An engineered strain of *Thiobacillus denitrification*, its preparation method and application

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a denitrifying thiobacillus (… T. denitrificans Engineered strains, their preparation methods, and applications. The *Thiobacillus denitrificationis* engineered strain described in this invention is an overexpression... act The denitrifying thiobacillus gene, the act The gene encodes an enzyme that synthesizes AHL signaling molecules, the nucleotide sequence of which is shown in SEQ ID NO:1. The produced signaling molecule is 3-OH-C. 14 -AHL. The engineered strain of *Thiobacillus denitrificationus* can sustainably synthesize 3-OH-C. 14 -AHL signaling molecules significantly accelerate the denitrification rate of denitrifying thiobacilli, shorten the denitrification start-up time, and improve the removal efficiency of nitrates or nitrites. They are applicable to various scenarios such as municipal sewage, aquaculture wastewater, and industrial nitrate wastewater, and have broad prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an engineered strain of Thiobacillus denitrification, its preparation method, and its application. Background Technology

[0002] Denitrifying Thiobacillus ( Thiobacillus denitrificans abbreviation T. denitrificans As a typical autotrophic denitrifying microorganism, it can use substances such as sulfides and thiosulfates as electron donors to convert nitrates (NO3) into nitrogen. - The sulfur-containing gas is reduced to nitrogen (N2), and is therefore widely used for the removal of nitrogen and sulfur from wastewater. Although this strain has been practically applied in the field of advanced nitrogen removal in wastewater, its sulfur autotrophic metabolism efficiency is relatively low, and its adaptability to environmental conditions is weak, resulting in treatment effects that are difficult to achieve as expected. Existing research indicates that optimizing sulfur metabolism pathways can indirectly promote nitrogen metabolism. At the same time, quorum sensing (QS) has also attracted attention as another key pathway for regulating nitrogen metabolism. Among various functional bacteria related to denitrification, the QS system, with acyl-homoserine lactones (AHLs) as signaling molecules, dominates. Its mechanism of action includes: promoting bacterial proliferation and biofilm formation by regulating the expression of specific genes, thereby improving bacterial activity and growth, enhancing biological denitrification, and promoting the formation of bioaggregates. It is considered an important regulatory strategy for optimizing the biological denitrification process.

[0003] Currently, there are no research reports on the identification of endogenous signaling molecules in *Thiobacillus denitrifyingus*, which greatly limits the application prospects of artificially regulating the denitrification process of this bacterium based on the QS system. In addition, exogenous signaling molecules are not easy to obtain and the process is complicated. There are no reports on the application of strains with quorum sensing behavior secreting signaling molecules to regulate the physiological characteristics of microbial communities and thereby improve the denitrification performance of *Thiobacillus denitrifyingus* itself. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an engineered strain of Thiobacillus denitrification, its preparation method and application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a denitrifying thiobacillus ( T. denitrificans The engineered strain of *Thiobacillus denitrificationus* is an acyltransferase overexpressing... act The denitrifying thiobacillus gene, the act The gene encodes an enzyme that synthesizes AHL signaling molecules; actThe gene sequence is shown in SEQ ID NO: 1; the AHLs signaling molecule is 3-hydroxytetradecanoyl-homoserine lactone (N-(3-Hydroxytetradecanoyl)-L-homoserinelactone, 3-OH-C 14 -AHL).

[0006] Furthermore, the denitrifying thiobacillus is T. denitrificans DSM 12475.

[0007] In a second aspect, the present invention provides a method for preparing the engineered strain of *Thiobacillus denitrificationus* as described in the first aspect, comprising the following steps: 1) Using the extracted genomic DNA of *Thiobacillus denitrificationus* as a template, PCR amplification was performed using primer pairs to obtain... act The target fragment; simultaneously, using the synthesized Ptac promoter fragment as a template, PCR amplification was performed using primer pairs to obtain the Ptac promoter fragment; the Ptac promoter fragment and the... act The target fragment is ligated into the plasmid via homologous recombination to obtain a recombinant expression plasmid. 2) Transform the recombinant expression plasmid described in step 1) into competent cells to obtain donor bacteria; use the donor bacteria and recipient bacteria... T. denitrificans The recombinant expression plasmid was introduced into the conjugation transfer process. T. denitrificans In this process, the engineered strain of Thiobacillus denitrification was obtained.

[0008] Furthermore, amplification act The primer pair used for the target fragment is the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4.

[0009] Furthermore, the primer pair used to amplify the Ptac promoter fragment is the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6.

[0010] Thirdly, the present invention provides the application of the engineered strain of *Thiobacillus denitrificationus* described in the first aspect in the preparation of products for accelerating the denitrification of *Thiobacillus denitrificationus*.

[0011] In some specific embodiments of the present invention, the product includes bacterial liquid, bacterial powder or biological agent, the active ingredient of which is obtained by fermentation culture of the engineered strain of Thiobacillus denitrification.

[0012] Fourthly, the present invention provides a method for deep denitrification of nitrogen-containing wastewater, the method comprising inoculating the nitrogen-containing wastewater with the engineered strain of denitrifying thiobacillus described in the first aspect.

[0013] The beneficial effects of this invention are: 1) This invention is the first to construct a signaling molecule (3-OH-C) capable of generating AHLs. 14 A denitrifying thiobacillus engineered strain (-AHL). This engineered strain was developed through overexpression... act Genes that can sustainably synthesize 3-OH-C 14 -AHL signaling molecules significantly accelerate the denitrification rate of denitrifying thiobacilli, shorten the denitrification start-up time, and improve the removal efficiency of nitrates or nitrites.

[0014] 2) This invention provides a method for preparing a highly efficient and stable strain of *Thiobacillus denitrifyingus* that produces AHL signaling molecules. Using plasmid pJRD215, the strain was successfully prepared via conjugation transfer. act Gene transfer to *Thiobacillus denitrificationus* provides a reliable operational platform for the molecular breeding and metabolic engineering of *Thiobacillus denitrificationus*.

[0015] 3) This invention uses the engineered strain of *Thiobacillus denitrificationus* that produces AHLs signaling molecules as the active ingredient, and obtains bacterial solution, bacterial powder, or biological agent after fermentation. 3-OH-C 14 -AHL can significantly promote T.denitrificans Proliferation of DSM 12475: OD on day 4 of 1 μM culture 600 The concentration reached 0.445, and the growth activity was significantly better than the 10 nM, 100 nM groups and the blank control group. Regarding nitrite degradation, 1 μM 3-OH-C... 14 - In the AHL group, nitrite levels dropped to 0 mg / L on day 4, achieving zero nitrite nitrogen one day earlier than other groups and the control group. Furthermore, 3-OH-C 14 The higher the concentration of -AHL, the more significant the promotion of nitrate nitrogen and total nitrogen metabolism. After 7 days of culture, the final concentrations of nitrate nitrogen and total nitrogen in the 1 μM group were much lower than those in the 10 nM, 100 nM groups and the blank control group. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 For the identification using LC-MS-MS technology in Embodiment 1 of the present invention act Synthesized AHLs plots; where, Figure 1 A is the standard 3-OH-C 14 -Schematic diagram of the secondary structure of AHL mass spectrometry; Figure 1 B is a wild-type (WT) strain. T. denitrificans Schematic diagram of AHL mass spectrometry generation; Figure 2The recombinant plasmid pJRD215-Ptac- T.denitrificans-act The results of promoter and target fragment assays determined by DNA gel electrophoresis, as well as colony PCR verification results of overexpressing strains, are shown in the figure. Figure 2 A is the banding result of the Ptac promoter (Lane M: DNA marker; Lanes 1-2: Ptac; Lane 3: control Ptac). Figure 2 B is the detection act Banding results of the target fragment (Lane M: DNA marker; Lanes 1-2: ...). act (Target segment); Figure 2 C is the result of colony PCR verification using primers 215-F3 / 215-R1; Figure 3 This is a diagram showing the PCR verification of the engineered strain using primer YZTD-F / 215-R2; among them, Figure 3 A is the validation overexpression strain. T. denitrificans DSM (pJRD215-Ptac-) T. denitrificans-act The banding results (Lane M: DNA marker; Lanes 1-2: overexpressing strains) T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act Lane 3: T. denitrificans DSM 12475; Lane 4: plasmid pJRD215-Ptac- T. denitrificans-act , Figure 3 B is the validation control strain. T. denitrificans Banding results for DSM 12475 (pJRD215) (Lane M: DNA marker; Lane 1: ...). T. denitrificans DSM 12475 (pJRD215); Lane 2: plasmid pJRD215); Figure 4 To add different concentrations of 3-OH-C 14 -AHL T. denitrificans The growth curve. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the specific embodiments described below are merely examples and are not intended to limit the invention.

[0019] Unless otherwise specified, the materials, reagents, strains, plasmids, etc. used in the following examples were obtained commercially.

[0020] This invention targets commercially available wastewater desulfurization and denitrification products. T. denitrificans DSM 12475 for the identification of signaling molecules, anti-addition, and quorum sensing systems. act Genetic engineering modifications to achieve faster denitrification rates and higher cultivation densities are beneficial for practical applications. The invention utilizes... T. denitrificans DSM 12475 was purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd.

[0021] Escherichia coli DH5α ( Escherichia coli DH5α, E.coli DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd.; Escherichia coli SM10 ( Escherichia coli SM10, E.coli SM10 (i.e., Escherichia coli SM10 competent cells) was published in (Simon et al. 1983), and the specific publication information is as follows: SIMON R, PRIEFER U AND PUHLER A. 1983. A broad host mobilizationsystem for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria. Bio / Technolgy 1:37-45. Acidophilic thermophilic bacteria ( Acidithiobacillus caldus , A. caldus MTH-04 is a strain previously disclosed in the applicant's patent and is deposited at the China General Microbiological Culture Collection Center (accession number CGMCC 1.15711).

[0022] First, this invention provides a denitrifying thiobacillus ( T. denitrificans The engineered strain, wherein the *Thiobacillus denitrificationus* engineered strain is an overexpressing strain. act The denitrifying thiobacillus gene, the act The gene encodes an enzyme that synthesizes AHL signaling molecules; act The gene sequence is shown in SEQ ID NO: 1; the AHLs signaling molecule is 3-OH-C. 14 -AHL.

[0023] In some specific embodiments of the present invention, the denitrifying thiobacillus is... T. denitrificans DSM12475.

[0024] In this invention, the term " actThe "gene" refers to the gene encoding the synthase of AHL signaling molecules, whose expression product can catalyze the synthesis of 3-OH-C. 14 -AHL signal molecules. act The gene can originate from *Thiobacillus denitrificationus* itself (i.e., the endogenous AHLs signaling molecule synthase gene of *Thiobacillus denitrificationus*) or from other microorganisms (i.e., the heterologous AHLs signaling molecule synthase gene). For example, the aforementioned act The gene can be endogenous in denitrifying thiobacterium. act The gene (whose nucleotide sequence is shown in SEQ ID NO: 1) can also be derived from other bacterial species (such as...). Acidithiobacillus caldus The heterogeneous source of MTH-04 act The gene (its nucleotide sequence is shown in SEQ ID NO: 11). Whether derived from the organism itself or from a foreign source, as long as overexpression of this gene in *Thiobacillus denitrificationis* enables the strain to produce 3-OH-C... 14 -AHL signaling molecules, and thereby accelerate the denitrification process of denitrifying thiobacilli, are all within the scope of protection of this invention.

[0025] This invention provides a method for preparing the engineered strain of *Thiobacillus denitrificationus*, comprising the following steps: 1) Using the extracted genomic DNA of *Thiobacillus denitrificationus* as a template, PCR amplification was performed using primer pairs to obtain... act The target fragment; simultaneously, using the synthesized Ptac promoter fragment as a template, PCR amplification was performed using primer pairs to obtain the Ptac promoter fragment; the Ptac promoter fragment and the... act The target fragment is ligated into the plasmid via homologous recombination to obtain a recombinant expression plasmid. 2) Transform the recombinant expression plasmid described in step 1) into competent cells to obtain donor bacteria; use the donor bacteria and recipient bacteria... T. denitrificans The recombinant expression plasmid was introduced into the conjugation transfer process. T. denitrificans In this process, the engineered strain of Thiobacillus denitrification was obtained.

[0026] Furthermore, amplification act The primer pair used for the target gene fragment is the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4.

[0027] Furthermore, the primer pair used to amplify the Ptac promoter fragment is the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6.

[0028] In some specific embodiments of the present invention, in step 1), the homologous recombination method is as follows: the plasmid pJRD215 is double-digested with restriction endonucleases Kpn I / Xba I and Xba I / Hind III to obtain a linearized plasmid; act The target fragment, the Ptac promoter fragment, and the linearized plasmid were ligated to construct the recombinant plasmid.

[0029] In some specific embodiments of the present invention, in step 1), the nucleotide sequence of the Ptac promoter fragment is shown in SEQ ID NO: 2.

[0030] In some specific embodiments of the present invention, in step 1), the PCR amplification conditions are as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s; annealing at 55°C for 15 s; extension at 72°C for 1 min / kb; 35 cycles; and final extension at 72°C for 10 min.

[0031] In some specific embodiments of the present invention, in step 2), the competent cells are either Escherichia coli SM10 competent cells or Escherichia coli S17-1 competent cells; in a preferred embodiment of the present invention, the competent cells are Escherichia coli SM10 competent cells.

[0032] In some specific embodiments of the present invention, in step 2), during the conjugation transfer process, the donor bacteria are vibrated and cultured to mid-logarithmic time, and the recipient bacteria are statically cultured to mid-logarithmic time. The vibration culture speed is 150-200 rpm, and the donor and recipient bacterial cells are obtained by centrifugation. The centrifugation speed is 4500-6000 rpm, and the centrifugation time is 5-10 min.

[0033] In some specific embodiments of the present invention, the conjugation ratio of the donor bacteria to the recipient bacteria is 1:1 to 5 by volume; in a preferred embodiment of the present invention, the conjugation ratio of the donor bacteria to the recipient bacteria is 1:1.

[0034] This invention provides the application of the engineered strain of *Thiobacillus denitrificationus* in the preparation of products for accelerating the denitrification of *Thiobacillus denitrificationus*; in some preferred embodiments of this invention, the product includes bacterial liquid, bacterial powder or biological agent, the active ingredient of which is obtained by fermentation culture of the engineered strain of *Thiobacillus denitrificationus*.

[0035] The present invention provides a method for deep denitrification of nitrogen-containing wastewater, the method comprising inoculating the nitrogen-containing wastewater with the engineered strain of denitrifying thiobacillus.

[0036] In some specific embodiments of the present invention, the inoculation amount of the denitrifying thiobacillus engineered strain is 1% to 3% of the volume of nitrogen-containing wastewater; in a preferred embodiment of the present invention, the inoculation amount of the denitrifying thiobacillus engineered strain is 2% of the volume of nitrogen-containing wastewater.

[0037] The present invention will be further described in detail below with reference to specific embodiments.

[0038] Example 1: T. denitrificans middle act Extraction and identification of signal molecules in population density sensing systems Should act The type of swarm induction system consists of glyQ, glyS, gph, act Four co-transcribed genes, including acyltransferases act Responsible for encoding and synthesizing signal molecules, among which, act The term "QS system" is an abbreviation for a quorum sensing system that uses acyl homoserine lactones (AHLs) as signal molecules.

[0039] To clarify T. denitrificans The types of signal molecules secreted by the strain were first extracted using dichloromethane extraction to obtain crude extracts of acylhomoserine lactones (AHLs); then, identification was performed using liquid chromatography-tandem mass spectrometry (LC-MS-MS), with commercially available AHLs standards serving as the control group.

[0040] The results are as follows Figure 1 As shown, commercially purchased AHLs standard products ( Figure 1 A) As a control test, the wild-type strain was found to be... T. denitrificans ( Figure 1 3-OH-C can be identified in B) 14 -AHL signaling molecules. Therefore: T. denitrificans of act The signal molecule generated by the type quorum sensing system is 3-OH-C. 14 -AHL.

[0041] Example 2: Overexpression strain T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act Construction and phenotypic characteristics of ) 1. Recombinant plasmid pJRD215-Ptac- T. denitrificans-act Construction 1) Extraction of genome and plasmids Genomic extraction kit from Qingke Biotechnology Co., Ltd. was used to extract... T. denitrificansThe genome is prepared for later use; plasmid pJRD215 is extracted using the same brand of plasmid extraction kit for later use.

[0042] 2) Obtaining the target fragment pJRD215-Ptac- T. denitrificans-act In act Target fragment with T. denitrificans Using the genome as a template, and the Ptac promoter fragment synthesized by Qingke Biotechnology as the original template, primers with homologous arms to plasmid pJRD215 were designed for PCR amplification. act The target fragment and the Ptac promoter fragment.

[0043] in, act The nucleotide sequence of the target fragment is as follows: ATGAATTTGCTCCGTTCGCTGATTTTCATGGTTCTGCAAACCGTGCTGACGGTTTTCTTCAGCCTGGTTGCCTTGCTCAGTTTTCCGTTTTCGGCGCATACGCGTTACCGGATGATTACCGGCTACAACCGCATCGTGATCTGGCTGGCGCGCTGGGTGCTCGGCATCCGCTATGTGGTGAAGGGGGCTGAAAACCTGCCGGAACAGCCTGCCGTCATTCTCGCCAAGCACCAGTCGGCGTGGGAAACCGTGGCGTTTCTGTTCCTGTTTCCGCCGGTCTCGCCGGTCATCAAGCAGGAGCTGCTGAACGTGCCGTTTTTTGGCTGGGCATTCCGGATGCTTTCTCCCATCGCCATCGACCGCGGTGCCGGGCGCGAAGCGCTCAAGCAGATCGTCGCCCGAGGCAAGGAAAAAATCGCGCAGGGATTCTGGGTGCTGGTGTTTCCCGAAGGCACGCGCGTCGCTCCCGGTGAAAAAGGCCGCTATGGCATAGGCGGCGGCTGGCTGGCGGCGGAAAGCGGCGCCCCCATCGTGCCGGTGGCGCACAACGCGGGCGAGGTGTGGCCAAAAAATGCCTTCATCAAATGTCCCGGCACCATCACCGTCAGCATCGGCCCGGCCATTGCCACGGCGGGCAAAAGTGCGGCCGAACTCACCCGCGCGGTCGAGGCCTGGATCGAGGCCGAAATGGCGAGGCTGCCCCCTGCTGCCGCACGCCAATAA (SEQ ID NO: 1); The nucleotide sequence of the Ptac promoter is: CGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAAATTC (SEQ ID NO: 2); PCR amplification act The target fragment and the Ptac promoter fragment were amplified using the following primers: T. denitrificans-actThe target fragment (lowercase parts are homologous arms): F-215-0293-TacMDPD:ttcacacaggaaacaaattcATGAATTTGCTCCGTTCGCT (SEQ IDNO: 3); 0293MDPD-R1:aaactaccgcattaaagcttTTATTGGCGTGCGGCAGCA (SEQ ID NO: 4); T. denitrificans-act promoter fragments (lowercase parts are homologous arms): 215-Ptac-F-BYB:tcttaaggcctaggtctagaCGACATCATAACGGTTCTGG (SEQ ID NO: 5); 215-PtacQDZ-R-0293: agcgaacggagcaaattcatGAATTTGTTTCCTGTGTGAA (SEQ IDNO: 6).

[0044] pJRD215-Ptac- T.denitrificans-act The verification primers are as follows: 215-F3: AACAGTGCGCCAACTA (SEQ ID NO: 7); 215-R1: ACCAAGCCTATGCCTACAG (SEQ ID NO: 8).

[0045] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 5 min; 98℃ for 10 s; annealing at 55℃ for 15 s; extension at 72℃ for 1 min / 1 kb; 35 cycles; extension at 72℃ for 10 min; and incubation at 4℃.

[0046] 3) Construction of recombinant plasmids Plasmid pJRD215 was linearized by double digestion with restriction endonucleases Kpn I / Xba I and Xba I / Hind III. The digestion products were recovered using a DNA purification and recovery kit, and their concentrations were determined. Following the instructions of the homologous recombinase, the digestion products were... act Ligate the target fragment, promoter fragment, and linearized plasmid. Follow the corresponding instruction manual for specific enzyme digestion and ligation steps. Transform the ligation buffer into... E.coli DH5α was plated on solid LB agar plates containing the corresponding antibiotics and incubated overnight. Colony PCR was then performed for verification, and positive clones were named... E.coli DH5α(pJRD215-Ptac- T.denitrificans- act ).

[0047] 4) DNA gel electrophoresis was used to analyze pJRD215-Ptac- T. denitrificans-act The promoter and target fragment, as well as the positive clone and plasmid pJRD215, were validated by PCR. The results are as follows: Figure 2 As shown.

[0048] in, Figure 2 A and Figure 2 Results B showed that PCR amplification yielded a 115 bp Ptac gene fragment and a 723 bp fragment, respectively. act Gene fragments. Figure 2 The results showed that the band size of the PCR amplification using the positive clone as a template was 1142 bp, while the PCR result of plasmid pJRD215 showed a band size of 300 bp.

[0049] 5) Sequencing analysis After extracting plasmids from positive clones, the recombinant plasmid pJRD215-Ptac- T. denitrificans-act The sequencing analysis was commissioned to Qingke Biotechnology Co., Ltd.

[0050] The above results indicate that the target plasmid pJRD215-Ptac- T. denitrificans-act Successfully built.

[0051] 2. Construction of overexpression strains The constructed plasmid pJRD215-Ptac- T. denitrificans-act Transformed into E. coli SM10 competent cells (donor bacteria), the donor bacteria were cultured with vibration to mid-log phase, and the recipient bacteria ( T. denitrificans DSM 12475 cells were statically cultured to mid-log phase, with shaking at 180 rpm. Donor and recipient bacterial cells were obtained by centrifugation at 5000 rpm for 8 minutes. The donor and recipient cells were then conjugated at a 1:1 volume ratio to obtain the overexpression strain. T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act Single colonies were randomly selected from the plates and verified by colony PCR using primer YZTD-F / 215-R2. The nucleotide sequence of primer YZTD-F / 215-R2 is as follows: YZTD-F: CTTTACCAGCATCCGGTT (SEQ ID NO: 9); 215-R2: GAAATATCCCGAATGTGCAG (SEQ ID NO: 10).

[0052] Control strains were constructed using the same operating procedures. T. denitrificans DSM 12475 (pJRD215).

[0053] like Figure 3 As shown, the overexpression strain T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act The band size was 1253 bp, consistent with the control band size. Figure 3 A), and T. denitrificans The PCR result for DSM 12475 (pJRD215) showed a band size of 608 bp. Figure 3 B). Based on the electrophoresis analysis results, it can be preliminarily determined that the engineered strain was successfully constructed and the plasmid was successfully transferred into the recipient bacteria.

[0054] 3. Degradation of total nitrogen, nitrite and nitrate nitrogen The constructed overexpression strain T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act ) and control strains T. denitrificans DSM 12475 (pJRD215) was used as the assay target to evaluate the degradation capabilities of both strains for total nitrogen, nitrite nitrogen, and nitrate nitrogen. The two strains were inoculated at a 2% inoculum into nitrogen-containing wastewater and cultured under identical conditions. The supernatant of the culture was collected periodically, and the concentrations of total nitrogen, nitrite nitrogen, and nitrate nitrogen were determined using the corresponding kits from Greencare and a Greencare GL-900 spectrophotometer. The results are shown in Table 1. Table 1. Detection results of total nitrogen, nitrite and nitrate nitrogen

[0055] As shown in Table 1: T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act OD 600 The growth was gradual, peaking at 0.235 on day 5, compared to the control strain. T. denitrificans DSM 12475 (pJRD215) Day 5 OD 600 The peak value was 0.185, an increase of 27%.

[0056] Regarding nitrite degradation, T. denitrificans DSM 12475 (pJRD215-Ptac- T. denitrificans-act On day 3, the nitrite level in the overexpression culture strain was only 0.56 mg / L, compared to the control strain.T. denitrificans The concentration of nitrite in DSM 12475 (pJRD215) was 0.835 mg / L on day 3, and the phased degradation rate of nitrite in the overexpressing strain was significantly better than that in the control strain.

[0057] Nitrate nitrogen removal rate is a key indicator of denitrification capacity, and overexpression strains T. denitrificans DSM12475 (pJRD215-Ptac-) T. denitrificans-act Compared with the control strain T. denitrificans The DSM 12475 (pJRD215) improved by 4.13%.

[0058] Total nitrogen removal efficiency is closely related to metabolic stability. Based on samples taken on day 7, the overexpression strain showed a 9.76% improvement compared to the control strain. These results indicate that overexpression... act Genes can significantly enhance T. denitrificans The growth performance of the strain, nitrite degradation rate, nitrate nitrogen removal rate and total nitrogen removal efficiency.

[0059] Example 3: Preparation and application of highly efficient denitrifying thiobacillus DSM 12475 bacterial agent 1, 3-OH-C 14 -AHL T. denitrificans DSM 12475 growth promoter To wild-type strains T. denitrificans Commercially available 3-OH-C was added to DSM 12475 at final concentrations of 10 nM, 100 nM, and 1 μM, respectively. 14 -AHL standard, with no added 3-OH-C 14 -Wild-type strains of AHL standard T.denitrificans DSM 12475 was used as a blank control.

[0060] 2. Experimental Results The results are as follows Figure 4 As shown, 10 nM, 100 nM and 1 μM 3-OH-C were added. 14 -The bacterial culture density of AHL standard was compared with that of no 3-OH-C. 14 -The AHL standard showed increases of 12.6%, 31.71%, and 51.5% on day 7. These results indicate that 3-OH-C 14 -AHL has a significant positive regulatory function on the growth of *Thiobacillus denitrifyingus*. Specifically, in the early stages of culture, this signaling molecule can shorten the lag phase, accelerate the logarithmic proliferation of the bacteria, and increase OD... 600 This allows the cells to enter the exponential growth phase more quickly, ultimately leading to a significant increase in the cell density of the culture system.

[0061] 3-OH-C 14 - Application of AHL in accelerating nitrogen metabolism in strains: As shown in Table 2, 3-OH-C 14 -AHL can promote T. denitrificans DSM 12475 proliferation, 1 μM group OD 600 The concentration reached 0.445 on day 4 of culture, and its growth viability was superior to the 10 nM, 100 nM groups and the blank control group. (1 μM 3-OH-C) 14 -AHL accelerated nitrite degradation, reducing it to 0 mg / L on day 4, achieving zero nitrite nitrogen one day earlier than other groups and the blank control group. 3-OH-C 14 The higher the concentration of -AHL, the more significant the promotion of nitrate nitrogen and total nitrogen metabolism. After 7 days of culture, the final concentrations of nitrate nitrogen and total nitrogen in the 1 μM group were much lower than those in the 10 nM, 100 nM groups and the blank control group.

[0062] Table 2. 3-OH-C at different concentrations 14 Effects of AHL on nitrogen metabolism in strains

[0063] Application of microbial agents in accelerating sulfur metabolism in bacterial strains: As shown in Table 3, 3-OH-C 14 -AHL accelerated the degradation of sodium thiosulfate. After 5 days of culture in the 1 μM group, the sodium thiosulfate concentration decreased to 1078.72 mg / L, significantly lower than the 10 nM, 100 nM groups and the blank control group. The degradation rate increased with increasing concentration. This molecule promoted the conversion of sodium thiosulfate to sulfate. After 7 days, the sulfate concentration in the 1 μM group reached 5893.2 mg / L, higher than the 10 nM, 100 nM groups and the blank control group. The conversion efficiency was positively correlated with the AHL concentration. 3-OH-C 14 -AHL enhances sulfur metabolism and acid production. The pH of the 1 μM group decreased to 6.8 after 7 days, which was more significant than that of the blank control (pH = 6.88) and the 10 nM and 100 nM groups. The metabolic activity increased with increasing concentration.

[0064] Table 3. 3-OH-C at different concentrations 14 The effect of AHL on sulfur metabolism in strains

[0065] Example 4: Heterogeneous act Construction and functional verification of engineered strains of genes By coming from Acidithiobacillus caldus MTH-04's alien source ac The t gene was constructed in the pJRD215-Ptac vector and introduced into the vector. In DSM 12475, the engineered bacterium WT(pJRD215-Ptac-) was successfully obtained. A T.denitrificans The preparation method is the same as in Example 2. The results are shown in Table 4. This strain can simultaneously promote host growth and optimize denitrification efficiency: the biomass of the engineered bacteria at the growth peak on day 5 was 36.63% higher than that of the control, and the bacterial growth level throughout the cycle was higher; in terms of denitrification, the intermediate degradation rate of nitrite in the strain was faster, and the removal efficiency of nitrate and total nitrogen on day 7 was 4.13% and 9.76% higher than that of the empty control group, respectively. The overall denitrification ability was significantly improved, and this strain also showed the ability to promote host growth and improve denitrification efficiency.

[0066] Among them, heterogeneous .caldus-act The nucleotide sequence of the gene is as follows: ATGGTGCTGCGCAGCACACTCTTTTATGTCGGGTTTACCCTCTGGACCCTGGCCTATGCGCCCCTGACGCCACTCGCCTGGCTGCTTCTGAGCCGAACCCAGCGCATTGCCTTCTGTGGTCTCTGGGCTCGGGTTGGGGCTCGCTGGTTATCCTGGAGCTGCGGCATCGGCGTCGACGTCCAAGGTGAGGCGCCCGCCCGGGATCGTCCGGTGGTACTCGTCTGCAATCACCAGTCGGCCCTGGAAACCCTGTTGTTGTGGCGCTATTTTCCGCGCATGGCGGTGGTCCTCAAGGCGGAGCTCCTGCGCCTGCCCGTGATCGGCTGGGCCCTGCCCCTGGCCTGGCCCATCGCCATCCAGCGCGAGGCCGGGCGCAAGGCTCTGGAGCAGGTCCTCAGCGAGGGCCGCAAACGCCTGGAAGCAGGACTGCCCGTACTCATCTTTCCCGAGGGTACCCGCCAGGCCTGTGGCGAGGTGGGCCGCTTCCACCAGAGCGCGGTCCAGCTTGCCCGGACCTGCGGCGTGCCCATTCAGACCATCGCCCTCAACAGCGGTTGCTTCTGGCCCAAGGGACAGTGGCGCAAGTATCCCGGCACGGTCCGGCTGGAGTTTGGCCCCGTCGTGCCCGTGAGCGACTCCAACGCCCGTTTCACGGCACTGCTGGAGGCACGCATCCGCAGCACCGTGGAGGCTATGCCGCGCGGCCCCGGCTTTCCAGTTGCAGATGCATCCAGGCCGCCACGGCGGCGGCGACATCCACCCCAGTAG (SEQ ID NO: 11).

[0067] Table 4 Denitrification performance of engineered strain WT(pJRD215-Ptac- act A .caldus-act )

[0068] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of denitrifying thiobacillus ( T. denitrificans The engineered strain is characterized by, The engineered strain of *Thiobacillus denitrification* is an overexpressing strain. act The denitrifying thiobacillus gene, the act The gene encodes an enzyme that synthesizes AHL signaling molecules; act The gene sequence is shown in SEQ ID NO: 1; The AHLs signaling molecule is 3-OH-C. 14 -AHL.

2. The engineered strain of *Thiobacillus denitrificationus* according to claim 1, characterized in that, The denitrifying thiobacillus is T. denitrificans DSM 12475.

3. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to any one of claims 1-2, characterized in that, Includes the following steps: 1) Using the extracted genomic DNA of *Thiobacillus denitrificationus* as a template, PCR amplification was performed using primer pairs to obtain... act The target fragment; simultaneously, using the synthesized Ptac promoter fragment as a template, PCR amplification was performed using primer pairs to obtain the Ptac promoter fragment; the Ptac promoter fragment and the... act The target fragment is ligated into the plasmid via homologous recombination to obtain a recombinant expression plasmid. 2) Transform the recombinant expression plasmid described in step 1) into competent cells to obtain donor bacteria; With the donor bacteria and T. denitrificans The recipient bacteria underwent conjugation transfer, introducing the recombinant expression plasmid. T. denitrificans In this process, the engineered strain of Thiobacillus denitrification was obtained; Preferably, amplification act The primer pair used for the target fragment is the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4; Preferably, the primer pair used to amplify the Ptac promoter fragment is the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO:

6.

4. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to claim 3, characterized in that, In step 1), the homologous recombination method is as follows: plasmid pJRD215 is double-digested with restriction endonucleases Kpn I / Xba I and Xba I / Hind III to obtain a linearized plasmid; act The target fragment, the Ptac promoter fragment, and the linearized plasmid were ligated to construct the recombinant expression plasmid.

5. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to claim 3, characterized in that, In step 1), the nucleotide sequence of the Ptac promoter fragment is shown in SEQ ID NO:

2.

6. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to claim 3, characterized in that, In step 1), the PCR amplification conditions are as follows: pre-denaturation at 98℃ for 5 min; denaturation at 98℃ for 10 s; annealing at 55℃ for 15 s; extension at 72℃ for 1 min / kb; 35 cycles; and final extension at 72℃ for 10 min.

7. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to claim 3, characterized in that, In step 2), the competent cells are either Escherichia coli SM10 competent cells or Escherichia coli S17-1 competent cells; preferably, Escherichia coli SM10 competent cells.

8. The method for preparing the engineered strain of *Thiobacillus denitrificationus* according to claim 3, characterized in that, In step 2), during the conjugation transfer process, the donor bacteria are cultured with vibration to mid-logarithmic phase, and the recipient bacteria are cultured statically to mid-logarithmic phase. The shaking culture speed is 150-200 rpm, and the donor and recipient bacterial cells are obtained by centrifugation. Preferably, the centrifugation speed is 4500~6000 rpm and the centrifugation time is 5~10 min; Preferably, the conjugation ratio of the donor bacteria to the recipient bacteria is 1:1 to 5 by volume.

9. The use of the engineered strain of Thiobacillus denitrification according to claim 1 in the preparation of products for accelerating Thiobacillus denitrification; Preferably, the product includes bacterial liquid, bacterial powder or biological agent, the active ingredient of which is obtained by fermentation culture of the engineered strain of Thiobacillus denitrification.

10. A method for deep denitrification of nitrogen-containing wastewater, characterized in that, The method includes inoculating nitrogen-containing wastewater with the engineered strain of Thiobacillus denitrification according to claim 1; Preferably, the inoculation amount of the denitrifying thiobacillus strain is 1% to 3% of the volume of nitrogen-containing wastewater.

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