Method for improving iron leaching rate of mineral leaching microorganisms, recombinant bacteria and application of recombinant bacteria

Abstract

The invention discloses a method for improving the iron leaching rate of mineral leaching microorganisms, recombinant bacteria and application of the recombinant bacteria, and belongs to the technical field of microbial mineral leaching. The method comprises the step of improving the expression quantity of c-di-GMP synthetase genes of mineral leaching microorganisms or / and improving the content of c-di-GMP synthetase so as to improve the leaching rate of the mineral leaching microorganisms to iron. According to the invention, c-di-GMP synthetase genes AFE-1379, AFE-0053 and AFE-1373 are respectively cloned to a vector pYDT, and strains S-1, S-2 and S-3 with different c-di-GMP concentration multiples increased are further respectively obtained. Compared with a control group, the bioleaching rates of S-1, S-2 and S-3 are respectively 1.43 mM / day, 1.62 mM / day and 1.91 mM / day, which are higher than 1.13 mM / day of the control group. Therefore, the concentration of the intracellular c-di-GMP is improved, and the biological leaching rate can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial leaching, and particularly relates to a method for improving the iron leaching rate of leaching microorganisms, a recombinant bacterium and an application thereof. Background Art

[0002] Bioleaching is an emerging green biotechnology method that uses microorganisms to extract metals from ores. It uses the metabolic activities of microorganisms to dissolve metal ions in ores or metal waste into a solution, thereby recovering the target metal in the solution. Compared with traditional physical and chemical mining methods, the microorganisms used in biomining are renewable energy, low-cost, and do not cause secondary pollution. They are environmentally friendly and have wide applicability. It is suitable for the treatment of low-grade and complex ores and is currently widely used in the extraction of metals such as iron, copper, gold, and uranium. However, bioleaching also has limitations such as long leaching cycles and susceptibility to environmental changes, which hinder its further promotion and application.

[0003] Second messengers are intermediate regulators of intracellular physiological and biochemical processes in response to environmental changes. The cyclic diguanylate (Bis-(3′-5′)-cyclic dimeric GMP, c-di-GMP) signaling network plays a crucial role in regulating bacterial biofilm formation and modulates multiple physiological and biochemical processes. It is one of the most common and important second messengers in bacteria. Summary of the Invention

[0004] The present invention aims to provide an acidic iron-oxidizing bacterium that can increase the bioleaching rate in arsenic-containing environments. The present invention is not limited to the technical subject matter described herein. Other technical subjects not described herein will be readily apparent to those skilled in the art through the following description.

[0005] In previous studies, the inventors discovered that the genome of Acidithiobacillus ferrooxidans contains genes related to regulating the intracellular concentration of c-di-GMP.

[0006] Therefore, by changing and increasing the intracellular c-di-GMP content of acidic iron-oxidizing bacteria, thereby promoting their biofilm formation, the leaching rate of acidic iron-oxidizing bacteria in an arsenic environment can be increased.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for improving the iron leaching rate of leaching microorganisms, comprising increasing the expression level of the second messenger cyclic diguanylate (c-di-GMP) gene of the leaching microorganisms or / and increasing the content of c-di-GMP synthetase synthase to improve the leaching rate of the leaching microorganisms.

[0009] In the above method, the leaching microorganism includes Acidithiobacillus ferrooxidans.

[0010] In the above method, the iron leaching rate refers to Fe 3+ and / or Fe 2+ leaching rate.

[0011] In a second aspect, the present invention provides a recombinant bacterium, which is obtained by increasing the content of c-di-GMP synthetase in a recipient bacterium or / and increasing the expression level of the c-di-GMP synthetase gene; the recipient bacterium is Acidithiobacillus ferrooxidans.

[0012] In the above method or recombinant bacteria, the increase in the c-di-GMP synthetase content of Acidithiobacillus ferrooxidans or / and the increase in the expression level of the c-di-GMP synthetase gene are achieved by introducing the c-di-GMP synthetase gene or its related biological materials into the recipient bacteria, or / and by replacing the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene in the recipient bacteria, or / and by inserting the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene into the genome of the recipient bacteria.

[0013] In the above method or recombinant bacteria, the c-di-GMP synthetase is protein AFE-1379, protein AFE-0053 or / and protein AFE-1373, and the protein AFE-1373 is any one of the following proteins:

[0014] A1-1) amino acid sequence is a protein consisting of 252 amino acid residues encoded by the coding sequence SEQ ID NO: 12;

[0015] A1-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A1-1) and having at least 70% identity with the protein and having the same function;

[0016] A1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A1-1) or A1-2);

[0017] The protein AFE-0053 is any one of the following proteins:

[0018] A2-1) the amino acid sequence is a protein consisting of 344 amino acid residues encoded by the coding sequence SEQ ID NO: 13;

[0019] A2-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A2-1) and having at least 70% identity with the protein and having the same function;

[0020] A2-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A2-1) or A2-2);

[0021] The protein AFE-1379 is any one of the following proteins:

[0022] A3-1) the amino acid sequence is a protein consisting of 54 amino acid residues encoded by the coding sequence SEQ ID NO: 14;

[0023] A3-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A3-1) and having at least 70% identity with the protein of the amino acid sequence and having the same function;

[0024] A3-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by either A3-1) or A3-2).

[0025] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0026] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained.

[0027] The 70% or greater identity may be 75% or greater identity. Specifically, the 75% or greater identity may be 80% or greater identity. Specifically, the 80% or greater identity may be 85% or greater identity. Specifically, the 85% or greater identity may be 90% or greater identity. Specifically, the 90% or greater identity may be 91% or greater identity, 92% or greater identity, 93% or greater identity, 94% or greater identity, 95% or greater identity, 96% or greater identity, 97% or greater identity, 98% or greater identity, or 99% or greater identity. More specifically, the 70% or greater identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0028] In the above method or recombinant bacteria, the gene encoding the protein AFE-1379 is SEQ ID NO: 1.

[0029] In the above method or recombinant bacteria, the gene encoding the protein AFE-0053 is SEQ ID NO: 2.

[0030] In the above method or recombinant bacteria, the gene encoding the protein AFE-1373 is SEQ ID NO: 3.

[0031] In the above method or recombinant bacteria, the biological material is any one of the following:

[0032] B1), a nucleic acid molecule encoding the aforementioned protein;

[0033] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0034] B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2).

[0035] In the above method or recombinant bacteria, the recipient bacteria is Acidithiobacillus ferrooxidans.

[0036] In the above methods or recombinant bacteria, the expression cassette containing a nucleic acid molecule described in B3) refers to a DNA capable of expressing the RNA molecule described above in a host cell. The expression cassette containing a nucleic acid molecule described in B9) refers to a DNA capable of expressing the protein described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary to express the nucleic acid molecule for any of the above proteins or the DNA for the RNA molecule. The regulatory sequences are capable of directing the expression of the coding sequence for any of the above proteins or the DNA for the RNA molecule in a suitable host cell under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligation of the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein or the DNA for the RNA molecule, a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. The promoter sequence contains transcriptional regulatory sequences that mediate expression of the protein or the DNA for the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence can be operably linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence can be operably linked to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the DNA encoding the protein or the RNA molecule into the cellular secretory pathway. Any signal peptide coding region that directs the expressed protein or the DNA of the RNA molecule into the secretory pathway of the selected host cell can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or RNA molecule according to the growth conditions of the host cell. Examples of regulatory sequences are systems that can turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification.

[0037] In the above-mentioned recombinant bacteria, the content of c-di-GMP synthetase or / and the expression level of c-di-GMP synthetase gene of the recombinant bacteria are higher than those of the recipient bacteria.

[0038] In a third aspect, the present invention provides applications, including applications of the aforementioned recombinant bacteria in increasing the leaching rate of pyrite in an arsenic environment or in preparing a product that increases the leaching rate of pyrite in an arsenic environment.

[0039] In the above application, the arsenic in the arsenic-containing environment includes but is not limited to trivalent arsenic and / or pentavalent arsenic, and the environment may specifically be a water environment and a mining environment.

[0040] In a fourth aspect, the present invention also provides a method for constructing a recombinant bacterium, comprising the following steps: introducing the aforementioned c-di-GMP synthetase gene or / and introducing a substance that regulates the expression of the c-di-GMP synthetase gene or / and introducing a substance that regulates the activity or content of the c-di-GMP synthetase into the recipient bacterium to obtain a recombinant bacterium.

[0041] In the above method or recombinant bacteria, the activity or content of c-di-GMP synthetase in the recombinant bacteria is higher than that in the recipient bacteria.

[0042] In the above method, the substance is any one of the following:

[0043] B1), a nucleic acid molecule encoding the aforementioned c-di-GMP synthetase;

[0044] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0045] B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2).

[0046] B1) The nucleotide sequence of the nucleotide molecule is SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 1.

[0047] The above method may specifically include the following steps:

[0048] Step S1, using the genome of the acidic iron-oxidizing bacteria as a template to amplify c-di-GMP synthase genes AFE-1379, AFE-0053, and AFE-1373, respectively, to obtain gene fragments of the corresponding proteins of AFE-1379, AFE-0053, and AFE-1373; the nucleotide sequence of the gene AFE-1379 is shown in SEQ ID NO: 1, the nucleotide sequence of the gene AFE-0053 is shown in SEQ ID NO: 2, and the nucleotide sequence of the gene AFE-1373 is shown in SEQ ID NO: 1;

[0049] Step S2: using the pYDT plasmid as the target DNA, double-digesting with Spe I and Sbf I to obtain a linearized vector fragment pYDT with double sticky ends;

[0050] Step S3, the amplified c-di-GMP synthetase gene AFE-1379, AFE-0053, and AFE-1373 fragments obtained in step S1 are double-digested with Xba I and Sbf I to obtain linear fragments, and then the digested AFE-1379, AFE-0053, and AFE-1373 genes are ligated to the vector fragment pYDT using T4 DNA ligase to obtain recombinant plasmids, namely, vectors pYDT-1379, pYDT-0053, and pYDT-1373;

[0051] Step S4, the vectors pYDT-1379, pYDT-0053, and pYDT-1373 obtained in the previous step were transformed into E. coli BL21, respectively. After verification by bacterial liquid PCR and DNA sequencing, plasmids pYDT-1379, pYDT-0053, and pYDT-1373 overexpressing c-di-GMP synthetase genes AFE-1379, AFE-0053, and AFE-1373 were obtained;

[0052] In step S5, the empty plasmid pYDT and the plasmids obtained in the previous step that overexpress the c-di-GMP synthetase genes AFE-1379, AFE-0053, and AFE-1373, respectively, are transferred to Acidithiobacillus ferrooxidans by combining with Escherichia coli and then cultured to obtain any one of the following strains S-1, S-2, and S-3 with increased leaching rate and biofilm thickness.

[0053] Furthermore, in step S1, the primers used for amplifying the gene AFE-1379 are 1379-F and 1379-R, the nucleotide sequence of the primer 1379-F is shown in SEQ ID NO:4, and the nucleotide sequence of the primer 1379-R is shown in SEQ ID NO:5.

[0054] Furthermore, in step S1, the primers used to amplify the gene AFE-0053 are 0053-F and 0053-R, the nucleotide sequence of the primer 0053-F is shown in SEQ ID NO: 6, and the nucleotide sequence of the primer 0053-R is shown in SEQ ID NO: 7.

[0055] Furthermore, in step S1, the primers used to amplify the gene AFE-1373 are 1373-F and 1373-R, the nucleotide sequence of the primer 1373-F is shown in SEQ ID NO: 8, and the nucleotide sequence of the primer 1373-R is shown in SEQ ID NO: 9.

[0056] Furthermore, in step S5, the culture method is to use 9K culture medium as the basic culture medium, the culture medium pH is 2, ferrous sulfate is used as the electron donor, and oxygen is used as the electron acceptor at 30° C. and 170 rpm.

[0057] The present invention has the following beneficial effects:

[0058] This study reports for the first time that altering the intracellular c-di-GMP content in acidic iron-oxidizing bacteria can promote biofilm formation, thereby increasing the leaching rate in the presence of arsenic. Using synthetic biology methods, the researchers overexpressed the c-di-GMP synthetase genes AFE-1379, AFE-0053, and AFE-1373 to generate engineered acidic iron-oxidizing bacteria strains S-1, S-2, and S-3, which significantly enhance biofilm thickness and pyrite bioleaching rates in the presence of arsenic. The strains S-1, S-2, and S-3 of the present invention formed biofilms of 220.03 μg, 261.45 μg, and 267.19 μg, respectively, which were 1.30 times, 1.54 times, and 1.58 times higher than the control strain PCA-C (169.52 μg). The strains S-1, S-2, and S-3 were able to achieve maximum bioleaching rates of 1.43 mM / day, 1.62 mM / day, and 1.91 mM / day, respectively, which were 1.27 times, 1.43 times, and 1.69 times higher than the control strain SC (1.13 mM / day). Changing the thickness of the biofilm of the leaching microorganisms can significantly increase the bioleaching rate of the bacteria in an arsenic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the structure of the overexpression plasmid of the present invention.

[0060] Figure 2 This is a comparison chart of colony PCR verification of the Acidithiobacillus ferrooxidans engineered strain with the plasmid transferred into it.

[0061] Figure 3 It is a comparison chart of the growth rates of the strains S-1, S-2, S-3 of the present invention and the control strain SC.

[0062] Figure 4 Graph comparing the intracellular c-di-GMP concentrations of the strains S-1, S-2, and S-3 of the present invention and the control strain SC.

[0063] Figure 5 The graph is a comparison of the well plate biofilm formation of the strains S-1, S-2, S-3 of the present invention and the control strain SC.

[0064] Figure 6 This is a comparison chart of the biofilm biomass on the pyrite surface of the strains S-1, S-2, S-3 of the present invention and the control strain SC.

[0065] Figure 7 This is a comparison chart of the iron ion generation rates in the leaching system of the strains S-1, S-2, S-3 of the present invention and the control strain SC.

[0066] Figure 8 This is a comparison chart of sulfate ion generation rates in the leaching system of the strains S-1, S-2, S-3 of the present invention and the control strain SC. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0068] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0069] The original strain (wild-type strain) Acidithiobacillus ferrooxidans ATCC 23270 used in the present invention is a product of ATCC.

[0070] 1. Source of the pYYDT plasmid: recorded in the non-patent literature "Yang, Y., Ding, Y., Hu, Y., et al. (2015). Enhancing bidirectional electron transfer of Shewanella oneidensis by asynthetic flavin pathway. ACS Synth Biol 4(7): 815-823. DOI: 10.1021 / sb500331x", which is publicly available from Peking University. This biological material was used only for repeating the experiments related to the present invention and was not used for other purposes.

[0071] 2. Source of Acidithiobacillus ferrooxidans genomic template: Use the Kangwei Bacterial Genomic DNA Extraction Kit according to the instructions to obtain 50uL of Acidithiobacillus ferrooxidans genomic DNA solution by taking 3mL of Acidithiobacillus ferrooxidans cultured in 9K basal medium with sulfur powder as an electron donor.

[0072] 3. Formula and preparation method of stock solution and culture medium:

[0073] 3.1 LB medium composition formula

[0074] Table 1

[0075] Element concentration yeast extract 5g / L Tryptone 10g / L Sodium chloride 10g / L

[0076] 3.2 9K basal medium composition formula

[0077] Table 2

[0078] Element concentration ammonium sulfate 3g / L Dipotassium hydrogen phosphate 0.5g / L Magnesium sulfate heptahydrate 0.5g / L potassium chloride 0.1g / L calcium nitrate 0.01g / L

[0079] The above 3.2 basic culture medium plus 0.5% sulfur powder is 9K-S culture medium; adding 44.5g / L ferrous sulfate is 9K-iron culture medium.

[0080] 3.3 Solid Binding Medium Components

[0081] Table 3

[0082] Element concentration Sodium thiosulfate pentahydrate 2g / L Ferrous sulfate heptahydrate 2g / L ammonium sulfate 9g / L potassium chloride 0.3g / L Magnesium sulfate heptahydrate 1.5g / L yeast extract 1g / L agar powder 12g / L

[0083] 3.4 Starkey solid medium composition formula

[0084] Table 4

[0085] Element concentration ammonium sulfate 3g / L Potassium dihydrogen phosphate 3g / L Magnesium sulfate heptahydrate 0.5g / L calcium chloride 0.2g / L Sodium thiosulfate pentahydrate 6g / L Ferrous sulfate heptahydrate 0.03g / L agar powder 12g / L

[0086] 3.5 Combined cleaning fluid formulation

[0087] Table 5

[0088] Element concentration ammonium sulfate 4.5g / L potassium chloride 0.15g / L Magnesium sulfate heptahydrate 0.75g / L

[0089] 3.6 Starkey cleaning fluid ingredients formula

[0090] Table 6

[0091] Element concentration ammonium sulfate 3g / L Potassium dihydrogen phosphate 3g / L Magnesium sulfate heptahydrate 0.05g / L calcium chloride 0.165g / L

[0092] Example 1: Construction of Acidithiobacillus ferrooxidans Strain S-1 with Increased Biofilm Thickness and Increased Bioleaching Rate in Arsenic Environments

[0093] The specific process includes:

[0094] Step 1: Amplify the AFE-1379 gene using the genome of the wild-type strain of Acidithiobacillus ferrooxidans as a template to obtain an amplified AFE-1379 gene fragment; the nucleotide sequence of the gene AFE-1379 is shown in SEQ ID NO: 1; the primers used to amplify the gene AFE-1379 are 1379-F and 1379-R, the nucleotide sequence of the primer 1379-F is shown in SEQ ID NO: 4, and the nucleotide sequence of the primer 1379-R is shown in SEQ ID NO: 5.

[0095] The PCR reaction system is:

[0096] Table 7

[0097] Element 50 μL reaction system 2×Es Taq Master Mix 25 μL Forward Primer 10μmol / L 2μL Reverse Primer 10μmol / L 2μL Template DNA 1 μL <![CDATA[dd H2O]]> 20 μL

[0098] PCR reaction conditions are:

[0099] Table 8

[0100] step temperature time Pre-denaturation 98℃ 2min transsexual 98℃ 30s annealing 55-65℃ 10s extend 72℃ 2kb / min Final extension 72℃ 5min

[0101] Step 2. Using the pYYDT plasmid as the target DNA, double enzyme digestion was performed with Spe I and Sbf I at 37°C for 2 h to obtain a linearized vector fragment pYDT with double sticky ends (the enzyme digestion system is shown in Table 9); the amplified c-di-GMP synthetase gene AFE-1379 fragment (no sticky ends) obtained in step 1 was double digested with Xba I (the sticky ends produced by Xba1 and Spe1 digestion are the same, so the fragments digested with spe1 and xba1 can be connected) and Sbf I to obtain a linearized fragment (with sticky ends at both ends). The enzyme digestion system is shown in Table 9, and the plasmid is replaced with the amplified c-di-GMP synthetase gene AFE-1379 fragment (no sticky ends) obtained in step 1.

[0102] The enzyme digestion reaction system is:

[0103] Table 9

[0104] Element 50 μL reaction system Restriction endonuclease I 2μL Restriction endonuclease II 2μL Buffer 5μL plasmids 1 μL <![CDATA[dd H2O]]> 40uL

[0105] Step 3: The AFE-1379 gene fragment with sticky ends obtained in the previous step is ligated with the linearized vector fragment pYDT with sticky ends using T4 DNA ligase to obtain a recombinant plasmid, namely, vector pYDT-1379.

[0106] The ligation reaction system is:

[0107] Table 10

[0108] Element 20 μL reaction system T4 DNA ligase 1 μL Buffer 2μL plasmids 0.02 pmol snippet 0.06 pmol <![CDATA[dd H2O]]> up to 20μL

[0109] Step 4: Transform the recombinant plasmid pYDT-1379 obtained in Step 3 into E. coli DH5α. After verification by PCR and DNA sequencing, plasmid pYDT-1379 overexpressing the c-di-GMP synthase gene AFE-1379 was obtained. The recombinant plasmid pYDT-1379 differs from plasmid pYDT in that the small fragment between the Spe I and Sbf I restriction enzyme recognition sites of plasmid pYDT is replaced with the AFE-1379 gene fragment (SEQ ID NO: 1), while the rest of the nucleotide sequence of plasmid pYDT remains unchanged.

[0110] In step 5, the recombinant plasmid pYDT-1379 obtained in step 3 was transformed into a wild-type strain of Acidithiobacillus ferrooxidans via conjugative transfer in E. coli, resulting in an Acidithiobacillus ferrooxidans strain S-1 that increases biofilm thickness and bioleaching rate in the presence of arsenic. The specific process is as follows: the E. coli strain constructed in step S4 was inoculated into LB medium and cultured at 170 rpm and 37°C until mid-to-late logarithmic growth, at which point the cells were harvested and centrifuged at 5000 rpm at 4°C for 10 minutes. The supernatant was discarded, representing the donor strain. The wild-type strain of Acidithiobacillus ferrooxidans was inoculated into 9K-S liquid medium and cultured at 170 rpm and 30°C until mid-to-late logarithmic growth, at which point the cells were harvested and centrifuged at 5000 rpm at 4°C for 10 minutes. The supernatant was discarded, representing the recipient strain. The donor and recipient strains were each washed twice with conjugated washing solution. After mixing the donor bacteria and the recipient bacteria in a ratio of 1:2, take 100 μl and apply it to the solid binding culture medium with a 0.45 μm binding filter membrane and spread it evenly with a coating rod, and place it in a 30°C incubator for five days. After removing the 0.45 μm binding filter membrane from the plate, place it in a 50 ml centrifuge tube and use 2 ml binding washing solution to blow and wash the bacteria on the filter membrane. Dilute the 2 ml binding washing solution containing the bacteria 10, 100 and 1000 times respectively, take 200 μl of the bacterial solution and apply it to the Starkey solid culture medium containing 200 μg / ml streptomycin, and place it in a 30°C incubator for fifteen days. The grown single colonies were PCR amplified and verified by DNA gel electrophoresis. The amplification primers were pYDT-F and pYDT-R. The nucleotide sequence of pYDT-F is shown in SEQ ID NO: 10, and the nucleotide sequence of pYDT-R is shown in SEQ ID NO: 11. The bacterial solution PCR results are shown in Figure 2The band that ran out was consistent with the target band (SEQ ID NO: 1), and the target S-1 engineered strain was obtained for strain preservation.

[0111] Example 2: Construction of Acidithiobacillus ferrooxidans Strain S-2 with Increased Biofilm Thickness and Increased Bioleaching Rate in Arsenic Environments

[0112] The specific process includes:

[0113] Step 1: Amplify the AFE-0053 gene using the genome of the wild-type strain of Acidithiobacillus ferrooxidans as a template to obtain an amplified AFE-0053 gene fragment; the nucleotide sequence of gene AFE-0053 is shown in SEQ ID NO: 2; primers used to amplify gene AFE-0053 are 0053-F and 0053-R, the nucleotide sequence of primer 0053-F is shown in SEQ ID NO: 6, and the nucleotide sequence of primer 0053-R is shown in SEQ ID NO: 7.

[0114] Step 2: Using the pYDT plasmid as the target DNA, double-digest it with SpeⅠ and SbfⅠ at 37°C for 2 hours to obtain a linearized vector fragment pYDT with double sticky ends; the amplified c-di-GMP synthetase gene AFE-0053 fragment obtained in step 1 was double-digested with XbaⅠ and SbfⅠ to obtain a linearized fragment.

[0115] Step 3: The AFE-0053 gene fragment with sticky ends obtained in the previous step is ligated with the linearized vector fragment pYDT with sticky ends using T4 DNA ligase to obtain a recombinant plasmid, namely, vector pYDT-0053.

[0116] Step S4, transforming the recombinant plasmid pYDT-0053 obtained in step 3 into E. coli DH5α, and verifying the bacterial liquid PCR and DNA sequencing to obtain the plasmid pYDT-0053 overexpressing the c-di-GMP synthase gene AFE-0053; the difference between the recombinant plasmid pYDT-0053 and the plasmid pYDT is that the small fragment between the SpeⅠ and SbfⅠ restriction enzyme recognition sites of the plasmid pYDT is replaced by the AFE-0053 gene fragment (SEQ ID NO: 2), and the other nucleotide sequences of the plasmid pYDT are kept unchanged to obtain the recombinant plasmid (structure as shown in FIG. Figure 1 shown).

[0117] Step 5: The recombinant plasmid pYDT-0053 obtained in step 3 was transformed into the wild-type strain of Acidithiobacillus ferrooxidans by E. coli conjugation transfer to obtain Acidithiobacillus ferrooxidans strain S-2 with increased biofilm thickness and bioleaching rate in an arsenic environment. The specific process is the same as step 5 of Example 1 (the PCR results of the bacterial solution are shown in FIG. Figure 2 ).

[0118] Example 3: Construction of Acidithiobacillus ferrooxidans Strain S-3 with Increased Biofilm Thickness and Increased Bioleaching Rate in Arsenic Environments

[0119] The specific process includes:

[0120] Step 1, amplifying the AFE-1373 gene using the genome of the wild-type strain of Acidithiobacillus ferrooxidans as a template to obtain an amplified AFE-1373 gene fragment; the nucleotide sequence of the gene AFE-1373 is shown in SEQ ID NO: 3; the primers used to amplify the gene AFE-1373 are 1373-F and 1373-R, the nucleotide sequence of the primer 1373-F is shown in SEQ ID NO: 8, and the nucleotide sequence of the primer 1373-R is shown in SEQ ID NO: 9.

[0121] Step 2: Using the pYDT plasmid as the target DNA, double-digest it with SpeⅠ and SbfⅠ at 37°C for 2 hours to obtain a linearized vector fragment pYDT with double sticky ends; the amplified c-di-GMP synthetase gene AFE-1373 fragment obtained in step 1 was double-digested with XbaⅠ and SbfⅠ to obtain a linearized fragment.

[0122] Step 3: The AFE-1373 gene fragment with sticky ends obtained in the previous step is ligated with the linearized vector fragment pYDT with sticky ends using T4 DNA ligase to obtain a recombinant plasmid, namely, vector pYDT-1373.

[0123] Step S4, transforming the recombinant plasmid pYDT-1373 obtained in step 3 into E. coli DH5α, and verifying the bacterial liquid PCR and DNA sequencing to obtain the plasmid pYDT-1373 overexpressing the c-di-GMP synthase gene AFE-1373; the difference between the recombinant plasmid pYDT-1373 and the plasmid pYDT is that the small fragment between the SpeⅠ and SbfⅠ restriction enzyme recognition sites of the plasmid pYDT is replaced by the AFE-1373 gene fragment (SEQ ID NO: 3), and the other nucleotide sequences of the plasmid pYDT are kept unchanged to obtain the recombinant plasmid (structure as shown in FIG). Figure 1 shown).

[0124] Step 5: The recombinant plasmid pYDT-1373 obtained in step 3 was transformed into the wild-type strain of Acidithiobacillus ferrooxidans by E. coli conjugation transfer to obtain Acidithiobacillus ferrooxidans strain S-3 with increased biofilm thickness and bioleaching rate in an arsenic environment. The specific process is the same as step 5 of Example 1 (the results of bacterial solution PCR are shown in Figure 2 ).

[0125] Comparative Example 1: Construction of a biofilm without increasing thickness and without increasing the bioleaching rate in an arsenic environment by Acidithiobacillus ferrooxidans strain SC

[0126] The specific process includes:

[0127] Step 1: The plasmid pYDT was transformed into E. coli DH5α, and the plasmid pYDT-C (structure as shown) without changes in biofilm thickness and electroactivity was obtained by PCR verification and DNA sequencing. Figure 1 shown).

[0128] Step 2: The recombinant plasmid pYDT-C obtained in step 1 was transformed into the wild-type strain of Acidithiobacillus ferrooxidans by E. coli conjugation transfer to obtain Acidithiobacillus ferrooxidans strain SC with no biofilm thickness and no change in bioleaching rate in the presence of arsenic. The specific process is the same as step 5 of Example 1 (the results of bacterial solution PCR are shown in Figure 2 ).

[0129] Example 4. Comparison of strains S-1, S-2, S-3 and SC

[0130] 1. Growth curve determination

[0131] Experimental method: The strain was inoculated into 9K-S liquid culture medium at a 10% inoculum volume ratio and cultured at 170 rpm and 30°C for 12 days. The bacterial concentration was detected by UV spectrophotometer at OD 600 nM on days 0, 2, 4, 6, 8, 10, and 12 after inoculation.

[0132] The experimental results are as follows Figure 3The figure shows the growth curve of the engineered strain of Acidithiobacillus ferrooxidans transformed with the plasmid in 9K-S liquid medium. The concentration changes of the growth medium of different strains in this figure prove that the gene transferred into the engineered strain constructed by the present invention does not affect the growth of the strain.

[0133] 2. Determination of intracellular c-di-GMP levels

[0134] Experimental Method: Bacterial culture was collected after reaching late logarithmic phase, and the OD600 nm was measured. The required volume of culture was calculated and concentrated in PBS to a 1 mL volume with an OD600 nm of 1.8. This 1 mL of culture was centrifuged at 16,000 rcf, 4°C for 2 minutes, the supernatant discarded, and the cells resuspended in 100 μL of ice-cold PBS. The cells were incubated in a 100°C waterbath for 5 minutes. Immediately after incubation, 186 μL of ice-cold ethanol (at this point, the ethanol concentration in the system was 65%) was added, vortexed for 10 seconds, and the cells were centrifuged at 16,000 rcf, 4°C for 2 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube and placed on ice. The cells were resuspended in 100 μL of ice-cold PBS in the original tube containing the pellet, and this step was repeated twice. The pellet from the third round was retained for protein analysis using a BCA kit. The collected supernatant was evaporated using a nitrogen purge device. A white substance containing c-di-GMP was observed adhering to the walls of the EP tube. Resuspend the dried material in 200 μL of ultrapure water and vortex for 10 seconds to dissolve. Filter the sample through a 0.22 μm filter into an HPLC vial. Measure the c-di-GMP concentration using high-performance liquid chromatography (HPLC). The HPLC settings include a gradient elution method. Solution A: 10 mM ammonium acetate in water; Solution B: 10 mM ammonium acetate in methanol. A flow rate of 0.2 mL / min was used throughout the elution. An Inertsustain C18 column (5 μm, 4.6 × 250 nm) was used. The elution gradient was as follows: 1% Solution B from 0 to 9 minutes, 15% Solution B from 9 to 14 minutes, 25% Solution B from 14 to 19 minutes, 90% Solution B from 19 to 26 minutes, and 1% Solution B from 26 to 40 minutes. Each cycle was 40 minutes, with an elution time between 14 and 15 minutes.

[0135] The experimental results are as follows Figure 4As shown, the intracellular c-di-GMP levels of the S-1 strain were 221.51±27.32μg / mg protein, the S-2 strain was 306.35.51±28.12μg / mg protein, and the S-3 strain was 651.44±15.48μg / mg protein, which were approximately 1.7, 2.5, and 5 times higher than the intracellular c-di-GMP levels of the control strain SC (136.91±9.63μg / mg protein), respectively. The differences among the S-1, S-2, and S-3 groups were significant (P>0.01 and P<0.05). These results indicate that the synthase genes AFE-1379, AFE-0053, and AFE-1373 are enzymatically active in Acidithiobacillus ferrooxidans and can effectively increase its intracellular c-di-GMP concentration.

[0136] 3. Comparison of flat biofilm characterization

[0137] 1. Preliminary treatment of biofilm growth on well plates

[0138] The strain was inoculated into 9K-S liquid medium with an inoculation density of 10% by volume. The culture was cultured at 170 rpm and 30°C until the late logarithmic phase. The bacterial solution was collected and the OD was measured. 600nm Calculate the required bacterial solution volume, concentrate and resuspend with PBS to obtain OD 600nm =0.5 resuspended bacteria solution. Inoculate into new 9K-iron medium with 10% inoculation volume (volume ratio) to make the initial inoculation volume OD 600nm =0.05, biofilm formation experiments were performed in 24-well plates, with 1 mL of 9K-iron medium added to each well. The plates were placed in a constant temperature incubator and the biofilm formation was measured at regular intervals.

[0139] Biofilm measurement: Remove the biofilm-grown plate and remove the supernatant. The biofilm will adhere to the bottom of the plate. Gently add 1 mL of 1% gentian violet stain along the edge of the plate. Let stand for 15 minutes, then remove by aspiration. Add 1 mL of PBS to wash the free cells. Repeat this process twice (be gentle to avoid breaking up the biofilm). Add 1 mL of 95% ethanol to dissolve the gentian violet stain bound to the biofilm. Dilute to the appropriate dilution and measure absorbance at OD = 585 nm using a microplate reader.

[0140] The results are as follows Figure 5 As shown in the figure, the biofilm biomass of the S-1, S-2 and S-3 strains were significantly higher than that of the control strain SC (P < 0.05).

[0141] 4. Comparative characterization of pyrite bioleaching in the presence of arsenic

[0142] 1. Pretreatment of pyrite

[0143] (1) The purchased pyrite was ground in a mortar to achieve a smaller particle size and remove the surface oxide layer, and then passed through a 200-mesh sieve to obtain pyrite slag.

[0144] (2) Soak the ground pyrite slag in 1M hydrochloric acid for 24 hours to remove the surface oxide layer to prevent the oxide layer from affecting the experiment.

[0145] (3) The pyrite slag was placed in an anaerobic glove box to prevent oxidation, and the acid-leached pyrite slag was washed twice with 75% ethanol and then washed twice with oxygen-free sterile water to remove the ethanol attached to the mineral.

[0146] (4) The cleaned pyrite slag is sterilized with ultraviolet light in an anaerobic glove box and then dried before use.

[0147] 2. Construction of pyrite leaching system

[0148] Sodium arsenite solution: Sodium arsenite powder (purchased from the Reagent Management Platform of Peking University) was dissolved in water to prepare a 5 M stock solution.

[0149] Collect the bacterial solution cultured in 9K-S medium until the late logarithmic phase and measure the OD 600nm Calculate the required bacterial solution volume, concentrate and resuspend with PBS to obtain OD 600nm =0.5 resuspended bacteria solution. Inoculate into new 9K basal medium with 10% (volume ratio) inoculation volume, so that the initial inoculation volume is OD 600nm =0.05. The pyrite slag described in "1. Pretreatment of Pyrite" was then added to the 9K basal medium at a slurry density of 10% (e.g., 10 g of pyrite slag per 100 ml of medium), and sodium arsenite solution was added. This established a pyrite water chemical leaching system in the presence of arsenic. The arsenic concentration in the pyrite water chemical leaching system in the presence of arsenic was 5 mM.

[0150] 3. Measurement of iron ions and sulfate ions

[0151] Pyrite is FeS2. As iron is oxidized and precipitated by microorganisms, sulfur ions are also oxidized and precipitated in the solution as sulfate ions. While the iron leaching rate mentioned above is affected by arsenic, the sulfate leaching rate here is not affected by precipitation, so the sulfate ion leaching rate is used to represent the bioleaching rate. Determination of sulfate ion content in solution: The supernatant from the leaching system was collected at fixed intervals (0 days, 6 days, 12 days, 18 days, 24 days, 30 days, 36 days, and 42 days) and filtered for impurities using a 0.22μm aqueous filter. A 10μL sample of the supernatant was diluted 500-fold with ultrapure water to 5ml and added to an ion chromatograph tube. The sulfate ion concentration was measured using an ion chromatograph. The leaching rate of sulfate ion = (sulfate concentration in the pyrite water chemical leaching system under the arsenic environment on the Nth day - sulfate concentration in the pyrite water chemical leaching system under the arsenic environment on the 0th day) / N, where N is 0, 6, 12, 18, 24, 30, 36, or 42.

[0152] The total iron ions leached into the solution (Fe 3+ and / or Fe 2+ Content Determination: At regular intervals (0 days, 6 days, 12 days, 18 days, 24 days, 30 days, 36 days, and 42 days), collect the supernatant from the leaching system and filter out impurities using a 0.22 μm aqueous filter. Transfer 38 μL of the supernatant to a 2 mL centrifuge tube, add 1.5 mL of phenanthroline solution containing hydroxylamine hydrochloride, mix thoroughly, and transfer to a cuvette. Measure the absorbance at 562 nm using a UV spectrophotometer. Calculate the total iron concentration using the standard curve. The iron ion leaching rate = sulfate ion leaching rate / 2.

[0153] 4. Measurement of biofilm protein content

[0154] Place the mineral sample to be tested in a 50ml centrifuge tube, add 5ml of 0.2M sodium hydroxide, and heat at 98°C for 1 hour. After heating, centrifuge and collect the supernatant as the test solution. After cooling to room temperature, use the BCA protein kit to measure the total amount of biofilm protein. Because iron-containing minerals can interfere with BCA quantitative analysis, acetone precipitation is required to remove interfering substances before measurement with the BCA kit. The specific steps are as follows:

[0155] (1) Pipette 50 μl of protein standards (including blanks) and samples into 1.5 ml centrifuge tubes in triplicate.

[0156] (2) Add 200 μl of pre-cooled acetone to each centrifuge tube, vortex to mix evenly, and incubate at -20°C for 30 min.

[0157] (3) Centrifuge at maximum speed in a microcentrifuge for 10 min and discard the supernatant.

[0158] (4) After evaporating the acetone at room temperature, the clean protein pellet in the centrifuge tube is added with 50 μl of ultrapure water and vortexed to dissolve the pellet. This is the purified sample.

[0159] (5) Place 50 μl of each protein standard (including blank) and purified sample in a centrifuge tube. Add 1 ml of BCA working solution and vortex to mix thoroughly. Incubate at 37°C for 30 min and measure the absorbance at 562 nm using a microplate reader.

[0160] (6) The sample protein concentration can be calculated based on the standard curve.

[0161] The results are as follows Figure 6 The figure shows a comparison of the biofilm protein amounts formed on pyrite by strains S-1, S-2, and S-3 of the present invention and the control strain SC. The biofilm protein amount of strain S-1 was 220.03±1.39μg; the biofilm protein amount of strain S-2 was 261.45±3.03μg; and the biofilm protein amount of strain S-3 was 267.19±10.15μg, which were approximately 1.30, 1.54, and 1.58 times higher than the biofilm protein amount of the control strain SC (169.52±6.00μg), respectively. The differences between the S-1, S-2, and S-3 groups were significant (P<0.05). The results showed that the transfer of the synthase genes AFE-1379, AFE-0053, and AFE-1373 into the wild-type strain of Acidithiobacillus ferrooxidans effectively promoted the formation of biofilms on pyrite surfaces in the presence of arsenic.

[0162] like Figure 7 The figure shows a comparison of the iron ion generation rates of the strains S-1, S-2, S-3 and the control strain SC in the leaching system. The S-1 strain catalyzed the leaching rate of iron ions in the leaching system at 0.58±0.05mM / day; the S-2 strain catalyzed the leaching rate of iron ions in the leaching system at 0.66±0.08mM / day; and the S-3 strain catalyzed the leaching rate of iron ions in the leaching system at 0.76±0.09mM / day, which were higher than the 0.47±0.03mM / day leaching rate of the control strain SC in the leaching system. The differences among the S-1, S-2 and S-3 groups were significant (P<0.05).

[0163] like Figure 8The figure shows a comparison of sulfate ion generation rates in the leaching system of the strains S-1, S-2, S-3 of the present invention and the control strain SC. The leaching rate of sulfate ions catalyzed by the S-1 strain in the leaching system was 1.43±0.11mM / day; the leaching rate of sulfate ions catalyzed by the S-2 strain in the leaching system was 1.62±0.24mM / day; and the leaching rate of sulfate ions catalyzed by the S-3 strain in the leaching system was 1.91±0.18mM / day, which were higher than the leaching rate of sulfate ions catalyzed by the control strain SC of 1.13±0.06mM / day in the leaching system, and the differences among the S-1, S-2 and S-3 groups were significant (P<0.05). The results showed that the transfer of synthase genes AFE-1379, AFE-0053, and AFE-1373 into Acidithiobacillus ferrooxidans could effectively promote the bioleaching rate of pyrite in the presence of arsenic.

[0164] In summary, the engineered strains S-1, S-2, and S-3 constructed in the present invention increased the formation of biofilm on the surface of pyrite and the bioleaching rate in an arsenic environment by overexpressing the c-di-GMP synthetase genes AFE-1379, AFE-0053, and AFE-1373, respectively.

[0165] Any matters not mentioned above shall be subject to the existing technology.

[0166] The above examples involve the following sequences:

[0167] SEQ ID NO: 1

[0168] 5'-ttaagccaatgaatgaccaaaatgttttcttttcgccaaggggaaaccgggacaccgttccggcatcgtgggcagtgattccggcatcgtgggcaccgattccggaaaacccacaaaagtgcccacgatcaatcgtaatggctgcccacgatcaatcggaatcac-3'.

[0169] SEQ ID NO: 2 (1035 bp)

[0170]

[0171] SEQ ID NO:3(759bp)

[0172] 5'-tcacgatgggtcaagttttggtgtttcgtgagcatcacgatgacggcgcagggagtcccctttcaaatcgagtcggtgggcactttgcagcaaccggtccagcaccgcatcggccacggtcggttcacccaagtattcgtgccaatggtccacagggagttggctagtaattagcgtggctcgttggttcacccgatcctcaatgatctccaacaaatctcgcacgtcttccccgttcatgggggcaagcccccagtcgtctaagataatcaaatccaccttggcgagcgtattcagatagcgaccgaagctgccgtcaccgtgacgaatccggagctcctcaaataggcgcggtaagcgcaaataacgaacgcttaatccctgacggcaggcctggtggcccagggcacagcccagccaggttttgccggtgccggtggcgccagtaatcagcagattatggccttgccgaatccaggtgttctggatcagtgccatcaccttggatcggtccagtccacgggagccgcgatagtcgatatcctccacacaggcattctgcttgagatgggcgagcttcagcaggcgcgtcagacggcgttggtcccgggcacagagttctcggtccaggagcagacccagacgctcctcgaaggagagatcctggatctgggggagacgagattgttcgtccagagcctcagccatggcgtagagattgagctgacgcagcgcgttcagagtgggttgttgtaacat-3'。

[0173] SEQ ID NO:4

[0174] 5'-cttctagacctactagagaaagaggagaaatactagagttaagccaatgaatgaccaaaatg-3'。

[0175] SEQ ID NO:5

[0176] 5'-accacctgcaggcggcgtgattccgattgatcgtggg-3'。

[0177] SEQ ID NO:6

[0178] 5'-cttctagacctactagagaaagaggagaaatactagagtcaacgggcttccatctcgt-3'。

[0179] SEQ ID NO:7

[0180] 5'-accacctgcaggcggcatgttcggggcgattctgc-3'。

[0181] SEQ ID NO:8

[0182] 5'-cttctagacctactagagaaagaggagaaatactagagtcacgatgggtcaagttttggt-3'。

[0183] SEQ ID NO:9

[0184] 5'-accacctgcaggcggctgttacaacaacccactctgaa-3'。

[0185] SEQ ID NO:10

[0186] 5'-gcctcaggcatttgagaagcaca-3'。

[0187] SEQ ID NO:11

[0188] 5'-cgctcagaagaactcgtcaa-3'。

[0189] SEQ ID NO:12

[0190] MLQQPTLNALRQLNLYAMAEALDEQSRLPQIQDLSFEERLGLLLDRELCARDQRRLTRLLKLAHLKQNACVEDIDYRGSRGLDRSKVMALIQNTWIRQGHNLLITGATGTGKTWLGCALGHQACRQ GLSVRYLRLPRLFEELRIRHGDGSFGRYLNTLAKVDLIILDDWGLAPMNGEDVRDLLEIIEDRVNQRATLITSQLPVDHWHEYLGEPTVADAVLDRLLQSAHRLDLKGDSLRRHRDAHETPKLDPS.

[0191] SEQ ID NO:13

[0192] MFGAILQYARLNRFNTIAGMAVLQNLAVAKTMAFTVGLGMLLLQSEILLGWADYHVKPLILVGVVVGGLLFGVGMAVLGYCPGTVAISLGQGNLDALVGIIGGLCGALVFAVVYPSLNPLLGPNLGALSVRSLLPDDILFWSVTSAVAILFMGIALYLQKLQHHDWRWLHAA VGLALLNCVLTLPFVAGHPMGASTAFPYAAMTITELGAESYQAAIAAPGAWELWFLTGAFLAGLVFALLHRSFRISSVPELWVRYHGPKPAKRFFWAFVGGFLLLFGARMAGGCTSGHVISGGMQLAISSLVFAVVVFAAFLTTGHYFYRIRQAVPIPPSPRIVEVNEMEAR.

[0193] SEQ ID NO:14

[0194] MIPIDRGQPLRLIVGTFVGFPESVPTMPESLPTMPERCPGFPLAKRKHFGHSLA.

[0195] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for improving the iron leaching rate of a leaching microorganism, comprising increasing the expression level of a c-di-GMP synthetase gene of the leaching microorganism or / and increasing the content of c-di-GMP synthetase to improve the leaching rate of the leaching microorganism.

2. The method according to claim 1, characterized in that The leaching microorganisms include acidithiobacillus ferrooxidans.

3. The method according to claim 1 or 2, characterized in that The increasing of the c-di-GMP synthetase content in the leaching microorganism or / and the increasing of the expression of the c-di-GMP synthetase gene are achieved by introducing the c-di-GMP synthetase gene or its related biological materials into the recipient bacteria, or / and by replacing the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene in the recipient bacteria, or / and by inserting the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene into the genome of the recipient bacteria.

4. The method according to claim 3, characterized in that The c-di-GMP synthetase is protein AFE-1379, protein AFE-0053 or / and protein AFE-1373, and the protein AFE-1373 is any one of the following proteins: A1-1) amino acid sequence is a protein consisting of 252 amino acid residues encoded by the coding sequence SEQ ID NO: 12; A1-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A1-1) and having at least 70% identity with the protein and having the same function; A1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A1-1) or A1-2); The protein AFE-0053 is any one of the following proteins: A2-1) the amino acid sequence is a protein consisting of 344 amino acid residues encoded by the coding sequence SEQ ID NO: 13; A2-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A2-1) and having at least 70% identity with the protein and having the same function; A2-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A2-1) or A2-2); The protein AFE-1379 is any one of the following proteins: A3-1) the amino acid sequence is a protein consisting of 54 amino acid residues encoded by the coding sequence SEQ ID NO: 14; A3-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A3-1) and having at least 70% identity with the protein and having the same function; A3-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by either A3-1) or A3-2).

5. A recombinant bacterium, characterized in that The recombinant bacteria are obtained by increasing the content of c-di-GMP synthetase in a recipient bacterium or / and increasing the expression level of a c-di-GMP synthetase gene; the recipient bacterium is a mineral leaching microorganism (Acidithiobacillus ferrooxidans).

6. The recombinant bacterium according to claim 5, characterized in that The increasing of the c-di-GMP synthetase content in the recipient bacteria or / and the increasing of the expression of the c-di-GMP synthetase gene are achieved by introducing the c-di-GMP synthetase gene or its related biological materials into the recipient bacteria, or / and by replacing the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene in the recipient bacteria, or / and by inserting the regulatory sequence that regulates the expression of the c-di-GMP synthetase gene into the genome of the recipient bacteria.

7. The recombinant bacterium according to claim 5 or 6, characterized in that The c-di-GMP synthetase is protein AFE-1379, protein AFE-0053 or / and protein AFE-1373, and the protein AFE-1373 is any one of the following proteins: A1-1) amino acid sequence is a protein consisting of 252 amino acid residues encoded by the coding sequence SEQ ID NO: 12; A1-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A1-1) and having at least 70% identity with the protein and having the same function; A1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A1-1) or A1-2); The protein AFE-0053 is any one of the following proteins: A2-1) the amino acid sequence is a protein consisting of 344 amino acid residues encoded by the coding sequence SEQ ID NO: 13; A2-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A2-1) and having at least 70% identity with the protein and having the same function; A2-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by any one of A2-1) or A2-2); The protein AFE-1379 is any one of the following proteins: A3-1) the amino acid sequence is a protein consisting of 54 amino acid residues encoded by the coding sequence SEQ ID NO: 14; A3-2) a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of A3-1) and having at least 70% identity with the protein and having the same function; A3-3) A fusion protein having the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid represented by either A3-1) or A3-2).

8. The recombinant bacterium according to claim 7, characterized in that The substance is any of the following: B1), a nucleic acid molecule encoding the protein according to claim 7; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2).

9. Application, characterized in that The application includes the use of the recombinant bacteria according to any one of claims 5 to 8 in increasing the leaching rate of pyrite in an arsenic environment or in preparing a product that increases the leaching rate of pyrite in an arsenic environment.

10. A method for constructing a recombinant bacterium, characterized in that: The method comprises the following steps: introducing the c-di-GMP synthetase gene according to claim 7 or / and introducing a substance that regulates the expression of the c-di-GMP synthetase gene or / and introducing a substance that regulates the activity or content of the c-di-GMP synthetase into a recipient bacterium to obtain a recombinant bacterium.

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