Antibiotic-free label inducible salt-tolerant rhodobacterium engineering bacteria and application thereof

Abstract

This invention relates to an antibiotic-label-free, induced, salt-tolerant Rhodotorula globulinii engineered strain and its applications, belonging to the field of microbial technology. Wild Rhodotorula globulinii is a dominant species for wastewater biological treatment, but natural strains grow extremely slowly at salt concentrations exceeding 3%, significantly inhibiting their ability to degrade pollutants. The induced, salt-tolerant Rhodotorula globulinii engineered strain constructed in this invention, after lactose induction, significantly improves the treatment capacity of ammonia nitrogen, phosphate, and acetate in 3%–5% high-salt wastewater compared to wild strains and constitutive overexpression engineered strains. This provides technical support for the application of Rhodotorula globulinii in coastal industrial wastewater purification, saline-alkali land ecological restoration, and seawater recycling, significantly enhancing the competitiveness of Rhodotorula globulinii in low-cost seawater cultivation, high-salt wastewater treatment, and biomanufacturing in special environments.

Description

Technical Field

[0001] This invention relates to an antibiotic-label-inducible salt-tolerant Rhodotorula glutinis engineered strain and its application, belonging to the field of microbial technology. Background Technology

[0002] Rhodopsylloides ( Rhodobacter sphaeroides It is a purple non-sulfur photosynthetic bacterium that possesses the abilities of photosynthetic carbon fixation, hydrogen production, organic matter degradation, and synthesis of high-value-added products (such as coenzyme Q). 10 Its ability to metabolize carotenoids and other substances has significant application value in environmental remediation, bioenergy, and biomanufacturing. However, its application and promotion face serious challenges: natural strains have poor salt tolerance and are difficult to adapt to high-salt environments (such as seawater culture media and high-salt wastewater), which limits its application scenarios.

[0003] To address the poor stress resistance of Rhodotorula glomeratus and further expand its application scenarios, Chinese patent document CN116926097A (application number CN202310685601.X) discloses the application of the small-molecule heat shock protein IbpA in improving the environmental tolerance of host bacteria. By overexpressing the small-molecule heat shock protein IbpA in host bacteria, the recombinant bacteria obtained show significantly increased resistance under different stress conditions, enhancing the survival ability of the host bacteria under various adverse environments. However, the survival and metabolic activity of the strain are easily affected by the synergistic influence of multiple complex environmental factors such as salinity and substrate. Although overexpression of IbpA has been proven to enhance the host bacteria's resistance to single or combined adverse conditions, whether this gene overexpression affects the photosynthetic carbon fixation efficiency, organic matter degradation pathways, and target product synthesis balance of Rhodotorula glomeratus in actual high-salinity wastewater treatment scenarios still lacks direct application data to support this.

[0004] Meanwhile, the expression level of IbpA is closely related to the survival status and function of the strain. Higher expression levels do not necessarily mean better results. Overexpression may consume a large amount of ribosomes, energy and material resources of the strain, increase the metabolic burden on cells, and inhibit the normal function of its core functions. Therefore, it is particularly important to choose a suitable overexpression method and precisely regulate the expression level of IbpA.

[0005] In conclusion, whether overexpression of IbpA can truly promote the practical application of Rhodopsycetes in the treatment of high-salt wastewater still requires further experimental exploration and scenario-based verification. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an antibiotic-tag-free, lactose-inducible, salt-tolerant Rhodotorula globulinii engineered strain and its applications. This invention focuses on constructing an antibiotic-tag-free, lactose-inducible, salt-tolerant engineered strain. Based on the crucial role of the key heat shock response protein IbpA in enhancing broad-spectrum cellular stress resistance, a synthetic biology strategy is employed, using the lactose-inducible promoter P...lac By replacing the promoter of the IbpA gene, an inducible switch for high salt concentration tolerance is established. This method achieves in-situ modification of the natural locus, enabling controllable and artificially modulated expression intensity under antibiotic-free conditions, while preserving broad-spectrum stress resistance and avoiding the lag in stress response. This solves the industry challenge of suppressed cell function under high salt stress, providing a safe, controllable, and functionally stable strain for the efficient biological treatment of high-salt wastewater.

[0007] The technical solution of the present invention is as follows:

[0008] An antibiotic-free, tag-inducible, salt-resistant Rhodotorula glutinis engineered bacterium, wherein the engineered bacterium is derived from heat shock response proteins in Rhodotorula glutinis. ibpA gene promoter P ibp A is replaced with the lactose promoter P. lac Thus, through lactose-induced regulation ibp Expression of gene A yields an antibiotic-tagged induced salt-tolerant Rhodotorula glutinis engineered strain.

[0009] According to a preferred embodiment of the present invention, the starting strain of the engineered bacteria is Rhodopseudomonas aeruginosa. R. sphaeroides 2.4.1, the strain number is ATCC 17023.

[0010] According to a preferred embodiment of the present invention, the gene ibp The nucleotide sequence of A is shown in SEQ ID NO.1, and the lactose promoter P lac The nucleotide sequence is shown in SEQ ID NO.2.

[0011] According to a preferred embodiment of the present invention, the method for constructing the engineered bacteria includes the following steps:

[0012] (1) Construction of recombinant plasmid: Select knockout plasmid pK18 mobsacB Used for the construction of the above-mentioned engineered bacteria; first, heat shock response protein... ibpA The left and right arms of the gene were amplified separately, and then the lactose promoter P was... lac First, overlap and extend with the left arm to obtain the product, then overlap and extend with the right arm. Finally, combine the amplified product with plasmid pK18. mobsacB After enzyme digestion and ligation, recombinant plasmid pK18 containing the lactose promoter was obtained. mobsacB -P lac ;

[0013] (2) Construction of engineered bacteria: The recombinant plasmid pK18 was constructed. mobsacB -P lac The cells were transferred into competent Escherichia coli S17-1, and then conjugated into Rhodopseudomonas aeruginosa for homologous recombination, thereby... ibpA Existing promoter P ibpAReplace with lactose promoter P lac An antibiotic-tagged induced salt-tolerant Rhodotorula salsa engineered strain with the lactose promoter integrated into the genome was obtained.

[0014] The above-mentioned antibiotic-free, label-inducible, salt-tolerant Rhodotorula glutinis engineered bacteria are used in the treatment of high-salt wastewater.

[0015] According to a preferred embodiment of the present invention, the NaCl concentration of the wastewater is 3% to 5% by mass.

[0016] The method for treating high-salinity wastewater using the aforementioned engineered bacteria includes the following steps:

[0017] (1) Select the above-mentioned engineered bacteria and inoculate them into NH1 liquid culture medium for activation culture for 48-72 h. Inoculate the activated bacterial solution into NH1 liquid culture medium at a volume ratio of 2%-10% for expansion culture for 24-48 h to obtain seed liquid; (2) Inoculate the seed liquid obtained in step (1) into NH1 liquid culture medium with added lactose at a volume ratio of 8%-20% for culture for 2-6 h; (3) Inoculate the obtained bacterial solution into sewage at a volume ratio of 8%-20% for sewage treatment at 25-32℃.

[0018] According to a preferred embodiment of the present invention, the culture conditions described in steps (1) and (2) are 25~32℃ and 150~200 rpm.

[0019] According to a preferred embodiment of the present invention, the amount of lactose added in step (3) is 0.1%~0.5% by mass-volume ratio, in g / mL.

[0020] According to a preferred embodiment of the present invention, the NH1 liquid culture medium is prepared as follows: 8 g yeast powder, 3 g glucose, 2 g NaCl, 1.3 g potassium dihydrogen phosphate, 0.125 g anhydrous magnesium sulfate, and 1 mL growth factor stock solution are added to 1 L of deionized water, and the pH value is adjusted to 7.2; the growth factor stock solution is prepared as follows: 4 mg biotin, 200 mg niacin, and 100 mg thiamine hydrochloride are added to 100 mL of deionized water, and the volume is adjusted to 200 mL, followed by filtration for sterilization.

[0021] Beneficial effects:

[0022] Wild-type Rhodopseudomonas globulus is a dominant species for wastewater biological treatment, but natural strains grow extremely slowly when the salt concentration exceeds 3%, and their ability to degrade pollutants is significantly inhibited. The inducible salt-tolerant Rhodopseudomonas globulus engineered strain constructed in this invention significantly improves the treatment capacity of ammonia nitrogen, phosphate and acetate in 3%~5% high-salt wastewater after lactose induction compared with wild strains and constitutive overexpression engineered strains. This provides technical support for the application of Rhodopseudomonas globulus in the fields of industrial wastewater purification, saline-alkali land ecological restoration and seawater recycling in coastal areas, and significantly enhances the competitiveness of Rhodopseudomonas globulus in low-cost seawater culture, high-salt wastewater treatment and special environment biomanufacturing. Attached Figure Description

[0023] Figure 1 For recombinant plasmid pK18 mobsacB -P lac Atlas;

[0024] Figure 2 for ibpA Gene promoter replacement strain △P ibp A / P lac The results of single-exchange screening electrophoresis are shown in the figure; where lane M is the marker, lane P is the positive control, and lanes 1-2 are the target bands.

[0025] Figure 3 for ibp A gene promoter replacement strain △P ibpA / P lac The results of double exchange screening electrophoresis are shown in the figure; where lane M is the marker, lane N is the negative control, lane P is the positive control, and lanes 1-5 are the target bands.

[0026] Figure 4 For △P ibp A / P lac Line graph showing the treatment effects of engineered bacteria and wild bacteria on synthetic wastewater with a salt concentration of 3%;

[0027] Figure 5 For △P ibp A / P ​ Line graph showing the treatment effects of engineered bacteria and wild bacteria on synthetic wastewater with a 5% salt concentration;

[0028] ​ For △P ​ A / P ​ engineered bacteria and ​ A / op engineered bacteria ​ A gene transcription level bar chart;

[0029] ​ For △P ​ A / P ​ engineered bacteria and ​Line graph showing the treatment effect of A / op engineered bacteria on 3% salt concentration synthetic wastewater;

[0030] ​ For △P ​ A / P ​ Engineered bacteria and ΔP ​ A / P ​ engineered bacteria ​ A gene transcription level bar chart;

[0031] ​ For △P ​ A / P ​ Engineered bacteria and ΔP ​ A / P ​ Line graph showing the treatment effect of engineered bacteria on artificially synthesized wastewater with a salt concentration of 3%. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] The following examples use Rhodopseudomonas stolonifer S1 ( ​ 2.4.1), purchased from the China Industrial Microbial Culture Collection Center, strain number ATCC 17023, now renamed *Cyclocarya cereus* on that website (…). ​ The strain has the preservation number CICC 10287.

[0035] Example 1

[0036] ​ A promoter replacement strain △P ​ A / P ​ Construction of engineered bacteria

[0037] Genomic DNA was extracted from Rhodopseudomonas aeruginosa S1 using a bacterial genome extraction kit. The results were obtained by searching the KEGG website. ​ Gene A sequence information (nucleotide sequence as shown in SEQ ID NO.1), ​ promoter P ​ The nucleotide sequence is shown in SEQ ID NO.5), designed using Primer 5. ​The amplification primers for the left arm (nucleotide sequence as shown in SEQ ID NO.3) and right arm (nucleotide sequence as shown in SEQ ID NO.4) of the gene are shown in Table 1.

[0038] The high expression plasmid pET-28a(+) and the knockout plasmid pK18 were amplified using E. coli DH5α. ​ Design a high-expression plasmid pET-28a(+) with the lactose promoter P ​ (The nucleotide sequence is shown in SEQ ID NO.2) Primers are shown in Table 2.

[0039] Table 1. Amplification ​ Primers used in the left and right arms and their names

[0040]

[0041] Table 2. Amplification of the lactose promoter P on pET-28a(+) ​ Primers used and their names

[0042]

[0043] Using the extracted Rhodopseudomonas spp. S1 genomic DNA as a template, primers were used... ​ AL-F and ​ AL-R, ​ AR-F and ​ AR-R ​ PCR amplification of nucleotide fragments from the left and right arms of cell A was performed according to the reaction system described in Table 3 and the reaction conditions described in Table 5. ​ A. Left arm ​ A right arm fragment. Using the high-expression plasmid pET-28a(+) as a template, primers were used... ​ A ​ -F、 ​ A ​ -R was used for PCR amplification, the reaction system is as shown in Table 4, and the reaction conditions are as shown in Table 5, to obtain P ​ Promoter segment.

[0044] Table 3. PCR reaction system

[0045]

[0046] Table 4. PCR reaction system

[0047]

[0048] Table 5. PCR reaction conditions

[0049]

[0050] The amplified product from the above step of the experiment ​ A left arm segment, P ​ Using the promoter fragment as a template, primers were used respectively. ​ AL-F / ​ A ​ -R, perform overlap PCR amplification, the reaction system is as shown in Table 6, and the reaction conditions are as shown in Table 5, to obtain "left arm-P ​ "Fragment".

[0051] Table 6. PCR reaction system

[0052]

[0053] The "left arm-P" amplified by the above step ​ "Fragments and" ​ Using the right arm fragment as a template, primers were used respectively. ​ AL-F / ​ AR-R overlap PCR amplification was performed, with the reaction system as shown in Table 7 and the reaction conditions as shown in Table 5, to obtain the PCR product (left arm-P). ​ -Right arm).

[0054] Table 7. PCR reaction system

[0055]

[0056] The PCR product (left arm-P) obtained in the previous experiment was used to... ​ -Right arm) and knockout plasmid pK18 ​ B was treated with restriction endonucleases. ​ RI / ​ dIII was subjected to double digestion (digestion system as shown in Tables 8 and 9), and digested in a water bath at 37°C for 2 h. Left arm-P ​ - The enzyme digestion product of the right arm was purified and recovered using a standard DNA product purification kit. After separation by agarose gel electrophoresis, the enzyme-digested plasmid was recovered by gel excision using a standard agarose gel DNA recovery kit.

[0057] Table 8. Enzyme digestion reaction system for PCR products

[0058]

[0059] Table 9. Knockout plasmid pK18 ​ B enzyme digestion reaction system

[0060]

[0061] The enzyme-digested PCR product (left arm-P) ​ -Right arm) and the enzyme-digested plasmid pK18 ​B was ligated overnight using T4 DNA ligase, the reaction system is shown in Table 10, and ligation was performed at 16℃ for 12 h. The ligated product pK18 ​ -P ​ Transformed into E. coli DH5α for amplification, the amplified recombinant plasmid was extracted and used... ​ RI / ​ Double digestion with dIII enzymes yielded the recombinant plasmid pK18. ​ -P ​ Plasmid map as follows ​ As shown.

[0062] Table 10. Connection Reaction System

[0063]

[0064] Highly competent E. coli S17-1 cells were prepared using a high-efficiency competent cell preparation kit, following the instructions in the kit's manual. The successfully constructed recombinant plasmid pK18 was then... ​ -P ​ Introduced into E. coli S17-1 competent cells, plasmid pK18 was processed using a high-performance competent cell kit. ​ -P ​ Transformation was performed following the instructions of the high-performance competent cell kit. After culturing on LB plates containing 100 μg / mL kanamycin for 16 h, the resulting white single colonies were the transformants to be validated.

[0065] Select transformants from LB plates and add them to LB liquid medium containing 100 μg / mL kanamycin. Incubate at 37°C and 150 rpm for 12 h. Once the bacterial culture becomes turbid, use... ​ LF and ​ RR was used as primers for colony PCR verification of the transformed cells. The reaction system is shown in Table 11, and the reaction conditions are shown in Table 5. The products obtained after the reaction were then analyzed by agarose gel electrophoresis. If the target fragment matched the positive control after agarose gel electrophoresis, it proved that pK18 was detected. ​ -P ​ Escherichia coli S17-1 positive transformant.

[0066] Table 11. PCR reaction system

[0067]

[0068] Rhodopseudomonas spp. S1 cultured to the logarithmic growth phase and successfully verified Escherichia coli S17-1 positive transformants were conjugated and transferred, followed by replacement strain ΔP. ​ / P ​Single-exchange and double-exchange screening verification. The corresponding experimental steps are as follows:

[0069] Culture of Rhodopseudomonas aeruginosa S1: A single colony of S1 strain was picked from a plate and inoculated into a shake flask containing 50 mL of NH1 liquid medium. The culture was incubated at 30℃ and 150 rpm for 72 h to rejuvenate the bacteria. 1 mL of the culture was then transferred to a shake flask containing 50 mL of NH1 liquid medium and incubated under the same conditions for 24 h to the logarithmic growth phase.

[0070] Culture of Escherichia coli S17-1 positive transformants: Select the verified S17-1 positive transformants and add them to LB liquid medium containing 100 μg / mL kanamycin. Incubate at 37℃ and 200 rpm for 16 h until the logarithmic growth phase.

[0071] Conjugation transfer culture: OD of bacterial cultures containing Rhodopseudomonas S1 and Escherichia coli S17-1 positive transformants cultured to the logarithmic growth phase was transferred separately. 600Adjust to between 0.8 and 1.0 as preparation for the bonding transfer. Step 1: In a clean bench, take 1 mL of Rhodopseudomonas spp. S1 bacterial suspension and 0.5 mL of Escherichia coli S17-1 positive transformant bacterial suspension and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 8000 rpm for 3 min, removing as much supernatant as possible. Step 2: Add 1 mL of 0.85% (w / w) physiological saline to each centrifuge tube for elution. Gently pipette to mix thoroughly and centrifuge at 8000 rpm for 3 min, removing as much supernatant as possible. Step 3: Add 1 mL of NH1 liquid culture medium to each centrifuge tube and gently pipette to mix thoroughly. Then, mix the Rhodopseudomonas spp. S1 and Escherichia coli S17-1 positive transformant bacterial suspensions at volume ratios of 3:1 and 7:1, respectively, into two 1.5 mL centrifuge tubes, with the Rhodopseudomonas spp. S17-1 having a higher proportion. Step 4: Using sterile forceps, place the filter membrane in the center of an NH1 solid plate and aspirate 400... The bacterial solution mixed in the previous step was slowly added dropwise to the center of the filter membrane, and the bacterial solution was dried in the ultra-clean bench. After the bacterial rings solidified on the filter membrane, it was placed in an incubator at 30℃ and incubated upside down for 24 hours. Step 5: After 24 hours, 200 μL of 0.85% (w / w) physiological saline was pipetted onto the membrane and the bacterial rings cultured in the previous step were gently mixed. One bacterial ring was mixed twice. The mixed bacterial solution was then spread on an NH1 solid plate containing 50 μg / mL kanamycin and 25 μg / mL potassium tellurite. The plate was placed in an incubator at 30℃ and incubated upside down until black transformants grew. Black transformants were picked and streaked in three zones on NH1 solid plates containing 50 μg / mL kanamycin. The plates were then inverted and placed in a 30°C incubator. After red transformants appeared, single colonies were picked and placed in NH1 liquid medium containing 50 μg / mL kanamycin and cultured at 30°C and 150 rpm for 48 h.

[0072] NH1 liquid medium: Add 8 g yeast extract, 3 g glucose, 2 g NaCl, 1.3 g potassium dihydrogen phosphate, 0.125 g anhydrous magnesium sulfate, and 1 mL of growth factor stock solution to 1000 mL of deionized water, adjusting the pH to 7.2. NH1 solid medium is prepared by adding 2% agar powder to the liquid medium and sterilizing at 121°C for 20 min. Add antibiotics of appropriate concentrations as needed. Growth factor stock solution: Add 4 mg biotin, 200 mg niacin, and 100 mg thiamine hydrochloride to 100 mL of deionized water, bringing the volume to 200 mL, and then filter sterilize.

[0073] Single exchange verification: in primers ​ AL-F and ​ The AR-R design site was extended by 400 bp at both ends to design and validate primers.​ AY-F and ​ AY-R, as shown in Table 12. Red transformants grown from the plates were picked and placed in NH1 liquid medium containing 50 μg / mL kanamycin. After incubation at 30℃ and 150 rpm for 24 h, 1 mL of bacterial culture was taken and bacterial DNA genome was extracted using a bacterial DNA genomics kit. Using the DNA genome as a template, [the following steps were performed]... ​ AR-R / ​ AY-F and ​ AL-F / ​ PCR was performed using AY-R primers. The reaction system was as shown in Table 11, and the reaction conditions were as shown in Table 5. The annealing temperature was 64℃. Agarose gel electrophoresis was then performed to verify the results. The electrophoresis results are shown below. ​ As shown.

[0074] Table 12. Primers and primer names used for single-exchange verification

[0075]

[0076] Double-exchange verification: The verified single-exchange strain was inoculated into a shake flask containing 50 mL of NH1 liquid medium and incubated at 30℃ and 150 rpm for 72 h for rejuvenation. 1 mL of the culture was then transferred to a shake flask containing 50 mL of NH1 liquid medium and incubated under the same conditions for 24 h. This process was repeated three times to obtain single-exchange bacterial solutions, which were then used for double-exchange strain screening. 500 μL of the single-exchange bacterial solution was serially diluted 10⁻⁶ times with 0.85% (w / w) physiological saline. 2 -10 5 100 μL of the diluted solution was spread onto SMM solid plates containing 5% (w / v, g / mL) sucrose. The plates were spread evenly using a sterile spreader and incubated upside down at 30°C. After the emergence of single red colonies, a single colony was picked and placed in NH1 liquid medium and incubated at 30°C and 150 rpm for 24 h. The resulting bacterial culture was serially diluted again, and the DNA genome was extracted from the resulting single red colonies. Using the DNA genome as a template, [the following steps were performed]... ​ AY-F and ​ PCR was performed using AY-R, with the reaction system as shown in Table 11 and the reaction conditions as shown in Table 5. The annealing temperature was 59℃. Agarose gel electrophoresis was then performed to verify the results. The electrophoresis results are as follows: ​ As shown. The strain that is verified to be correct is △P. ​ A / P ​ Engineered bacteria, with the verified strains stored at -80°C.

[0077] SMM liquid medium: First, add 20 mL of CB stock solution and 1 mL of growth factor stock solution to 80 mL of deionized water, then add 8 g of succinic acid, 3.5 g of dipotassium hydrogen phosphate, 0.5 g of ammonium sulfate, 0.5 g of NaCl and 2 g of potassium hydroxide, and finally adjust the volume to 1 L and adjust the pH to 7.2; for solid medium, add 15 g / L of agar to the liquid medium; sterilize in an autoclave at 121℃ for 20 min.

[0078] CB stock solution: Add 10 g aminotriacetic acid and 8 g KOH to 500 mL of deionized water. After complete dissolution, add 14.6 g anhydrous magnesium sulfate, 1.7 g CaCl2•2H2O, and 50 mL of M44 stock solution in sequence, and adjust the pH to 7.0. M44 stock solution: Add 2 g EDTA, 11 g ZnSO4•7H2O, 5 g FeSO4•7H2O, 1.5 g MnSO4•H2O, 0.4 g CuSO4•5H2O, 0.12 g H3BO3, and 0.37 g CoCl4•6H2O to 800 mL of deionized water, and bring the volume to 1 L.

[0079] Example 2

[0080] △P ​ / P ​ Practical application of engineered bacteria in high-salinity wastewater

[0081] Pick △P respectively ​ / P ​ Single colonies from both engineered and wild-type bacterial plates were inoculated into shake flasks containing 50 mL of NH1 liquid medium and incubated at 30°C and 150 rpm for 72 h for rejuvenation. Subsequently, 2 mL of each of the engineered and wild-type bacterial cultures was separately inoculated into shake flasks containing ΔP. ​ A / P ​ The engineered bacterial culture was transferred to a shake flask containing 100 mL of NH1 liquid medium and cultured at 30℃ and 150 rpm for 24 h to prepare a seed culture. The experimental group then used the prepared ΔP... ​ / P ​ Engineered bacterial seed culture was inoculated at a 20% (v / v) inoculum into NH1 liquid medium supplemented with 0.5% (w / v, g / mL) lactose and cultured at 30℃ and 150 rpm for 6 h. The control group consisted of wild-type bacterial seed culture inoculated into NH1 liquid medium and cultured at 30℃ and 150 rpm for 6 h. After centrifugation, the bacterial cells were collected, washed three times with physiological saline, and resuspended in an equal volume. The resuspended bacterial solutions were then inoculated at a 10% (v / v) inoculum into artificially synthesized wastewater culture media with different NaCl concentrations and placed in a constant temperature incubator for the experiment. CH3COO in the wastewater was measured every 24 h. - PO43- and NH4 + The concentration changes were observed. The culture conditions were a constant temperature of 30℃. To simulate a real ecological environment, light (2300 Lux) was applied during the culture process, with a light cycle of 12 h in darkness followed by 12 h in light.

[0082] The above-mentioned artificially synthesized wastewater culture medium consists of: 900 mg anhydrous sodium acetate, 60 mg K₂HPO₄, 25 mg NH₄Cl, 4.5 mg MgSO₄·7H₂O, 2.0 mg KCl, 30 mg KNO₃, 20 mg CaCl₂, 100 μL of CB stock solution, 1 mL of growth factor, and deionized water to a final volume of 1 L. The artificially synthesized wastewater culture medium with different NaCl concentrations is based on the above culture medium with the addition of different mass fractions of NaCl.

[0083] like ​ As shown, in artificially synthesized wastewater containing 3% NaCl, the experimental group CH3COO... - When the concentration was reduced from 600 mg / L to 222 mg / L, the degradation rate in the first 24 h was 15.75 ± 0.55 mg / (L·h), and the degradation rate was 63.16 ± 0.35%. After the system had run for 96 h, CH3COO - The concentration was below 125 mg / L and tended to stabilize; for the control group, under the same operating conditions, CH3COO content was lower than 125 mg / L after 24 hours of system startup. - The concentration only decreased to 328 mg / L, the degradation rate in the first 24 hours was 11.36 ± 0.62 mg / (L·h), and the degradation rate was 45.53 ± 0.17%; the results showed ΔP ​ A / P ​ Engineered bacteria on CH3COO in the first 24 hours - The degradation rate was 1.39 times that of the control group, indicating that the engineered bacteria could promote the degradation of CH3COO. - The removal of CH3COO was observed during the experimental run. - It exhibits higher stability in removing PO4. 3- After 24 hours of system startup, a significant difference in processing efficiency was already evident between the two groups. After 96 hours, the experimental group showed a lower PO4 content. 3- The concentration decreased from the initial 30 mg / L to 6.65 mg / L, while the control group only decreased to 19.2 mg / L, indicating a 2.16-fold increase in treatment efficiency in the experimental group compared to the control group. (From NH4) + The concentration changes show that after 96 hours of operation, NH4 + The concentration was 5 mg / L, lower than the control group's 7.5 mg / L. In summary, under 3% NaCl conditions, ΔP​ A / P ​ Engineered bacteria have a stronger wastewater purification capacity than wild bacteria.

[0084] like ​ As shown, in wastewater containing 5% NaCl, the experimental group CH3COO... - When the concentration was reduced from 600 mg / L to 312 mg / L, the degradation rate in the first 24 h was 12.00 ± 0.25 mg / (L·h), and the degradation rate was 52 ± 0.12%. After the system had run for 96 h, CH3COO - The concentration was below 215 mg / L and tended to stabilize; for the control group, under the same operating conditions, CH3COO content was lower than 215 mg / L after 24 hours of system startup. - The concentration only dropped to 435 mg / L, and the degradation rate in the first 24 hours was 6.88±0.62 mg / (L·h), with a degradation rate of 27.5±0.11%. This indicates that the degradation of pollutants by wild bacteria in wastewater containing 5% NaCl was significantly inhibited. The results showed that the experimental group exhibited significantly reduced degradation of CH3COO in the first 24 hours. - The degradation rate was 1.74 times that of the control group, indicating that the modified engineered bacteria can promote the degradation of CH3COO in high-salt wastewater. - The removal of PO4. 72 hours after system startup, the experimental group showed PO4 removal. 3- The concentration decreased from the initial 30 mg / L to 12.39 mg / L, while the control group only decreased to 22.8 mg / L, indicating that the treatment efficiency of the experimental group was 2.44 times higher than that of the control group. (From NH4) + The concentration changes show that after 96 hours of operation, NH4 + The concentration was 5.15 mg / L, lower than the control group's 8.63 mg / L. This demonstrates that even under high-salinity wastewater conditions, ΔP ​ A / P ​ Engineered bacteria also have a high degradation efficiency for pollutants in wastewater.

[0085] Comparative Example 1

[0086] IbpA-overexpressing Rhodotorula glutinis ​ The A / op engineered bacteria were constructed according to the method described in patent CN116926097A (application number 202310685601.X).

[0087] Comparative Example 2

[0088] △P ​ A / P ​ engineered bacteria and ​ A / op engineered bacteria ​ Comparison of A gene transcription levels

[0089] Select △P constructed in Example 1 respectively ​ A / P ​ Engineered bacteria and Comparative Example 1 constructed ​ A single colony from an A / op engineered bacteria plate was inoculated into a shake flask containing 50 mL of NH1 liquid medium. ​ During the culture of strain A / op, 50 mg / L kanamycin should be added to the NH1 liquid medium to ensure that the recombinant plasmid pBBR1MCS-2 is not lost during bacterial passage and is continuously overexpressed. ​ Gene A), cultured at 30℃ and 150 rpm for 72 h for rejuvenation. 2 mL of ΔP was then transferred. ​ A / P ​ The engineered bacterial culture was transferred to 100 mL of NH1 liquid medium, and 2 mL was taken out. ​ The A / op engineered bacterial culture was transferred to 100 mL of NH1 liquid medium containing 50 mg / L kanamycin and cultured at 30°C and 150 rpm for 24 h to prepare seed culture. The experimental group then used the prepared ΔP... ​ A / P ​ The engineered bacterial seed culture was inoculated at a 20% (v / v) inoculum into NH1 liquid medium supplemented with 0.5% (w / v, g / mL) lactose and cultured for 6 h; the control group was... ​ The A / op engineered bacterial seed culture was inoculated at a 20% (v / v) inoculation rate into NH1 liquid medium supplemented with 50 mg / L kanamycin and 0.5% (w / v, g / mL) lactose and cultured for 6 h. Subsequently, ΔP was extracted. ​ A / P ​ engineered bacteria and ​ cDNA was prepared from the mRNA of A / op engineered bacteria and detected by quantitative real-time PCR.

[0090] The results are as follows ​ As shown, △P ​ A / P ​ engineered bacteria ​ The relative expression level of gene A mRNA was significantly higher than that of gene A. ​ A / op engineered bacteria.

[0091] Comparative Example 3

[0092] ΔP in high-salinity wastewater ​ A / P ​ engineered bacteria and ​ Comparison of A / op engineered bacteria's ability to degrade pollutants

[0093] ΔP was prepared according to the method described in Comparative Example 2. ​ A / P ​ engineered bacteria and ​A / op engineered bacteria seed culture, the experimental group prepared ΔP ​ A / P ​ The engineered bacterial seed culture was inoculated at a 20% (v / v) inoculum into NH1 liquid medium supplemented with 0.5% (w / v, g / mL) lactose and cultured for 6 h; the control group was... ​ The A / op engineered bacterial seed culture was inoculated at a 20% (v / v) inoculum into liquid medium supplemented with 50 mg / L kanamycin NH1 and cultured for 6 h. The bacterial culture was then washed three times with physiological saline and resuspended in equal volumes. The resuspended bacterial culture was then inoculated at a 10% (v / v) inoculum into 3% NaCl synthetic wastewater medium and placed in a constant temperature incubator for the experiment. The culture conditions were the same as in Example 2, and the CH3COO content in the wastewater was measured every 24 h. - PO4 3- and NH4 + The concentration change.

[0094] The results are as follows ​ As shown, under 3% NaCl conditions, ΔP 96 h after the experiment started ​ / P ​ Engineered bacteria against CH3COO - PO4 3- NH4 + The degradation rates were 89.55±0.89%, 75.86±0.21%, and 70.58±0.82%, respectively. ​ / op engineered bacteria against CH3COO - PO4 3- and NH4 + The degradation rates were 45.96±0.77%, 27.55±0.62%, and 47.05±0.89%, respectively. △P ​ A / P ​ Engineered bacteria showed significantly better treatment efficiency than synthetic wastewater under high-salt conditions. ​ A / op engineered bacteria.

[0095] Comparative Example 4

[0096] ​ Promoter replacement strain △P ​ / P ​ The engineered bacteria were constructed in accordance with the method described in Example 1, except that the engineered bacteria were constructed by introducing endogenous genes from Rhodopseudomonas stolonifer S1. ​ A's promoter P ​ A is replaced by a constitutive promoter P. ​ (Synthesized by Beijing Qingke Biotechnology Co., Ltd., nucleotide sequence as shown in SEQ ID NO.6), used ​The primer sequences for the left and right arms are shown in Table 13, and the other steps are the same as in Example 1.

[0097] Table 13. Amplification ​ Primers used in the left and right arms and their names

[0098]

[0099] Comparative Example 5

[0100] △P ​ A / P ​ Engineered bacteria and ΔP ​ A / P ​ engineered bacteria ​ Comparison of A gene transcription levels

[0101] Select △P constructed in Example 1 respectively ​ A / P ​ The engineered bacteria and the ΔP constructed from Comparative Example 4 ​ A / P ​ Single colonies from engineered bacterial plates were inoculated into shake flasks containing 50 mL of NH1 liquid medium and incubated at 30°C and 150 rpm for 72 h for rejuvenation. 2 mL of ΔP was then transferred to each flask. ​ A / P ​ Engineered bacteria, ΔP ​ A / P ​ The engineered bacterial culture was transferred to 100 mL of NH1 liquid medium and cultured at 30 °C and 150 rpm for 24 h to prepare seed culture. The prepared ΔP... ​ A / P ​ Engineered bacteria, ΔP ​ A / P ​ The engineered bacterial seed culture was inoculated at a 20% (v / v) inoculum into NH1 liquid medium supplemented with 0.5% (w / v, g / mL) lactose and cultured for 6 h. Subsequently, ΔP was extracted. ​ A / P ​ Engineered bacteria and ΔP ​ A / P ​ cDNA was prepared from the mRNA of engineered bacteria and detected by real-time PCR.

[0102] The results are as follows ​ As shown, ​ Gene A in ΔP ​ A / P ​ The transcription level in engineered bacteria is approximately ΔP ​ A / P ​ The engineered bacteria were 10 times more potent than ΔP, significantly higher than ΔP. ​ A / P ​ Engineered bacteria.

[0103] Comparative Example 6

[0104] △P was prepared according to the method described in Comparative Example 5. ​ A / P ​ Engineered bacteria and ΔP ​ A / P ​ The engineered bacterial seed culture, the experimental group prepared ΔP ​ A / P ​ The engineered bacterial seed culture was inoculated at a 20% (v / v) inoculation rate into NH1 liquid medium supplemented with 0.5% (w / v, g / mL) lactose and cultured for 6 h; the control group was inoculated with ΔP ​ A / P ​ The engineered bacterial seed culture was inoculated into NH1 liquid medium at a 20% (v / v) inoculum and cultured for 6 h. The bacterial culture was then washed three times with physiological saline and resuspended in equal volumes. The resuspended bacterial culture was then inoculated into 3% NaCl synthetic wastewater medium at a 10% (v / v) inoculum and placed in a constant temperature incubator for the experiment. The culture conditions were the same as in Example 2. CH3COO in the wastewater was measured every 24 h. - PO4 3- and NH4 + The concentration change.

[0105] The results are as follows ​ As shown, under 3% NaCl conditions, ΔP 96 h after the experiment started ​ A / P ​ Engineered bacteria against CH3COO - PO4 3- NH4 + The degradation rates were 89.55±0.89%, 75.86±0.21%, and 70.58±0.82%, respectively, ΔP ​ A / P ​ Engineered bacteria against CH3COO - PO4 3- NH4 + The degradation rates were 50.01±0.09%, 44.82±0.75%, and 64.70±0.57%, respectively, ΔP ​ A / P ​ Engineered bacteria showed significantly better treatment efficiency than ΔP for synthetic wastewater under high-salt conditions. ​ A / P ​ The results of the transcriptional level analysis of the engineered bacteria, combined with those of Comparative Example 5, indicate that a higher IbpA expression level does not necessarily mean better wastewater treatment capabilities.

[0106] Finally, it should be noted that the embodiments disclosed in this invention are merely preferred embodiments of this invention and are only used to illustrate the technical solutions of this invention, not to limit it. Although this invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this invention.

Claims

1. An antibiotic-free label inducible salt-tolerant Rhodobacter sphaeroides engineered bacteria, characterized in that, The engineering bacteria is to replace the promoter P ibpA of the heat shock response protein gene in Rhodobacter sphaeroides ibp A is replaced by lactose promoter P lac , so as to regulate the expression of A gene by lactose induction ibp , obtain antibiotic label-free inducible salt-tolerant Rhodobacter sphaeroides engineering bacteria, and the starting strain of the engineering bacteria is Rhodobacter sphaeroides R. sphaeroides 2.4.1, and the strain number is ATCC 17023.

2. The engineered bacterium of claim 1, wherein, The gene ibp The nucleotide sequence of A is shown in SEQ ID NO. 1, the lactose promoter P lac The nucleotide sequence of A is shown in SEQ ID NO. 1, the lactose promoter P 3. The engineered bacterium of claim 1, wherein, The method for constructing the engineering bacteria comprises the following steps: (1) Construction of recombinant plasmid: knockout plasmid pK18 mobsacB for the construction of the engineering bacteria of claim 1; the left and right arms of the heat shock protein ibpA gene were amplified respectively, and then the lactose promoter P lac was inserted between the left and right arms; the product was obtained by overlap extension with the left arm, and then overlap extension was performed with the right arm; and then the amplification product was inserted into the plasmid pK18 mobsacB after enzyme digestion and ligation, to obtain the recombinant plasmid pK18 mobsacB with the lactose promoter lac ; (2) Construction of engineered bacteria: The recombinant plasmid pK18 was constructed. mobsacB -P lac The cells were transferred into competent Escherichia coli S17-1, and then conjugated into Rhodopseudomonas aeruginosa for homologous recombination, thereby... ibpA Existing promoter P ibpA Replace with lactose promoter P lac An antibiotic-tagged induced salt-tolerant Rhodotorula salsa engineered strain with the lactose promoter integrated into the genome was obtained.

4. The application of the engineering bacteria of claim 1 in treating high-salt sewage.

5. The use according to claim 4, wherein the compound is ###00002### The NaCl concentration of the sewage is 3-5% by mass.

6. The method for treating high-salinity wastewater by using the engineered bacteria according to claim 1, characterized in that, comprises the following steps: (1) picking the engineering bacteria of claim 1 to inoculate in NH1 liquid medium for activation culture for 48-72 h, inoculating the activated bacteria liquid into NH1 liquid medium for expansion culture for 24-48 h to obtain a seed liquid at a volume ratio of 2-10%; (2) inoculating the seed liquid obtained in step (1) into NH1 liquid medium added with lactose at a volume ratio of 8-20% for culture for 2-6 h; and (3) inoculating the obtained bacteria liquid into sewage at a volume ratio of 8-20% for sewage treatment at 25-32 °C.

7. The method of claim 6, wherein, The culture conditions in steps (1) and (2) are both 25-32 °C and 150-200 rpm.

8. The method of claim 6, wherein, The addition amount of lactose in step (3) is 0.1-0.5% by mass volume ratio, unit g / mL.

9. The method of claim 6, wherein, The preparation method of the NH1 liquid medium is as follows: adding 8 g of yeast powder, 3 g of glucose, 2 g of NaCl, 1.3 g of potassium dihydrogen phosphate, 0.125 g of anhydrous magnesium sulfate and 1 mL of growth factor mother liquor into 1 L of deionized water, and adjusting the pH value to 7.2; the preparation method of the growth factor mother liquor is as follows: adding 4 mg of biotin, 200 mg of nicotinic acid and 100 mg of thiamine hydrochloride into 100 mL of deionized water, making up to 200 mL, and filtering to remove bacteria.

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