear in the culture solution, collect 4 mL of the culture solution into two 2 mL EP tubes, centrifuge at 5000g, 4°C for 5 min, discard the supernatant, and wash the bacteria twice with 0.9% NaCl solution. 600The bacterial solution with a value of 1 was placed in an EP tube, centrifuged at 5000g, 4°C for 5min, the supernatant was discarded, 800μL of 2.5% pentanediol was added, the bacteria were resuspended, and the solution was allowed to stand at 4°C for 12h. The fixed bacterial solution was centrifuged, the supernatant was discarded, the bacteria were washed twice with 0.9% NaCl solution, and gradient eluted with 30%, 50%, 70%, 90% and 100% anhydrous ethanol in sequence, and each gradient was dehydrated twice. After each elution, anhydrous ethanol of the corresponding concentration was added to resuspend, the solution was allowed to stand for 5min, and centrifuged at 5000g for 3min. Finally, 1mL of anhydrous ethanol was added to resuspend, the solution was allowed to stand for 2h, and 4μL of the bacterial solution was dropped on a silicon wafer to make a SEM shooting sample ( Fig.10 ).

[0104] The results showed that the surface of wild-type E. coli DH5α was smooth without any particles. cadC -cdsH-cysE 768 On the surface of the bacteria, obvious granular precipitation appeared. In order to verify the composition of the particles, EDS measurement was carried out, and the test report showed that the granular precipitation on the surface of the bacteria was indeed CdS precipitation ( Fig.10 ).

[0105] ⑤After culturing for 24 hours as in step ② above, collect 5 mL of bacterial solution in a 10 mL EP tube, centrifuge at 5000 g, 4 °C for 5 min, and collect the supernatant. Pass the supernatant through a 0.22 μm microporous filter membrane to remove macromolecular organic matter and remaining bacteria. Collect the filtrate, use a 3 kD ultrafiltration tube to remove the remaining metal sulfide nanoparticles in the solution, and prepare a sample for ICP-MS determination of the remaining heavy metal ion concentration. According to the results of the ICP-MS determination, the removal rate of Cd(Ⅱ) [removal rate = (initial concentration of heavy metals in the culture medium - remaining concentration of heavy metals in the culture medium) / initial concentration of heavy metals in the culture medium] was calculated to be 71.4% ( Fig.11 ).

[0106] The test results showed that the engineered strain E. coli DH5α / pBF-cadC-P cadC -cdsH-cysE 768 Able to produce S under aerobic conditions 2- When Cd(Ⅱ) was added to the culture medium, after 24 hours of culture, the bacterial solution changed color and yellow CdS precipitate appeared. The results of SEM and EDS tests showed that the yellow granular precipitate on the surface of the bacteria was indeed CdS metal sulfide. The results of ICP-MS test also showed that the removal rate of Cd(Ⅱ) by the experimental strain reached more than 70%.

[0107] Example 2

[0108] In this embodiment, a "sensing-control-in-one" heavy metal remediation engineering strain that specifically responds to Cu is constructed for accurate perception and precise remediation of environmental heavy metal Cu.

[0109] (1) Construction of shuttle plasmid pBF-cysE

[0110] Plasmid pBF-P was stored in the laboratory lac -B0035-egfp was used as a template, and primers pBF-linear-F and pBF-liner-R were used to amplify the plasmid backbone; the E. coli MG1655 genome was used as a template, and primers cysE-F and cysE-R were used to amplify the cysE gene sequence.

[0111] (2) Construction of shuttle plasmid pBF-cysE 768

[0112] The shuttle plasmid pBF-cysE was used as a template and the plasmid sequence was amplified using primers cysE-mu-F and cysE-mu-R.

[0113] (3) Construction of recombinant plasmid pBF-P lac -cdsH-cysE 768

[0114] shuttle plasmid pBF-cysE 768 Primers pBF-cysE-linear-F and pBF-cysE-linear-R were used as templates to amplify the backbone sequence of the plasmid; primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence using pUC57-cdsH plasmid as a template.

[0115] (4) Construction of recombinant plasmid pBF-P lac -cdsH-ΔgspA-cysE 768

[0116] pBF-P lac -cdsH-cysE 768 The plasmid was used as a template and the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R.

[0117] (5) Construction of recombinant plasmid pBF-cueR-P copA -cdsH-ΔgspA-cysE 768

[0118] pBF-P lac -cdsH-ΔgspA-cysE 768The plasmid was used as a template, and the primers pBF-cueR-linear-F and pBF-cueR-linear-R were used to amplify the backbone sequence of the plasmid; the E. coli MG1655 genome was used as a template, and the primers cueR-F and cueR-R were used to amplify the cueR gene and its downstream P copA Promoter sequence.

[0119] (6) Using shake flask fermentation, the constructed engineered strain was tested for assimilation of sulfate reduction pathway under aerobic conditions to evaluate its S production 2- and the ability to precipitate Cu(II).

[0120] The strains and plasmids used in this example are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0121] The specific implementation steps are as follows:

[0122] (1) Expression of CysE insensitive to cysteine ​​negative feedback inhibition in E. coli DH5α 768 protein and can convert excess cysteine ​​into S 2- CdsH protein, construct S 2- strain ( Figure 1 ). The specific construction steps are as follows:

[0123] ①Preserve plasmid pBF-P in the laboratory lac -B0035-egfp was used as a template, and the plasmid backbone was amplified using primers pBF-linear-F and pBF-liner-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E.coli MG1655 genome as a template, the cysE gene sequence was amplified using primers cysE-F and cysE-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0124] ② The above two fragments were excised, recovered and purified, and the linear vector and target fragment were assembled by Gibson. The process of Gibson assembly of linear vector and target fragment is as follows: 20μL assembly system, including Gibson reaction solution, target fragment, linear vector and ultrapure water. When performing recombination reaction, the amount of vector is generally 50-100ng, and the target fragment and vector are generally mixed in a 5:1 molar ratio. React in a PCR instrument at a constant temperature of 50℃ for 1h, then electrotransform into E.coli DH5α, and spread on a 50mg / L kanamycin resistance plate. The positive transformant E.coli DH5α / pBF-cysE ( Figure 1 ).

[0125] ③ Using the shuttle plasmid pBF-cysE as a template, primers cysE-mu-F and cysE-mu-R were used to amplify the plasmid sequence; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 1 min, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0126] ④Same as step ② above. Screen the successful integration positive transformant DH5α / pBF-cysE by colony PCR and sequencing 768 ( Figure 1 ).

[0127] ⑤ With shuttle plasmid pBF-cysE 768 As a template, primers pBF-cysE-linear-F and pBF-cysE-linear-R were used to amplify the backbone sequence of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 3min, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-cdsH plasmid as a template, primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0128] ⑥Same as step ② above. Screen the successful integration positive transformant E.coliDH5α / pBF-P by colony PCR and sequencing. lac -cdsH-cysE 768 ( Figure 1 ).

[0129] ⑦ Using the recombinant plasmid pBF-Plac-cdsH-cysE768 As a template, the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 3 min 30 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0130] ⑧Same as step ② above. Screen the successful integration positive transformant E.coliDH5α / pBF-P by colony PCR and sequencing lac -cdsH-ΔgspA-cysE 768 ( Figure 1 ).

[0131] (2) Integrate the copper heavy metal sensing protein gene and its corresponding inducible promoter into pBF-P lac -cdsH-ΔgspA-cysE 768 The cdsH gene upstream of the plasmid was electroporated into E. coli DH5α to form an engineered strain that can sense copper ( Figure 1 ). The specific construction steps are as follows:

[0132] ① Plasmid pBF-P lac -cdsH-ΔgspA-cysE 768 As a template, primers pBF-cueR-linear-F and pBF-cueR-linear-R were used to amplify the backbone of the plasmid; PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E. coli MG1655 genome as a template, primers cueR-F and cueR-R were used to amplify the cueR gene and P copA The promoter sequence was amplified; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3

[0133] ② The above DNA fragments were excised and purified, and the linear vector and target fragments were assembled and electrotransformed into E. coli DH5α as shown in step (1) ②. The positive transformants E. coli DH5α / pBF-cueR-P that were successfully integrated were screened by colony PCR and sequencing. copA -cdsH-cysE 768 ( Figure 1 ).

[0134] (3) E. coli DH5α / pBF-cueR-P copA -cdsH-cysE 768 S 2- And the ability to precipitate Cu(Ⅱ) is evaluated. The specific steps are as follows:

[0135] ① Store E. coli DH5α / Wide-type and E. coli DH5α / pBF-cueR-P at -80℃ copA -cdsH-cysE 768 The strains were inoculated into 5mL LB medium and cultured overnight at 37°C and 200rpm. The strains cultured overnight were expanded and inoculated into 100mL LB medium and cultured at 37°C and 200rpm for 150min-180min. When the bacterial concentration reached about 0.6-0.8, the bacterial solution was poured into two 50mL centrifuge tubes and centrifuged at 4°C and 5000g for 5min to collect the bacteria. The bacteria were resuspended in M9 medium, added to 50mL of medium, the bacterial concentration was adjusted to about 0.6, and 50μM CuCl2 was added. Lead acetate test paper was attached to the bottle mouth and the bottle mouth was sealed with plastic wrap to prevent the generated H2S gas from escaping. After 6h of shaking culture at 37°C and 200rpm, the lead acetate test paper was successfully observed to turn black ( Figure 4 ).

[0136] ② As in step ①, the growth curve of the engineered strain was measured. The addition of 50 μM CuCl2 had no effect on the growth of the engineered strain ( Figure 8 ).

[0137] ③ As in step ①, after 24 hours of shaking culture at 37°C and 200 rpm, the bacterial solution was successfully observed to change. After centrifugation, a brown precipitate was clearly found ( Figure 4 ).

[0138] ④After culturing for 24 hours as in step ② above, collect 5 mL of bacterial solution in a 10 mL EP tube, centrifuge at 5000 g, 4°C for 5 minutes, and collect the supernatant. Pass the supernatant through a 0.22 μm microporous filter membrane to remove macromolecular organic matter and remaining bacteria. Collect the filtrate, use a 3 kD ultrafiltration tube to remove the remaining metal sulfide nanoparticles in the solution, and prepare a sample for ICP-MS determination of the remaining heavy metal ion concentration. According to the results of the ICP-MS determination, the removal rate of Cu(Ⅱ) [removal rate = (initial concentration of heavy metals in the culture medium - remaining concentration of heavy metals in the culture medium) / initial concentration of heavy metals in the culture medium] was calculated to be 79.8% ( Fig.12 ).

[0139] The test results showed that the engineered strain E. coli DH5α / pBF-cueR-P copA -cdsH-cysE 768 Able to produce S under aerobic conditions 2- When Cu(Ⅱ) was added to the culture medium, after 24 hours of culture, the bacterial solution changed color and brown CuS precipitate appeared. The results of ICP-MS detection also showed that the removal rate of Cu(Ⅱ) by the experimental strain reached about 80%.

[0140] Example 3

[0141] In this embodiment, a "sensing-control-in-one" heavy metal remediation engineering strain that specifically responds to Pb is constructed for accurate perception and precise remediation of environmental heavy metal Pb.

[0142] (1) Construction of shuttle plasmid pBF-cysE

[0143] Plasmid pBF-P was stored in the laboratory lac -B0035-egfp was used as a template, and primers pBF-linear-F and pBF-liner-R were used to amplify the plasmid backbone; the E. coli MG1655 genome was used as a template, and primers cysE-F and cysE-R were used to amplify the cysE gene sequence.

[0144] (2) Construction of shuttle plasmid pBF-cysE 768

[0145] The shuttle plasmid pBF-cysE was used as a template and the plasmid sequence was amplified using primers cysE-mu-F and cysE-mu-R.

[0146] (3) Construction of recombinant plasmid pBF-P lac -cdsH-cysE 768

[0147] shuttle plasmid pBF-cysE 768 Primers pBF-cysE-linear-F and pBF-cysE-linear-R were used as templates to amplify the backbone sequence of the plasmid; primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence using pUC57-cdsH plasmid as a template.

[0148] (4) Construction of recombinant plasmid pBF-P lac -cdsH-ΔgspA-cysE 768

[0149] pBF-P lac -cdsH-cysE 768The plasmid was used as a template and the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R.

[0150] (5) Construction of recombinant plasmid pBF-pbrR-P pbr -cdsH-ΔgspA-cysE 768

[0151] pBF-P lac -cdsH-ΔgspA-cysE 768 The plasmid was used as a template, and the primers pBF-pbrR-linear-F and pBF-pbrR-linear-R were used to amplify the backbone sequence of the plasmid; the pUC57-pbrR plasmid was used as a template, and the primers pbrR-F and pbrR-R were used to amplify the pbrR gene and its downstream P pbr Promoter sequence.

[0152] (6) Using shake flask fermentation, the constructed engineered strain was tested for assimilation of sulfate reduction pathway under aerobic conditions to evaluate its S production 2- And the ability to precipitate Pb(Ⅱ).

[0153] The strains and plasmids used in this example are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0154] The specific implementation steps are as follows:

[0155] (1) Expression of CysE insensitive to cysteine ​​negative feedback inhibition in E. coli WM3064 768 protein and can convert excess cysteine ​​into S 2- CdsH protein, construct S 2- strain ( Figure 1 ). The specific construction steps are as follows:

[0156] ①Preserve plasmid pBF-P in the laboratory lac -B0035-egfp was used as a template, and the plasmid backbone was amplified using primers pBF-linear-F and pBF-liner-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E.coli MG1655 genome as a template, the cysE gene sequence was amplified using primers cysE-F and cysE-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0157] ② The above two fragments were cut and recovered and purified, and the linear vector and the target fragment were assembled by Gibson. The process of Gibson assembly of linear vector and target fragment is as follows: 20μL assembly system, including Gibson reaction solution, target fragment, linear vector and ultrapure water. When performing recombination reaction, the amount of vector is generally 50-100ng, and the target fragment and vector are generally mixed in a 5:1 molar ratio. React in a PCR instrument at a constant temperature of 50℃ for 1h, then electrotransform into E.coli WM3064, and spread on a 50mg / L kanamycin resistance plate. The positive transformant E.coliWM3064 / pBF-cysE( Figure 1 ).

[0158] ③ Using the shuttle plasmid pBF-cysE as a template, primers cysE-mu-F and cysE-mu-R were used to amplify the plasmid sequence; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 1 min, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0159] ④Same as step ② above. Screen the successful integration positive transformant WM3064 / pBF-cysE by colony PCR and sequencing 768 ( Figure 1 ).

[0160] ⑤ With shuttle plasmid pBF-cysE 768 As a template, primers pBF-cysE-linear-F and pBF-cysE-linear-R were used to amplify the backbone sequence of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 3min, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-cdsH plasmid as a template, primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0161] ⑥Same as step ② above. Screen the positive transformants with successful integration by colony PCR and sequencing. lac -cdsH-cysE 768 ( Figure 1 ).

[0162] ⑦ Using the recombinant plasmid pBF-Plac-cdsH-cysE 768 As a template, the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 3 min 30 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0163] ⑧Same as step ② above. Screen the successful integration positive transformant E.coliWM3064 / pBF-P by colony PCR and sequencing. lac -cdsH-ΔgspA-cysE 768 ( Figure 1 ).

[0164] (2) Integrate the lead heavy metal sensing protein gene and its corresponding inducible promoter into pBF-P lac -cdsH-ΔgspA-cysE 768 The cdsH gene upstream of the plasmid was transferred by conjugation to form an engineered strain capable of sensing lead ( Figure 1 ). The specific construction steps are as follows:

[0165] ① Using plasmid pBF-P lac -cdsH-ΔgspA-cysE 768 As a template, primers pBF-pbrR-linear-F and pBF-pbrR-linear-R were used to amplify the backbone of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-pbrR as a template, primers pbrR-F and pbrR-R were used to amplify the pbrR gene and P pbr The promoter sequence was amplified; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0166] ② The above DNA fragments were excised and purified, and the linear vector and target fragment were assembled and electroporated into E. coli WM3064 as shown in step (1) ②. The positive transformants E. coli WM3064 / pBF-pbrR-P with successful integration were screened by colony PCR and sequencing. pbr -cdsH-cysE 768 ( Figure 1 ).

[0167] ③Transfer E.coli WM3064 / pBF-pbrR-P pbr -cdsH-cysE 768 pBF-pbrR-P pbr -cdsH-cysE 768 The plasmid was transferred into S. oneidensis MR-1 to obtain the engineered strain S. oneidensis MR-1 / pBF-pbrR-P pbr -cdsH-cysE 768 .

[0168] (3)S.oneidensis MR-1 / pBF-pbrR-P pbr -cdsH-cysE 768 S 2- And the ability to precipitate Pb(Ⅱ) is evaluated. The specific steps are as follows:

[0169] ① Store S. oneidensis MR-1 / Wide-type and S. oneidensis MR-1 / pBF-pbrR-P at -80℃ pbr -cdsH-cysE 768 The strains were inoculated into 5 mL of LB medium and cultured overnight at 37°C and 200 rpm. The strains cultured overnight were expanded and inoculated into 100 mL of LB medium and cultured at 37°C and 200 rpm for 150 min-180 min. When the bacterial concentration reached about 0.6-0.8, the bacterial solution was poured into two 50 mL centrifuge tubes and centrifuged at 4°C and 5000 g for 5 min to collect the bacteria. The bacteria were resuspended in mineral salt medium, added to 50 mL of medium, the bacterial concentration was adjusted to about 0.6, and 50 μM PbCl2 was added. A lead acetate test paper was attached to the bottle mouth, and the bottle mouth was sealed with plastic wrap to prevent the generated H2S gas from escaping. After 6 hours of shaking culture at 37°C and 200 rpm, the lead acetate test paper was successfully observed to turn black ( Figure 5 ).

[0170] ② As in step ①, the growth curve of the engineered strain was measured. The addition of 50 μM PbCl2 had no effect on the growth of the engineered strain ( Fig. 9 ).

[0171] ③ As in step ①, after 24 hours of shaking culture at 37°C and 200 rpm, the bacterial solution was successfully observed to change. After centrifugation, a black precipitate was clearly found ( Figure 5 ).

[0172] ④After culturing for 24 hours as in step ② above, collect 5 mL of bacterial solution in a 10 mL EP tube, centrifuge at 5000 g, 4°C for 5 min, and collect the supernatant. Pass the supernatant through a 0.22 μm microporous filter membrane to remove macromolecular organic matter and remaining bacteria. Collect the filtrate, use a 3 kD ultrafiltration tube to remove the remaining metal sulfide nanoparticles in the solution, and prepare a sample for ICP-MS determination of the remaining heavy metal ion concentration. According to the results of the ICP-MS determination, the removal rate of Pb (Ⅱ) [removal rate = (initial concentration of heavy metals in the culture medium - remaining concentration of heavy metals in the culture medium) / initial concentration of heavy metals in the culture medium] was calculated to be 99.8% ( Fig.13 ).

[0173] The test results showed that the engineered strain S. oneidensis MR-1 / pBF-pbrR-P pbr -cdsH-cysE 768 Able to produce S under aerobic conditions 2- When Pb(Ⅱ) was added to the culture medium, after 24 hours of culture, the bacterial solution changed color and black PbS precipitate appeared. The results of ICP-MS detection also showed that the removal rate of Pb(Ⅱ) by the experimental strain reached 99.8%.

[0174] Example 4

[0175] This embodiment constructs a "sensing-control-in-one" heavy metal remediation engineering strain that specifically responds to Hg, which is used for the accurate perception and precise remediation of the environmental heavy metal Hg.

[0176] (1) Construction of shuttle plasmid pBF-cysE

[0177] Plasmid pBF-P was stored in the laboratory lac -B0035-egfp was used as a template, and primers pBF-linear-F and pBF-liner-R were used to amplify the plasmid backbone; the E. coli MG1655 genome was used as a template, and primers cysE-F and cysE-R were used to amplify the cysE gene sequence.

[0178] (2) Construction of shuttle plasmid pBF-cysE 768

[0179] The shuttle plasmid pBF-cysE was used as a template and the plasmid sequence was amplified using primers cysE-mu-F and cysE-mu-R.

[0180] (3) Construction of recombinant plasmid pBF-P lac -cdsH-cysE 768

[0181] shuttle plasmid pBF-cysE768 Primers pBF-cysE-linear-F and pBF-cysE-linear-R were used as templates to amplify the backbone sequence of the plasmid; primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence using pUC57-cdsH plasmid as a template.

[0182] (4) Construction of recombinant plasmid pBF-P lac -cdsH-ΔgspA-cysE 768

[0183] pBF-P lac -cdsH-cysE 768 The plasmid was used as a template and the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R.

[0184] (5) Construction of recombinant plasmid pBF-merR-P merT -cdsH-ΔgspA-cysE 768

[0185] pBF-P lac -cdsH-ΔgspA-cysE 768 The plasmid was used as a template, and the primers pBF-merR-linear-F and pBF-merR-linear-R were used to amplify the backbone sequence of the plasmid; the pUC57-merR plasmid was used as a template, and the primers merR-F and merR-R were used to amplify the merR gene and its downstream P merT Promoter sequence.

[0186] (6) Using shake flask fermentation, the constructed engineered strain was tested for assimilation of sulfate reduction pathway under aerobic conditions to evaluate its S production 2- and the ability to precipitate Hg(II).

[0187] The strains and plasmids used in this example are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0188] The specific implementation steps are as follows:

[0189] (1) Expression of CysE insensitive to cysteine ​​negative feedback inhibition in E. coli WM3064 768 protein and can convert excess cysteine ​​into S 2- CdsH protein, construct S 2- strain ( Figure 1 ). The specific construction steps are as follows:

[0190] ①Preserve plasmid pBF-P in the laboratory lac-B0035-egfp was used as a template, and the plasmid backbone was amplified using primers pBF-linear-F and pBF-liner-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E.coli MG1655 genome as a template, the cysE gene sequence was amplified using primers cysE-F and cysE-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0191] ② The above two fragments were cut and recovered and purified, and the linear vector and the target fragment were assembled by Gibson. The process of Gibson assembly of linear vector and target fragment is as follows: 20μL assembly system, including Gibson reaction solution, target fragment, linear vector and ultrapure water. When performing recombination reaction, the amount of vector is generally 50-100ng, and the target fragment and vector are generally mixed in a 5:1 molar ratio. React in a PCR instrument at a constant temperature of 50℃ for 1h, then electrotransform into E.coli WM3064, and spread on a 50mg / L kanamycin resistance plate. The positive transformant E.coliWM3064 / pBF-cysE( Figure 1 ).

[0192] ③ Using the shuttle plasmid pBF-cysE as a template, primers cysE-mu-F and cysE-mu-R were used to amplify the plasmid sequence; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 1 min, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0193] ④Same as step ② above. Screen the successful integration positive transformant WM3064 / pBF-cysE by colony PCR and sequencing 768 ( Figure 1 ).

[0194] ⑤ With shuttle plasmid pBF-cysE 768As a template, primers pBF-cysE-linear-F and pBF-cysE-linear-R were used to amplify the backbone sequence of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 3min, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-cdsH plasmid as a template, primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0195] ⑥Same as step ② above. Screen the positive transformants with successful integration by colony PCR and sequencing. lac -cdsH-cysE 768 ( Figure 1 ).

[0196] ⑦ Using the recombinant plasmid pBF-Plac-cdsH-cysE 768 As a template, the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 3 min 30 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0197] ⑧Same as step ② above. Screen the successful integration positive transformant E.coliWM3064 / pBF-P by colony PCR and sequencing. lac -cdsH-ΔgspA-cysE 768 ( Figure 1 ).

[0198] (2) Integrate the mercury heavy metal sensing protein gene and its corresponding inducible promoter into pBF-P lac -cdsH-ΔgspA-cysE 768 The cdsH gene upstream of the plasmid was transferred by conjugation to form an engineered strain capable of sensing mercury ( Figure 1 ). The specific construction steps are as follows:

[0199] ① Using plasmid pBF-P lac -cdsH-ΔgspA-cysE 768As a template, primers pBF-merR-linear-F and pBF-merR-linear-R were used to amplify the backbone of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-merR as a template, primers merR-F and merR-R were used to amplify the merR gene and P merT The promoter sequence was amplified; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0200] ② The above DNA fragments were excised and purified, and the linear vector and target fragment were assembled and electroporated into E. coli WM3064 as shown in step (1) ②. The positive transformants with successful integration, E. coli WM3064 / pBF-merR-P were screened by colony PCR and sequencing. merT -cdsH-cysE 768 ( Figure 1 ).

[0201] ③Transfer E.coli WM3064 / pBF-merR-P merT -cdsH-cysE 768 pBF-merR-P merT -cdsH-cysE 768 The plasmid was transferred into S. oneidensis MR-1 to obtain the engineered strain S. oneidensis MR-1 / pBF-merR-P merT -cdsH-cysE 768 .

[0202] (3)S.oneidensis MR-1 / pBF-merR-P merT -cdsH-cysE 768 S 2- And the ability to precipitate Hg(Ⅱ) is evaluated. The specific steps are as follows:

[0203] ① Store S. oneidensis MR-1 / Wide-type and S. oneidensis MR-1 / pBF-merR-P at -80℃ merT -cdsH-cysE 768The strains were inoculated into 5 mL of LB medium and cultured overnight at 37°C and 200 rpm. The strains cultured overnight were expanded and inoculated into 100 mL of LB medium and cultured at 37°C and 200 rpm for 150 min-180 min. When the bacterial concentration reached about 0.6-0.8, the bacterial solution was poured into two 50 mL centrifuge tubes and centrifuged at 4°C and 5000 g for 5 min to collect the bacteria. The bacteria were resuspended in mineral salt medium, added to 50 mL of medium, the bacterial concentration was adjusted to about 0.6, and 50 μM HgCl2 was added. Lead acetate test paper was attached to the bottle mouth and the bottle mouth was sealed with plastic wrap to prevent the generated H2S gas from escaping. After 6 hours of shaking culture at 37°C and 200 rpm, the lead acetate test paper was successfully observed to turn black ( Figure 6 ).

[0204] ② As in step ①, the growth curve of the engineered strain was measured. The addition of 50 μM HgCl2 had no effect on the growth of the engineered strain ( Fig. 9 ).

[0205] ③ As in step ①, after culturing at 37°C and 200 rpm for 24 hours, the bacterial solution was successfully observed to change. After centrifugation, an apricot-colored precipitate was clearly found ( Figure 6 ).

[0206] ④After culturing for 24 hours as in step ② above, collect 5 mL of bacterial solution in a 10 mL EP tube, centrifuge at 5000 g, 4°C for 5 min, and collect the supernatant. Pass the supernatant through a 0.22 μm microporous filter membrane to remove macromolecular organic matter and remaining bacteria. Collect the filtrate, use a 3 kD ultrafiltration tube to remove the remaining metal sulfide nanoparticles in the solution, and prepare a sample for ICP-MS determination of the remaining heavy metal ion concentration. According to the results of the ICP-MS determination, the removal rate of Hg (Ⅱ) [removal rate = (initial concentration of heavy metals in the culture medium - remaining concentration of heavy metals in the culture medium) / initial concentration of heavy metals in the culture medium] was calculated to be 82.6% ( Fig.14 ).

[0207] The test results showed that the engineered strain S. oneidensis MR-1 / pBF-merR-P merT -cdsH-cysE 768 Able to produce S under aerobic conditions 2- When Hg(Ⅱ) was added to the culture medium, after 24 hours of culture, the bacterial solution changed color and apricot-colored HgS precipitate appeared. The results of ICP-MS detection also showed that the removal rate of Hg(Ⅱ) by the experimental strain reached 82.6%.

[0208] Example 5

[0209] In this embodiment, a "sensing-control-in-one" heavy metal remediation engineering strain that specifically responds to As is constructed for accurate perception and precise remediation of environmental heavy metal As.

[0210] (1) Construction of shuttle plasmid pBF-cysE

[0211] Plasmid pBF-P was stored in the laboratory lac -B0035-egfp was used as a template, and primers pBF-linear-F and pBF-liner-R were used to amplify the plasmid backbone; the E. coli MG1655 genome was used as a template, and primers cysE-F and cysE-R were used to amplify the cysE gene sequence.

[0212] (2) Construction of shuttle plasmid pBF-cysE 768

[0213] The shuttle plasmid pBF-cysE was used as a template and the plasmid sequence was amplified using primers cysE-mu-F and cysE-mu-R.

[0214] (3) Construction of recombinant plasmid pBF-P lac -cdsH-cysE 768

[0215] shuttle plasmid pBF-cysE 768 Primers pBF-cysE-linear-F and pBF-cysE-linear-R were used as templates to amplify the backbone sequence of the plasmid; primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence using pUC57-cdsH plasmid as a template.

[0216] (4) Construction of recombinant plasmid pBF-P lac -cdsH-ΔgspA-cysE 768

[0217] pBF-P lac -cdsH-cysE 768 The plasmid was used as a template and the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R.

[0218] (5) Construction of recombinant plasmid pBF-arsR-P arsR -cdsH-ΔgspA-cysE 768

[0219] pBF-P lac -cdsH-ΔgspA-cysE 768The plasmid was used as a template, and the primers pBF-arsR-linear-F and pBF-arsR-linear-R were used to amplify the backbone sequence of the plasmid; the pUC57-arsR plasmid was used as a template, and the primers arsR-F and arsR-R were used to amplify the arsR gene and its downstream P arsR Promoter sequence.

[0220] (6) Using shake flask fermentation, the constructed engineered strain was tested for assimilation of sulfate reduction pathway under aerobic conditions to evaluate its S production 2- and the ability to precipitate As(III).

[0221] The strains and plasmids used in this example are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0222] The specific implementation steps are as follows:

[0223] (1) Expression of CysE insensitive to cysteine ​​negative feedback inhibition in E. coli WM3064 768 protein and can convert excess cysteine ​​into S 2- CdsH protein, construct S 2- strain ( Figure 2 ). The specific construction steps are as follows:

[0224] ①Preserve plasmid pBF-P in the laboratory lac -B0035-egfp was used as a template, and the plasmid backbone was amplified using primers pBF-linear-F and pBF-liner-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using the E.coli MG1655 genome as a template, the cysE gene sequence was amplified using primers cysE-F and cysE-R; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0225] ② The above two fragments were cut and recovered and purified, and the linear vector and the target fragment were assembled by Gibson. The process of Gibson assembly of linear vector and target fragment is as follows: 20μL assembly system, including Gibson reaction solution, target fragment, linear vector and ultrapure water. When performing recombination reaction, the amount of vector is generally 50-100ng, and the target fragment and vector are generally mixed in a 5:1 molar ratio. React in a PCR instrument at a constant temperature of 50℃ for 1h, then electrotransform into E.coli WM3064, and spread on a 50mg / L kanamycin resistance plate. The positive transformant E.coliWM3064 / pBF-cysE( Figure 2 ).

[0226] ③ Using the shuttle plasmid pBF-cysE as a template, primers cysE-mu-F and cysE-mu-R were used to amplify the plasmid sequence; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 1 min, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0227] ④Same as step ② above. Screen the successful integration positive transformant WM3064 / pBF-cysE by colony PCR and sequencing 768 ( Figure 2 ).

[0228] ⑤ With shuttle plasmid pBF-cysE 768 As a template, primers pBF-cysE-linear-F and pBF-cysE-linear-R were used to amplify the backbone sequence of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 3min, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-cdsH plasmid as a template, primers cdsH-F and cdsH-R were used to amplify the cdsH gene sequence; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. The PCR reaction system is shown in Table 3.

[0229] ⑥Same as step ② above. Screen the positive transformants with successful integration by colony PCR and sequencing. lac -cdsH-cysE 768 ( Figure 2 ).

[0230] ⑦ Using the recombinant plasmid pBF-Plac-cdsH-cysE768 As a template, the plasmid sequence was amplified using primers move-gpsA-F and move-gpsA-R; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 3 min 30 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0231] ⑧Same as step ② above. Screen the positive transformants with successful integration by colony PCR and sequencing. lac -cdsH-ΔgspA-cysE 768 ( Figure 2 ).

[0232] (2) Integrate the arsenic heavy metal sensing protein gene and its corresponding inducible promoter into pBF-P lac -cdsH-ΔgspA-cysE 768 The cdsH gene upstream of the plasmid was transferred by conjugation to form an engineered strain that can sense arsenic ( Figure 2 ). The specific construction steps are as follows:

[0233] ① Plasmid pBF-P lac -cdsH-ΔgspA-cysE 768 As a template, primers pBF-arsR-linear-F and pBF-arsR-linear-R were used to amplify the backbone of the plasmid; the PCR amplification conditions were: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 2min 30s, a total of 31 cycles, and finally 72℃ extension for 10min. Using pUC57-arsR as a template, primers arsR-F and arsR-R were used to amplify the arsR gene and P arsR The promoter sequence was amplified; the PCR amplification conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, a total of 31 cycles, and finally 72°C extension for 10 min. The PCR reaction system is shown in Table 3.

[0234] ② The above DNA fragments were excised and purified, and the linear vector and target fragment were assembled and electroporated into E. coli WM3064 as shown in step (1) ②. The positive transformants E. coli WM3064 / pBF-arsR-P with successful integration were screened by colony PCR and sequencing. arsR -cdsH-cysE 768 ( Figure 2 ).

[0235] ③Transfer E.coli WM3064 / pBF-arsR-P arsR -cdsH-cysE 768 pBF-arsR-P arsR -cdsH-cysE 768 The plasmid was transferred into S. oneidensis MR-1 to obtain the engineered strain S. oneidensis MR-1 / pBF-arsR-P arsR -cdsH-cysE 768 .

[0236] (3)S.oneidensis MR-1 / pBF-arsR-P arsR -cdsH-cysE 768 S 2- And the ability to precipitate As(III) is evaluated. The specific steps are as follows:

[0237] ① Store S. oneidensis MR-1 / Wide-type and S. oneidensis MR-1 / pBF-arsR-P at -80℃ arsR -cdsH-cysE 768 The strains were inoculated into 5 mL of LB medium and cultured overnight at 37°C and 200 rpm. The strains cultured overnight were expanded and inoculated into 100 mL of LB medium and cultured at 37°C and 200 rpm for 150 min-180 min. When the bacterial concentration reached about 0.6-0.8, the bacterial solution was poured into two 50 mL centrifuge tubes and centrifuged at 4°C and 5000 g for 5 min to collect the bacteria. The bacteria were resuspended in mineral salt medium, added to 50 mL of medium, the bacterial concentration was adjusted to about 0.6, and 50 μM NaAsO2 was added. Lead acetate test paper was attached to the bottle mouth and the bottle mouth was sealed with plastic wrap to prevent the generated H2S gas from escaping. After 6 hours of shaking culture at 37°C and 200 rpm, the lead acetate test paper was successfully observed to turn black ( Figure 7 ).

[0238] ② As in step ①, the growth curve of the engineered strain was measured. The addition of 50 μM NaAsO2 had no effect on the growth of the engineered strain ( Fig. 9 ).

[0239] ③ As in step ①, after 24 hours of shaking culture at 37°C and 200 rpm, the bacterial solution was successfully observed to change. After centrifugation, an orange precipitate was clearly found ( Figure 7 ).

[0240] ④After culturing for 24 hours as in step ② above, collect 5 mL of bacterial solution in a 10 mL EP tube, centrifuge at 5000 g, 4°C for 5 min, and collect the supernatant. Pass the supernatant through a 0.22 μm microporous filter membrane to remove macromolecular organic matter and remaining bacteria. Collect the filtrate, use a 3 kD ultrafiltration tube to remove the remaining metal sulfide nanoparticles in the solution, and prepare a sample for ICP-MS determination of the remaining heavy metal ion concentration. According to the results of the ICP-MS determination, the removal rate of As(Ⅲ) [removal rate = (initial concentration of heavy metals in the culture medium - remaining concentration of heavy metals in the culture medium) / initial concentration of heavy metals in the culture medium] was calculated to be 88.7% ( Fig.15 ).

[0241] The test results showed that the engineered strain S. oneidensis MR-1 / pBF-arsR-P arsR -cdsH-cysE 768 Able to produce S under aerobic conditions 2- When AsO2 is added to the culture medium - After 24 hours of culture, the bacterial solution changed color and orange As2O3 precipitate appeared. The results of ICP-MS also showed that the As(Ⅲ) removal rate of the experimental strain reached 88.7%.

[0242] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for coupling a heavy metal sensing module and an aerobic sulfate reduction module to achieve precise green remediation of environmental heavy metal pollutants, characterized in that: Through synthetic biology strategies, the heavy metal perception module and the aerobic sulfate reduction module are coupled to each other to construct a heavy metal "sensing and controlling integrated" module, which is then used to transform environmental microorganisms and construct heavy metal remediation engineering strains; this engineering strain can specifically activate the expression of the aerobic sulfate reduction module according to the type of environmental heavy metal pollution, thereby converting heavy metal ions into stable metal sulfides, thereby achieving in situ green and low-carbon remediation of environmental heavy metal pollutants.

2. The method according to claim 1, characterized in that The specific steps are as follows: (1) Through gene editing, CysE, which is not affected by cysteine ​​negative feedback inhibition, is 768 The protein combines with the CdsH protein that can convert cysteine ​​into H2S to construct an aerobic sulfate reduction module; (2) Different types of heavy metal sensing protein genes and their corresponding inducible promoters were inserted into recombinant plasmids to construct heavy metal sensing modules to respond to different environmental heavy metal pollutants; (3) The constructed heavy metal sensing module and aerobic sulfate reduction module are coupled into a "sensing and controlling integrated" remediation module, which is then introduced into environmental microorganisms to construct a new sulfate reduction engineering strain; (4) Using this engineered strain, S2- can be efficiently produced under aerobic conditions, thereby converting heavy metal pollutants into insoluble metal sulfides, achieving accurate perception and precise remediation of heavy metals.

3. The method according to claim 2, characterized in that The genetic construction method of the aerobic sulfate reduction module is as follows: cloning the cysE gene from the E. coli genome to construct a shuttle plasmid pBF-cysE; designing primers to mutate the cysE gene, mutating the base G at 768 bp to A, and constructing the shuttle plasmid pBF-cysE 768 ; Synthesize the cdsH gene and integrate it into the shuttle plasmid pBF-cysE 768 , construct the recombinant plasmid pBF-P lac -cdsH-cysE 768 , and removed the endogenous promoter gspA of the cdsH gene to obtain the assimilation sulfate reduction module; the recombinant plasmid was introduced into E. coli DH5α or S. oneidensis MR-1 by electroporation and conjugation transfer for functional verification.

4. The method according to claim 3, characterized in that The genetic construction method of the heavy metal sensing module is: synthesizing or amplifying heavy metal sensing protein genes from different sources and corresponding inducible promoters, constructing a recombinant plasmid pBF-P lac -XP X -egfp, to obtain the heavy metal sensing module; where X represents different types of heavy metal sensing protein genes, P X Represents an inducible promoter regulated by heavy metal sensing protein; the recombinant plasmid was introduced into E.coliDH5α by electroporation and conjugation transfer, and the green fluorescence signal was used to detect the specificity and sensitivity of the sensing protein.

5. The method according to claim 4, characterized in that The constructed heavy metal sensing module can specifically respond to environmental heavy metals such as cadmium, copper, lead, arsenic and mercury.

6. The method according to claim 4, characterized in that The genetic construction method of the heavy metal "sensing and controlling integrated" module is as follows: using homologous recombination method, the constructed heavy metal sensing module and aerobic sulfate reduction module are coupled into a "sensing and controlling integrated" repair module; the recombinant plasmid is introduced into E. coli DH5α or S. oneidensis MR-1 by electroporation and conjugation transfer method, and the integrated repair module is verified; After successful verification, it was transferred to environmental microorganisms through CRISPR gene editing technology to construct a new sulfate-reducing engineered strain.

7. The method according to claim 6, characterized in that The construction method of the heavy metal remediation engineering strain is: using a new sulfate reduction engineering strain to controllably and efficiently generate S2- under aerobic conditions, thereby converting heavy metal pollutants into insoluble metal sulfides, and detecting the removal effect of heavy metal ions.

8. A heavy metal remediation engineering strain constructed according to the method described in any one of claims 1 to 7.

9. The heavy metal remediation engineering strain as claimed in claim 8 is used for the remediation of heavy metals in soil, heavy metals in water bodies, and industrial heavy metal wastewater.

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