Engineering strain for efficiently degrading new pollutant triclosan and application thereof

Abstract

The invention relates to the field of environmental microbiology and microbial genetic engineering, in particular to an engineering strain for efficiently degrading a new pollutant triclosan and application of the engineering strain. According to the invention, pBBR1MCS2 is selected as a skeleton, tcsAB from Sphingomonas sp.RD1 and tfdB from Sphingomonas sp.YL-M2C are inserted, a resistance gene kanR of the expression vector is replaced by FabV from Pseudomonas triclosanedens ZM23, an obtained plasmid is introduced into Pseudomonas knackmussii B13 after endA is knocked out, the obtained engineering strain not only can efficiently degrade triclosan, but also can avoid the introduction of additional antibiotics, so that the triclosan can be efficiently degraded, the production cost is reduced, and the production efficiency is improved. Abuse of antibiotics and spreading of antibiotic resistance genes can be effectively restrained, and the method has a good application prospect in bioremediation of new pollutants.

Description

Technical Field

[0001] The invention relates to the fields of environmental microbiology and microbial genetic engineering, and in particular to an engineering strain capable of efficiently degrading a new pollutant triclosan and application thereof. Background Art

[0002] In recent years, a large number of new pollutants have been discharged into the water environment, posing a potential threat to aquatic life and human health. Their control has become a major strategic need for my country's sustainable development. New pollutants mainly include persistent organic pollutants, endocrine disruptors, antibiotics, etc. that are controlled by international conventions; strengthening process control, reducing new pollutant emissions, especially standardizing the use of antibiotics and pesticides, strengthening the connection between new pollutant control and environmental management systems such as pollutant discharge permits, and promoting the construction of new pollutant control pilot projects are urgent issues to be addressed in the control of new pollutants.

[0003] Triclosan is one of the new pollutants frequently detected in rural and urban sewage treatment systems. It is also a difficult-to-degrade organic pollutant in the category of Pharmaceuticals and Personal Care Products (PPCPs). Its toxicity to organisms and its environmental remediation research have received extensive attention. As one of the typical PPCPs-type difficult-to-degrade organic pollutants, triclosan can harm the digestive, endocrine and reproductive development systems of aquatic organisms in the water environment, and can also promote the spread of antibiotic resistance genes, posing a potential threat to human health. At present, some physical and chemical remediation methods have been applied to the removal of triclosan in the environment, but they are difficult to be applied in practice because they are easy to produce intermediates and have high maintenance costs. Using microbial metabolism to degrade triclosan in wastewater has become an economical and effective means to control such new pollutants.

[0004] Microorganisms play a major role in the material cycle in nature and can degrade almost all artificial or natural compounds. At present, a total of 21 strains of microorganisms with triclosan degradation ability have been reported, mainly Sphingomonas, Pseudomonas and Rhodococcus. These microorganisms can biodegrade or transform triclosan through direct metabolism or cometabolism. At present, although more and more triclosan-degrading bacteria have been isolated and screened, some strains of these degrading bacteria cannot completely mineralize triclosan and will accumulate the toxic intermediate metabolite 2,4-dichlorophenol; while the degrading bacteria that have been screened to completely mineralize triclosan have problems such as low degradation efficiency and poor stress resistance, and it is difficult to play a long-term role in the actual treatment process.

[0005] Synthetic biology can systematically reshape the phenotype of organisms based on the "bottom-up" design concept, and has become an important research field in life sciences. According to the pollution scenario of a specific environment, functional strains are selected in a targeted manner to obtain new pollutant degradation engineered strains with high degradation efficiency, thereby providing better strain resources for the practical application of environmental microbial technology, breaking through research bottlenecks, and promoting the development of related fields. Compared with traditional technologies, this research strategy has a short cycle, high efficiency, and clear goals, which brings major opportunities for the innovation of environmental microbial remediation technology. Therefore, it is of great practical significance to provide an engineered strain that can efficiently degrade the new pollutant triclosan. Summary of the invention

[0006] In view of this, the present invention provides an engineering strain for efficiently degrading a new pollutant triclosan and its application, wherein a broad-host expression vector pBBR1MCS2 is selected as a skeleton, and the triclosan dioxygenase encoding gene tcsAB from the triclosan-degrading bacteria Sphingomonas sp.RD1 and the 2,4-dichlorophenol monooxygenase encoding gene tfdB from Sphingomonas sp.YL-M2C are inserted, and the kanamycin resistance gene kan of the expression vector is converted into R The triclosan resistance gene fabV from Pseudomonas triclosanedensZM23 is replaced to construct a functional gene plasmid pBBR-tcsAB-tfdB-fabV expressing the initial biodegradation pathway of triclosan. The present invention selects Pseudomonas knackmussii B13 (LMG 23759) as a chassis cell, knocks out the restriction endonuclease gene endA encoded in its genome, and introduces the expression vector pBBR-tcsAB-tfdB-fabV to construct an efficient degradation engineering strain with a complete mineralization pathway of triclosan. And detect its degradation activity on triclosan, in order to provide important scientific support and technical accumulation for the further development of the field of triclosan pollution remediation in water environment.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a genetically engineered strain, the chassis of which is Pseudomonas knackmussii (P. knackmussii), which:

[0009] (i), endA deletion;

[0010] (ii), having tcsA derived from Sphingomonas sp. RD1;

[0011] (iii), having tcsB derived from Sphingomonas sp. RD1;

[0012] (iv) having tfdB derived from Sphingomonas sp. YL-JM2C;

[0013] (v) having fabV derived from P. triclosanedens ZM23.

[0014] In some specific embodiments of the present invention, the Pseudomonas knackmussii of the above genetically engineered strain is Pseudomonas knackmussii B13.

[0015] In some specific embodiments of the present invention, the Pseudomonas knackmussii B13 (P. knackmussii B13) of the above-mentioned genetically engineered strain was purchased from the Belgian Co-ordinated Collections of Micro-organisms (BCCM) Collection Center in Belgium on April 25, 2023, with the collection number LMG 23759.

[0016] In some specific embodiments of the present invention, the expression vector introduced into the above-mentioned genetically engineered strain does not have kan R .

[0017] In some specific embodiments of the present invention, the P. knackmussii B13 of the above-mentioned genetically engineered strain is sensitive to commonly used antibiotics such as streptomycin, chloramphenicol, kanamycin, tetracycline, etc., and the genome does not contain genes encoding a type III secretion system, and has good biosafety.

[0018] In some specific embodiments of the present invention, the restriction endonuclease gene endA encoding the genetically engineered strain P. knackmussii B13 is knocked out to produce a mutant strain P. knackmussii B13ΔendA.

[0019] In some specific embodiments of the present invention, the genetically engineered strain uses P. knackmussii B13ΔendA as a chassis cell to heterologously express triclosan dioxygenase and 2,4-dichlorophenol monooxygenase.

[0020] In some specific embodiments of the present invention, the triclosan dioxygenase of the above genetically engineered strain is derived from Sphingomonas sp. RD1.

[0021] In some specific embodiments of the present invention, the 2,4-dichlorophenol monooxygenase of the above genetically engineered strain is derived from Sphingomonas sp. YL-M2C.

[0022] In some specific embodiments of the present invention, the heterologous expression of the above genetically engineered strain is to insert the triclosan dioxygenase gene tcsAB and the 2,4-dichlorophenol monooxygenase gene tfdB into the wide host expression vector pBBR1MCS2, and to insert the kanamycin resistance gene kan R The triclosan resistance gene fabV from P.triclosanedens ZM23 was replaced to construct an expression vector pBBR-tcsAB-tfdB-fabV, which was introduced into the chassis cell P.knackmussii B13ΔendA to obtain the genetically engineered strain P.knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV, thereby achieving heterologous expression.

[0023] The present invention also provides a method for constructing a genetically engineered strain, comprising: knocking out endA of Pseudomonas knackmussii, introducing an expression vector having tcsA derived from Sphingomonas sp.RD1, tcsB derived from Sphingomonas sp.RD1, tfdB derived from Sphingomonas sp.YL-JM2C, and fabV derived from P.triclosanedens ZM23, to obtain the genetically engineered strain.

[0024] In some specific embodiments of the present invention, the Pseudomonas knackmussii (P. knackmussii) in the above construction method is Pseudomonas knackmussii B13 (P. knackmussii B13).

[0025] In some specific embodiments of the present invention, the introduction of the above construction method does not include the introduction of kan R .

[0026] In some specific embodiments of the present invention, the expression vector in the above construction method is pBBR-tcsAB-tfdB-fabV.

[0027] In some specific embodiments of the present invention, the above genetically engineered strain or the above construction method:

[0028] The nucleotide sequence of endA is SEQ ID NO: 1; and / or

[0029] The nucleotide sequence of tcsA is SEQ ID NO: 2; and / or

[0030] The nucleotide sequence of tcsB is SEQ ID NO: 3; and / or

[0031] The nucleotide sequence of tfdB is SEQ ID NO: 4; and / or

[0032] The nucleotide sequence of fabV is SEQ ID NO:6.

[0033] In some specific embodiments of the present invention, the promoter sequence of the fabV in the genetically engineered strain is SEQ ID NO:5.

[0034] In some specific embodiments of the present invention, the genetically engineered strain has a faster triclosan degradation ability.

[0035] In some specific embodiments of the present invention, the genetically engineered strain can rapidly degrade triclosan with an initial concentration of 10 mg / L within 4 hours at 30°C, and can grow with triclosan as the sole carbon source and energy source.

[0036] The present invention also provides a method for degrading triclosan, including degradation based on the above-mentioned genetically engineered strain.

[0037] In some specific embodiments of the present invention, the method for degrading triclosan comprises: inoculating the genetically engineered strain into sewage to achieve degradation.

[0038] In some specific embodiments of the present invention, the above method for degrading triclosan does not introduce additional antibiotics, and can effectively curb the abuse of antibiotics and the spread of antibiotic resistance genes.

[0039] The strain of the present invention and its application have the following effects:

[0040] The genetically engineered strain provided by the present invention can rapidly degrade the new pollutant triclosan. The engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV was first activated in LB liquid medium, cultured to the logarithmic growth phase, and then cultured at an initial OD 600 The inoculum size is 0.05 and inoculated into an inorganic salt medium containing 10 mg / L triclosan, and the shake flask culture is carried out at 30°C and 200 rpm. The engineered strain has a good degradation effect on triclosan, and can completely degrade triclosan with an initial concentration of 10 mg / L after 4 hours of reaction. No additional antibiotics are introduced during the degradation test as a selective pressure to retain the plasmid, which can effectively curb the abuse of antibiotics and the spread of antibiotic resistance genes. The genetically engineered strain provided by the present invention can be used for rapid and efficient bioremediation of new pollutants, with good economic and environmental benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0042] Figure 1 Annotation of genes encoding secretion system of P. knackmussii B13, where green indicates functional genes annotated in the genome of P. knackmussii B13;

[0043] Figure 2 Schematic diagram of the construction of a scarless knockout vector encoding the restriction endonuclease gene endA in P. knackmussii B13;

[0044] Figure 3 Schematic diagram for the construction of chassis cell P. knackmussii B13ΔendA;

[0045] Figure 4 The electrophoresis verification diagram of the knockout of the restriction endonuclease gene endA encoding P. knackmussii B13, wherein A is the electrophoresis verification diagram of the upstream and downstream homology arms of endA, B is the electrophoresis verification diagram of the endA specific primer amplification, M represents Marker, ΔendA represents the P. knackmussii B13 ΔendA knockout strain, and WT represents the P. knackmussii B13 wild strain;

[0046] Figure 5 Schematic diagram of constructing the expression vector pBBR-tcsAB-tfdB-fabV;

[0047] Figure 6 The electrophoresis verification diagram for constructing the expression vector pBBR-tcsAB-tfdB-fabV, wherein A is the electrophoresis verification diagram of the amplification of each gene of tcsA, tcsB, and tfdB and the connection of tcsAB-tfdB with the linearized plasmid pBBR1MCS2, B is the electrophoresis verification diagram of the connection of fabV with the linearized plasmid pBBR-tcsAB-tfdB, M represents Marker, 1 represents the amplification band of tcsA, 2 represents the amplification band of tcsB, 3 represents the amplification band of tfdB, 4 represents the PCR verification band for verifying the connection of tcsAB-tfdB with the linearized vector pBBR1MCS2, and 5 represents the PCR verification band for the connection of fabV with the linearized vector pBBR-tcsAB-tfdB;

[0048] Figure 7 This is a curve graph of the degradation of triclosan by the engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV, wherein the solid line is the degradation curve graph and the dotted line is the growth curve graph. DETAILED DESCRIPTION

[0049] The present invention discloses an engineered strain for efficiently degrading a new pollutant triclosan and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The present invention provides a genetically engineered strain with high efficiency in degrading triclosan, which is transformed from P. knackmussii B13. P. knackmussii B13 is deposited in the BCCM Collection Center of Belgium with the deposit number LMG 23759. The P. knackmussii B13 used in the present invention was purchased from the collection center on April 25, 2023.

[0051] Furthermore, the P. knackmussii B13 is sensitive to commonly used antibiotics such as streptomycin, chloramphenicol, kanamycin, tetracycline, etc., and is only resistant to ampicillin and penicillin. Its genome contains only one antibiotic resistance gene and lacks genes encoding a type III secretion system, and has good biosafety.

[0052] Furthermore, the restriction endonuclease gene endA encoded by the genetically engineered strain is seamlessly knocked out to construct a mutant strain P. knackmussii B13ΔendA, which can prevent the exogenous gene from being cut.

[0053] Furthermore, the broad host expression vector pBBR1MCS2 was selected as the backbone, and the triclosan dioxygenase gene tcsAB from Sphingomonas sp.RD1 and the 2,4-dichlorophenol monooxygenase gene tfdB from Sphingomonas sp.YL-M2C were inserted, and the kanamycin resistance gene kan R The triclosan resistance gene fabV from P.triclosanedens ZM23 was replaced to construct the expression vector pBBR-tcsAB-tfdB-fabV. The expression vector was introduced into the chassis cell P.knackmussii B13ΔendA to obtain the genetically engineered strain P.knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV.

[0054] The specific embodiment involves the culture medium and reagent formula as follows:

[0055] LB liquid medium: 10.0 g / L tryptone; 5.0 g / L yeast extract; 10.0 g / L NaCl.

[0056] LB solid medium: 10.0 g / L tryptone; 5.0 g / L yeast extract; 10.0 g / L NaCl; agar 2.0 g / 100 mL.

[0057] LBS solid medium: 10.0 g / L tryptone; 5.0 g / L yeast extract; 10.0 g / L NaCl; 15.0 g / 100 mL sucrose; 2.0 g / 100 mL agar.

[0058] Inorganic salt liquid culture medium: 0.66g / L (NH4)2SO4; 1.00g / L MgSO4·7H2O; 0.015g / L CaCl2·2H2O; 0.46g / L K2HPO4; 2.36g / L KH2PO4; 1mL / L trace elements.

[0059] Trace element mother solution: 0.5g / L FeSO4·7H2O; 0.4g / L ZnSO4·7H2O; 0.02g / L MnSO4·H2O; 0.015g / L H3BO3; 0.01g / L NiCl2·6H2O; 0.25g / L EDTA; 0.05g / L CoCl2·6H2O; 0.005g / L CuSO4·2H2O. Store at 4℃.

[0060] 50 mg / mL kanamycin (Km): Dissolve 0.5 g Km in sufficient water, make up to 10 mL, filter and sterilize using a 0.22 μm sterile filter, and dispense into 1.5 mL EP tubes and store at -20°C.

[0061] 75 mM 2,6-Diaminopimelic acid (2,6-DAP): Dissolve 0.1426 g 2,6-DAP in sufficient water to make up to 10 mL. Filter and sterilize using a 0.22 μm sterile filter and dispense into 1.5 mL EP tubes and store at -20°C.

[0062] 10g / L triclosan stock solution: Dissolve 1.0g triclosan in sufficient methanol, make up to 100mL, filter and sterilize using a 0.22μm sterile filter, and dispense into 1.5mL EP tubes and store at 4℃.

[0063] The sequence information involved in the specific embodiment is as follows:

[0064] endA nucleotide sequence:

[0065]

[0066] tcsA nucleotide sequence:

[0067]

[0068]

[0069] tcsB nucleotide sequence:

[0070]

[0071] tfdB nucleotide sequence:

[0072]

[0073] fabV-promoter nucleotide sequence:

[0074]

[0075] fabV nucleotide sequence:

[0076]

[0077]

[0078] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0079] The present invention will be further described below in conjunction with the embodiments.

[0080] Example 1: Safety Assessment of P. knackmussii B13

[0081] 1. Antibiotic resistance spectrum of P. knackmussii B13

[0082] P.knackmussii B13 was inoculated in LB liquid medium for activation, and 200 μL of fresh bacterial liquid in the logarithmic growth phase was evenly spread on LB solid medium, and different types of antibiotic paper discs (streptomycin, kanamycin, penicillin, ampicillin, polymyxin, gentamicin, tetracycline, erythromycin, cephalexin, rifampicin and chloramphenicol) were pasted on the surface of the medium, and cultured at 30°C for 24 to 48 hours to observe the sensitivity of P.knackmussii B13 to various antibiotics. Among them, the appearance of an inhibition zone around the antibiotic paper disc indicates that P.knackmussii B13 is sensitive to this antibiotic; if not, it means that P.knackmussii B13 is tolerant to this antibiotic. The results of the antibiotic tolerance test of P.knackmussii B13 are shown in Table 1. The antibiotic paper discs were purchased from Hangzhou Microbiological Reagent Co., Ltd.

[0083] Table 1: Antibiotic resistance spectrum test results of P. knackmussii B13

[0084] antibiotic Concentration (μg / tablet) Sensitive / Tolerant Streptomycin 10 sensitive Kanamycin 30 sensitive penicillin 10 Tolerance Ampicillin 10 Tolerance Polymyxin 30 sensitive Gentamycin 10 sensitive tetracycline 30 sensitive Erythromycin 15 Tolerance Cephalexin 30 Tolerance Rifampin 5 sensitive Chloramphenicol 30 sensitive

[0085] The results showed that P. knackmussii B13 was sensitive to commonly used antibiotics such as streptomycin, kanamycin, tetracycline, gentamicin, chloramphenicol, etc., which also provided convenient conditions for the subsequent molecular biology experiments.

[0086] 2. Annotation of antibiotic resistance genes in P. knackmussii B13

[0087] The full genome sequence of P. knackmussii B13 (GenBank accession number: HG322950.1) was downloaded and obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ) and uploaded to the Comprehensive Antibiotic Resistance Database (CARD, https: / / card.mcmaster.ca / ) for annotation of antibiotic resistance genes. The results showed that only one antibiotic resistance gene rsmA annotated as "RND antibiotic efflux pump" was detected in the genome of P. knackmussii B13, indicating that there is a lack of antibiotic resistance genes in its genome. P. knackmussii B13 is not a multidrug-resistant strain and has good biosafety.

[0088] 3. Annotation of genes encoding secretion system in P. knackmussii B13

[0089] The genome sequence of P. knackmussii B13 was uploaded to the Kyoto Encyclopedia of Genes and Genomes (KEGG) online analysis website (https: / / www.genome.jp / tools / kofamkoala / ) for functional gene annotation. The gene annotation results of the secretion system encoding genome of P. knackmussii B13 are shown in Figure 2. Figure 1 As shown. The results showed that the P. knackmussii B13 genome lacks functional genes encoding type III, type IV and type V secretion. The bacterial secretion system is closely related to the survival and pathogenicity of bacteria. Among them, the type III secretion system is particularly common in various pathogenic Gram-negative bacteria. It can secrete infection-related proteins and has a strong toxic tendency. No functional genes encoding these proteins were detected in the P. knackmussii B13 genome, indicating that it has good biosafety.

[0090] Example 2: Construction of P. knackmussii B13 restriction endonuclease gene endA knockout vector pK18mobsacB-endA

[0091] 1. Linearization of plasmid pK18mobsacB

[0092] The laboratory-stored plasmid pK18mobsacB (GenBank accession number: FJ437239) was cleaved with restriction endonucleases QuickCut TM EcoR I and QuickCut TM Hind III was used for double digestion at 37°C for 30 min. After digestion, the linearized plasmid was electrophoresed to confirm that the plasmid was completely digested. The nucleic acid purification kit OMEGA GelExtraction Kit was used for purification. After obtaining the linearized plasmid, its concentration and purity were determined by Nanodrop. Restriction endonucleases were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0093] 2. Amplification of upstream and downstream homology arms of P. knackmussii B13 endA

[0094] The construction of knockout vector adopts seamless cloning technology, and the primer design principle refers to the seamless cloning kit MultiS One Step Cloning Kit Instructions. The upstream and downstream homologs of the P. knackmussii B13 restriction endonuclease gene endA with a length of about 1,000 bp were amplified using the high-fidelity enzyme 2×Phanta FlashMaster Mix. The primer sequences are shown in Table 2, and the PCR reaction procedures are shown in Table 3. The PCR amplification products were identified by electrophoresis and the DNA fragments were purified using a nucleic acid purification kit, and finally the concentration was determined using Nanodrop. Seamless cloning kit MultiS One Step Cloning Kit and 2×Phanta Flash Master Mix were purchased from Nanjing Novozyme Biotech Co., Ltd.

[0095] Table 2: Primer sequences for knockout vector construction

[0096] 3. Ligation of the upstream and downstream homology arms of P. knackmussii B13 endA with the linearized plasmid pK18mobsacB

[0097] The linearized plasmid and the upstream and downstream homology arms were connected using a seamless cloning kit MultiS One Step Cloning Kit. Mix the reaction system and place it in a PCR instrument, react at 37℃ for 30 minutes, and store it on ice. Take Escherichia coli DH5α competent cells and place them on ice to melt. Take 10μL of the seamless cloning reaction solution and add it to 100μL competent cells. Mix gently and place it on ice for 30 minutes. Heat shock at 42℃ for 90s, then place it on ice for 3 minutes. Add 700μL LB liquid culture medium and activate it at 37℃ and 180rpm for 1h. Then centrifuge at 8,000rpm for 2 minutes and discard 600μL of the upper

[0098]

[0099] Table 3: PCR program for 2×Phanta Flash Master Mix

[0100] Reaction temperature Reaction time describe Number of cycles 98℃ 30s Pre-denaturation 1 98℃ 10s transsexual 9 55～65℃ 5s Renaturation 9 72℃ 5s / kb extend 9 98℃ 10s transsexual 32 55℃ 5s Renaturation 32 72℃ 5s extend 32 72℃ 1min Complete extension 1 4℃ 1min cool down 1

[0101] The remaining bacterial solution was cleared and resuspended, 150 μL of bacterial solution was spread on LB plates containing 50 mg / L Km, and cultured overnight at 37° C. E. coli DH5α competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0102] After a single colony grows on the plate, pick up multiple single colonies and transfer them to 600 μL LB liquid culture medium containing 50 mg / L Km. After overnight culture at 37°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform bacterial solution PCR verification. The PCR amplification primers select the verification primers pK18-verification-F / pK18-verification-R on both sides of the knockout vector pK18mobsacB restriction site. The sequences of the knockout vector PCR verification primers are shown in Table 2, and the PCR reaction system is shown in Table 4. The PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were analyzed by sequence alignment using SnapGene software, and finally a bacterial solution containing a knockout vector without mutation sites was obtained. Take 200 μL of bacterial solution and transfer it to 6 mL of Km-resistant LB liquid culture medium containing 50 mg / L and culture it at 37°C overnight. The successfully constructed knockout vector pK18mobsacB-endA was used in OMEGA Plasmids were extracted using Plasmid DNA Mini Kit, and their concentration and purity were detected using Nanodrop and stored in a -20°C refrigerator. 2×T5 Super PCR Mix was purchased from Beijing Qingke Biotechnology Co., Ltd. The schematic diagram of the construction of the knockout vector pK18mobsacB-endA is shown in Figure 2 shown.

[0103] Table 4: PCR program for 2×T5 Super PCR Mix

[0104] Reaction temperature Reaction time describe Number of cycles 98℃ 3min Pre-denaturation 1 98℃ 10s transsexual 9 55-65℃ 10s Renaturation 9 72℃ 10s / kb extend 9 98℃ 10s transsexual 30 55℃ 10s Renaturation 30 72℃ 10s extend 30 72℃ 2min Complete extension 1 4℃ 1min cool down 1

[0105] Example 3: Construction of knockout strain P. knackmussii B13ΔendA

[0106] 1. Knockout vector pK18mobsacB-endA was introduced into E.coli WM3064

[0107] Take the E.coli WM3064 competent cells stored in -80℃ refrigerator and put them on ice to dissolve. Melt on ice, take 10μL knockout vector pK18mobsacB-endA and add it to 100μL competent cells, mix gently and put it on ice for 30min, heat shock at 42℃ for 90s, then put it on ice for 3min, add 700μL LB liquid culture medium, and activate it at 37℃ and 180rpm for 1h. Then centrifuge at 8,000rpm for 2min, discard 600μL supernatant, resuspend the remaining bacterial solution, take 150μL bacterial solution and spread it on LB plate containing 50mg / LKm and 0.3mM 2,6-DAP, and culture it at 37℃ overnight. After a single colony grows on the plate, pick several single colonies and transfer them to 600 μL LB liquid medium containing 50 mg / L Km and 0.3 mM 2,6-DAP. After overnight culture at 37°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform PCR verification on the bacterial solution. The PCR amplification primers select the verification primers pK18-verification-F / pK18-verification-R on both sides of the knockout vector pK18mobsacB restriction site.

[0108] 2. Conjugation of E. coli WM3064 containing knockout vector with P. knackmussii B13

[0109] After confirming that the knockout vector pK18mobsacB-endA was successfully introduced into E.coli WM3064, the E.coli WM3064 containing the knockout vector was cultured in LB medium containing 50 mg / L Km and 0.3 mM 2,6-DAP to the late logarithmic phase or the stable phase, and P.knackmussii B13 was cultured to the logarithmic phase or the stable phase. After washing twice with LB medium, the E.coli WM3064 containing the knockout vector and P.knackmussii B13 were mixed at a volume ratio of 2:1, dropped on LB solid medium containing 0.3 mM 2,6-DAP, and cultured at 30°C overnight. The mixed cells were washed with LB medium and washed twice with 10 -1 and 10 -2 The diluted product was spread onto LB solid medium containing 50 mg / L Km and cultured at 30°C overnight.

[0110] 3. Screening of single-crossover mutants

[0111] After a single colony grows on the plate, pick a few single colonies and transfer them to 600 μL LB liquid medium containing 50 mg / L Km. After overnight culture at 30°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform bacterial solution PCR to verify whether a single exchange occurs in the upstream and downstream homology arms. Among them, the upstream homology arm PCR verification primer is endA-Up-Verification-F / endA-Up-Verification-R, and the downstream homology arm PCR verification primer is endA-Down-Verification-F / endA-Down-Verification-R. The primer sequences are shown in Table 5. Among them, the size of the mutant upstream homology arm PCR product is 1,198 bp, and the size of the downstream homology arm PCR product is 1,176 bp. If the size of the PCR product on either side of the upstream and downstream is consistent, it is considered a single exchange mutant.

[0112] 4. Screening of double-crossover mutant P. knackmussii B13ΔendA

[0113] After obtaining the single exchange mutant, it was inoculated into LB liquid medium and cultured at 30°C for 24 h. -3 and 10 -4 The dilution was spread on LBS solid medium and cultured at 30℃ for 48h. After a single colony grew on the plate, multiple single colonies were picked and transferred to LB liquid medium containing 50mg / L Km and without antibiotics for culture. Single colonies that did not grow in the medium containing Km were selected, and the bacterial liquid was used for bacterial liquid PCR using 2×T5 Super PCR Mix to verify whether the double exchange mutant strain P.knackmussii B13ΔendA and whether the endA gene was knocked out. Among them, the primers endA-Up-verification-F / endA-Down-verification-R were used to verify whether the double exchange mutant strain was obtained. The PCR product size of the double exchange mutant strain was 2,332bp, and the PCR product size of the wild-type strain was 3,017bp; endA-F / endA-R was used to verify whether the endA gene was knocked out. The mutant strain could not amplify the band, and the PCR product size of the wild-type strain was 727bp. The primer sequences are shown in Table 5. The schematic diagram of the construction of the mutant strain P. knackmussii B13ΔendA is shown in Figure 3 The electrophoresis verification diagram of P. knackmussii B13ΔendA is shown in Figure 4 As shown. Figure 4 The results showed that the mutant strain P. knackmussii B13ΔendA in which the restriction endonuclease gene endA was knocked out was successfully obtained.

[0114] Table 5: P. knackmussii B13ΔendA single and double exchange verification primer sequences

[0115]

[0116] Example 4: Construction of expression vector pBBR-tcsAB-tfdB-fabV

[0117] 1. Linearization of plasmid pBBR1MCS2

[0118] The laboratory-preserved plasmid pBBR1MCS2 was amplified using primers pBBR-linearized-F / pBBR-linearized-R and high-fidelity enzyme 2×Phanta Flash Master Mix, and the circular plasmid pBBR1MCS2 was removed using restriction endonuclease Dpn I. After restriction digestion at 37°C for 2 h, the linearized plasmid PCR product was identified by electrophoresis and purified using the nucleic acid purification kit OMEGA Gel Extraction Kit was used for purification. After obtaining the linearized plasmid, its concentration and purity were determined by Nanodrop. Restriction endonucleases were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. PCR linearization primers are shown in Table 6.

[0119] Table 6: Primer sequences for expression vector construction

[0120]

[0121]

[0122] 2. In vitro synthesis of genes related to triclosan degradation

[0123] The triclosan dioxygenase encoding gene tcsAB provided by the present invention is derived from triclosan degrading bacteria Sphingomonas sp.RD1 (Kagle, JM; Paxson, C.; Johnstone, P.; Hay, A. Identification of a gene cluster associated with triclosan catabolism. Biodegradation, 2015, 26 (3), 235-246.). Since only the 16S rRNA sequence and the triclosan dioxygenase large subunit gene tcsA sequence of Sphingomonas sp.RD1 are disclosed in the GenBank database, and the small subunit tcsB gene sequence is lacking; and Sphingopyxis sp.MC1, which has a 100.00% identity with the TcsA amino acid sequence of Sphingomonas sp.RD1, has its genome data disclosed in GenBank (GenBank accession number: GCA_000371385). Therefore, tcsA and tcsB sequences of 1,377 bp and 963 bp were obtained from the genome of Sphingopyxis sp.MC1, and tcsA and tcsB were synthesized in vitro by primer synthesis, Overlap PCR, cloning into pET28a vector, and sequencing quality inspection. The genome of P.knackmussii B13 was annotated and analyzed, and it was found that the genome of P.knackmussii B13 had a complete gene cluster encoding the chlorocatechol degradation pathway, but its genome lacked the 2,4-dichlorophenol degradation gene tfdB. 2,4-Dichlorophenol is the product of triclosan degradation catalyzed by dioxygenase TcsAB, so the 2,4-dichlorophenol monooxygenase gene tfdB encoding in the genome of triclosan-degrading bacteria Sphingomonas sp.YL-JM2C (Mulla, SI; Wang, H.; Sun, Q.; Hu, AY; Yu, CP Characterization of triclosan metabolism in Sphingomonas sp. strain YL-JM2C. Scientific Reports, 2016, 6, 21965.) was synthesized in vitro. The gene synthesis was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0124] 3. Ligation of tcsAB-tfdB with linearized vector pBBR1MCS2

[0125] The expression vector was constructed using seamless cloning technology, and the primer design principles refer to the seamless cloning kit MultiS One Step Cloning Kit Instructions. 2×Phanta FlashMaster Mix was used to amplify tcsA, tcsB and tfdB sequences. The primer sequences are shown in Table 6. The PCR amplification products were identified by electrophoresis and purified by a nucleic acid purification kit. Finally, the concentration was determined by Nanodrop.

[0126] The linearized plasmid and each functional gene were connected using a seamless cloning kit MultiS OneStep Cloning Kit. Mix the reaction system and place it in a PCR instrument, react at 37℃ for 30min, and store it on ice. Take E.coli DH5α competent cells and place them on ice to melt. Take 10μL of the seamless cloning reaction solution and add it to 100μL competent cells. Mix gently and place it on ice for 30min. Heat shock at 42℃ for 90s, then place it on ice for 3min, add 700μL LB liquid culture medium, and activate it at 37℃ and 180rpm for 1h. Then centrifuge at 8,000rpm for 2min, discard 600μL supernatant, resuspend the remaining bacterial solution, take 150μL bacterial solution and spread it on LB plate containing 50mg / L Km, and culture it at 37℃ overnight.

[0127] After a single colony grows on the plate, pick multiple single colonies and transfer them to 600 μL LB liquid culture medium containing 50 mg / L Km. After overnight culture at 37°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform bacterial solution PCR verification. The PCR amplification primers select the verification primers pBBR-verification-F / pBBR-verification-R on both sides of the restriction site of the expression vector pBBR1MCS2. The PCR verification primer sequences are shown in Table 6. The PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were analyzed by sequence alignment using SnapGene software, and finally the bacterial solution containing the expression vector without mutation sites was obtained.

[0128] 4. Linearization of Plasmid pBBR-tcsAB-tfdB

[0129] The bacterial solution containing the vector pBBR-tcsAB-tfdB was used as a template, and primers pBBR-tcsAB-tfdB-linearized-F / pBBR-tcsAB-tfdB-linearized-R and high-fidelity enzyme 2×Phanta Flash Master Mix were used for amplification, and restriction endonuclease Dpn I was used to remove the circular plasmid pBBR-tcsAB-tfdB. After restriction digestion at 37℃ for 2h, the PCR product of the linearized plasmid was identified by electrophoresis and purified using the nucleic acid purification kit OMEGA Gel Extraction Kit was used for purification. After obtaining the linearized plasmid, its concentration and purity were determined by Nanodrop. The PCR linearization primers are shown in Table 6.

[0130] 5. Replace the Km resistance of the expression vector with triclosan resistance

[0131] The isozyme gene fabV encoding enoyl acetyltransferase is a triclosan resistance gene. Studies have shown that fabV is highly conserved in the Pseudomonas genus (McFarland, AG; Bertucci, HK; Littman, E.; Shen, JX; Huttenhower, C.; Hartmann, EM Triclosan tolerance is driven by a conserved mechanism diverse Pseudomonas species. Applied and Environmental Microbiology, 2021, 87 (7), e02924-20.). However, the genome annotation results found that the triclosan resistance gene fabV was not annotated in P. knackmussii B13, and P. knackmussii B13 could not grow in LB medium containing triclosan. Therefore, fabV can be replaced by kan in the expression vector pBBR-tcsAB-tfdB. R As a screening marker for the vector. P.triclosanedens ZM23 is a triclosan-degrading bacterium isolated and screened in the early stage (the strain was publicly deposited in the China Marine Microbial Culture Collection Center on December 20, 2022, with the deposit number MCCC 1K08497), and its genome contains the triclosan resistance gene fabV. The GenBank accession number of the genome sequence of P.triclosanedens ZM23 is CP113432.

[0132] The Km resistance gene kan in pBBR-tcsAB-tfdB R The primers pBBR-tcsAB-tfdB-linearization-F / pBBR-tcsAB-tfdB-linearization-R and high-fidelity enzyme 2×Phanta Flash Master Mix were used for PCR linearization amplification on both sides, and the circular plasmid pBBR-tcsAB-tfdB was removed using restriction endonuclease Dpn I. After restriction digestion at 37℃ for 2h, the linearized plasmid PCR product was identified by electrophoresis and purified using the nucleic acid purification kit OMEGA GelExtraction Kit was used for purification. After obtaining the linearized plasmid, its concentration and purity were determined by Nanodrop. Restriction endonucleases were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. Using P.triclosanedens ZM23 bacterial culture as a template, the triclosan resistance gene fabV and its promoter sequence were amplified using the high-fidelity enzyme 2×Phanta Flash Master Mix. The PCR amplification product was identified by electrophoresis and the DNA fragment was purified using a nucleic acid purification kit, and finally the concentration was determined by Nanodrop. The primer sequences are shown in Table 6.

[0133] The linearized plasmid pBBR-tcsAB-tfdB and the triclosan resistance gene fabV were connected using the seamless cloning kit IIOne Step Cloning Kit. Mix the reaction system and place it in a PCR instrument, react at 37℃ for 30min, and store it on ice. Take E.coli DH5α competent cells and place them on ice to melt. Take 10μL of the seamless cloning reaction solution and add it to 100μL competent cells. Mix gently and place it on ice for 30min. Heat shock at 42℃ for 90s, then place it on ice for 3min, add 700μL LB liquid culture medium, and activate it at 37℃ and 180rpm for 1h. Then centrifuge at 8,000rpm for 2min, discard 600μL supernatant, resuspend the remaining bacterial solution, take 150μL bacterial solution and spread it on LB plate containing 1mg / L triclosan, and culture it at 37℃ overnight.

[0134] After a single colony grows on the plate, pick up multiple single colonies and transfer them to 600 μL LB liquid culture medium containing 1 mg / L triclosan. After overnight culture at 37°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform bacterial solution PCR verification. The PCR amplification primers select the verification primers fabV-verification-F / fabV-verification-R at 200 bp upstream and downstream of the inserted fabV sequence on the expression vector pBBR1MCS2 backbone. The PCR verification primer sequences are shown in Table 6. The PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were sequenced using SnapGene software to obtain a bacterial solution containing an expression vector without mutation sites. Take 200 μL of bacterial solution and transfer it to 6 mL LB liquid culture medium containing 1 mg / L triclosan and culture it at 37°C overnight. The successfully constructed expression vector pBBR-tcsAB-tfdB-fabV was used in OMEGA Plasmids were extracted using Plasmid DNA Mini Kit, their concentration and purity were detected using Nanodrop, and the plasmids were stored in a -20°C refrigerator.

[0135] The construction process of the expression vector pBBR-tcsAB-tfdB-fabV is as follows Figure 5 shown. Figure 6 The electrophoresis verification diagram of each fragment, among which the tcsA sequence length is 1,377bp, the tcsB sequence length is 963bp, the tfdB sequence length is 1,803bp, the linearized plasmid pBBR1MCS2 connected with tcsAB-tfdB is verified to have a band size of 4,432bp, and the fabV and its promoter connected with the linearized plasmid pBBR-tcsAB-tfdB is verified to have a band size of 1,578bp.

[0136] Example 5: Construction of engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV

[0137] 1. Expression vector pBBR-tcsAB-tfdB-fabV was introduced into E.coli WM3064

[0138] Take the E.coli WM3064 competent cells stored in -80℃ refrigerator and put them on ice to dissolve. Melt on ice, take 10μL expression vector pBBR-tcsAB-tfdB-fabV and add it to 100μL competent cells, mix gently and put it on ice for 30min, heat shock at 42℃ for 90s, then put it on ice for 3min, add 700μL LB liquid culture medium, and activate it at 37℃ and 180rpm for 1h. Then centrifuge at 8,000rpm for 2min, discard 600μL supernatant, resuspend the remaining bacterial solution, take 150μL bacterial solution and spread it on LB plate containing 1mg / L triclosan and 0.3mM 2,6-DAP, and culture it at 37℃ overnight. After a single colony grows on the plate, pick several single colonies and transfer them to 600 μL LB liquid medium containing 1 mg / L triclosan and 0.3 mM 2,6-DAP. After overnight culture at 37°C and 180 rpm, take the bacterial solution and use 2×T5 Super PCR Mix to perform PCR verification on the bacterial solution. PCR amplification primer verification primer pBBR-verification-F / pBBR-verification-R.

[0139] 2. Conjugation of E. coli WM3064 containing the expression vector pBBR-tcsAB-tfdB-fabV and P. knackmussii B13ΔendA

[0140] After confirming that the expression vector pBBR-tcsAB-tfdB-fabV was successfully introduced into E. coli WM3064, the E. coli WM3064 containing the knockout vector was cultured in LB medium containing 1 mg / L triclosan and 0.3 mM 2,6-DAP to the late logarithmic phase or the stable phase, and P. knackmussii B13ΔendA was cultured to the logarithmic phase or the stable phase. After washing twice with LB medium, the E. coli WM3064 containing the expression vector and P. knackmussii B13ΔendA were mixed at a volume ratio of 2:1, dropped on LB solid medium containing 0.3 mM 2,6-DAP, and cultured at 30°C overnight. The mixed bacteria were washed with LB medium and washed twice with 10 -1 and 10 -2 The diluted samples were spread onto LB solid medium containing 1 mg / L triclosan and cultured at 30°C overnight.

[0141] After a single colony grows on the plate, pick several single colonies and transfer them to 600 μL LB liquid culture medium containing 1 mg / L triclosan. After culturing overnight at 30°C and 180 rpm, take the bacterial solution and use the verification primers pBBR-verification-F / pBBR-verification-R and 2×T5 Super PCR Mix to perform bacterial solution PCR to verify whether the expression vector pBBR-tcsAB-tfdB-fabV is successfully introduced into P. knackmussii B13ΔendA.

[0142] Example 6: Degradation of triclosan by the engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV

[0143] After obtaining the engineered strain P.knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV, P.knackmussii B13ΔendA-pBBR and P.knackmussii B13ΔendA-pBBR were activated using LB containing 10 mg / L Km and LB containing 1 mg / L triclosan, respectively. Take 2 mL of the activated bacterial solution, wash the cells three times with sterile water, resuspend the cells with 2 mL of sterile water, and inoculate them in an inorganic salt liquid containing 10 mg / L triclosan, and adjust the initial OD 600 The OD value was 0.05, cultured at 30℃ and 200rpm, and 1 mL of culture was taken every 1h to detect OD 600The concentration of triclosan was measured by HPLC (Agilent, Germany). The detection conditions are shown in Table 7.

[0144] Table 7: Testing conditions for triclosan concentration

[0145] project describe Mobile phase 76% methanol + 24% water Detection wavelength 281nm Chromatographic columns Eclipse XDB-C18 column(5μm 4.6×250mm) Column temperature 30℃ Flow rate 2.0mL / min Liquid intake 20μL

[0146] The results showed that the engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV had the ability to tolerate and degrade triclosan. It could rapidly degrade triclosan at an initial concentration of 10 mg / L within 4 h, and could grow and degrade triclosan using triclosan as the sole carbon source and energy source ( Figure 7 and Table 8).

[0147] Table 8: Degradation of triclosan by engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV

[0148]

[0149]

[0150] Note: The values ​​in the table are mean ± SD.

[0151] At present, a total of 21 strains of triclosan-degrading microorganisms have been reported. The degradation abilities of different triclosan-degrading bacteria vary, and most of them can only degrade triclosan through co-metabolism, requiring the provision of other carbon sources, or the degrading bacteria cannot completely mineralize triclosan, resulting in the accumulation of toxic intermediate metabolites. Table 9 shows the degradation characteristics of the triclosan-degrading bacteria that have been reported so far.

[0152] Table 9: Degradation characteristics of reported triclosan-degrading bacteria

[0153]

[0154] Compared with the triclosan-degrading bacteria reported so far, the triclosan-degrading engineered strain P. knackmussii B13ΔendA-pBBR-tcsAB-tfdB-fabV provided by the present invention can completely degrade triclosan with an initial concentration of 10 mg / L within 4 hours, and its degradation rate is higher than all the degrading bacteria reported so far, and has the potential to efficiently repair triclosan-contaminated scenes. P. knackmussii B13 has good biosafety, and the triclosan degradation process does not introduce additional antibiotics, thus avoiding the abuse of antibiotics and the spread of antibiotic resistance genes, and has good application prospects.

[0155] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A genetically engineered strain, the chassis of which is Pseudomonas knackmussii, characterized in that: include: (i), endA deletion; (ii), having tcsA derived from Sphingomonas sp. RD1; (iii), having tcsB derived from Sphingomonas sp. RD1; (iv) having tfdB derived from Sphingomonas sp. YL-JM2C; (v) having fabV derived from Pseudomonas triclosanedens ZM23.

2. The genetically engineered strain according to claim 1, characterized in that The Pseudomonas knackmussii is Pseudomonas knackmussii B13.

3. The genetically engineered strain according to claim 1 or 2, characterized in that: No resistance gene kan R expression vector.

4. The genetically engineered strain according to any one of claims 1 to 3, characterized in that include: The nucleotide sequence of endA is SEQ ID NO: 1; The nucleotide sequence of tcsA is SEQ ID NO: 2; The nucleotide sequence of tcsB is SEQ ID NO: 3; The nucleotide sequence of tfdB is SEQ ID NO: 4; The nucleotide sequence of fabV is SEQ ID NO:

6.

5. The genetically engineered strain according to any one of claims 1 to 4, characterized in that The promoter sequence of fabV is SEQ ID NO:

5.

6. A method for constructing a genetically engineered strain, characterized in that: include: The endA of Pseudomonas knackmussii B13 (P. knackmussii B13) is knocked out, and an expression vector having tcsA derived from Sphingomonas sp. RD1, tcsB derived from Sphingomonas sp. RD1, tfdB derived from Sphingomonas sp. YL-JM2C and fabV derived from P. triclosanedens ZM23 is introduced to obtain the genetically engineered bacteria.

7. The construction method according to claim 6, characterized in that: The expression vector does not include the resistance gene kan R .

8. A method for degrading triclosan, characterized in that: Degradation based on the genetically engineered strain according to any one of claims 1 to 5.

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