Rhodococcus ruber SD3 locen-2 protease-like protein 2 gene enhanced strain as well as construction method and application of rhodococcus ruber SD3 locen-2 protease-like protein 2 gene enhanced strain

Abstract

The invention relates to a Rhodococcus ruber SD3 locen-2 protease-like protein 2 gene enhanced strain, which is a recombinant strain obtained by improving the expression of a gene s2plp2 of R.ruber SD3 in a R.ruber SD3 wild strain. The Rhodococcus ruber SD3 locen-2 protease-like protein 2 gene enhanced strain has the advantages that the expression of the gene s2plp2 of the R.ruber SD3 is improved; the method for constructing the enhanced strain comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a gene s2plp2 of R.ruber SD3, and constructing a recombinant plasmid pNV18-s2plp2 carrying the gene s2plp2; the method comprises the following steps: carrying out recombinant escherichia coli expression on recombinant escherichia coli to obtain plasmids, transforming the plasmids into E. coli Top 10 competent cells, culturing a large amount of recombinant escherichia coli, extracting the plasmids, and electrically transforming the extracted plasmids into R.ruber SD3 competent cells to obtain the R.ruber s2plp2 gene enhanced strain R.ruber s2plp2-E. ruber s2plp2-E. ruber s2plp2-E. ruber s2plp2-E. ruber s2plp2-E. ruber s2plp2-E. ruber s2plp2. Compared with an R.ruber SD3 wild strain, the expression quantity of the s2plp2 gene in the R.ruber SD3 s2plp2 gene enhanced strain is obviously enhanced by 3.35 times, and the R.ruber SD3 s2plp2 gene enhanced strain shows stronger tolerance to phenol stress.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to an enhanced strain of Rhodococcus SD3 site-2 protease-like protein 2 gene, its construction method, and its application. Background Technology

[0002] Although microbial-mediated bioremediation of organic solvents offers significant advantages, its molecular regulatory mechanisms remain unclear, and the function of transmembrane signal transduction proteins in organic solvent stress responses is lacking validation. Existing research indicates that *Rhodococcus ruber* SD3 (R. ruber SD3) achieves a 99.73% phenol degradation rate after 72 hours of shaking in an inorganic phenol medium (phenol concentration 1.0 g / L) at 35°C and 200 rpm. *Rhodococcus ruber* SD3 can also degrade isooctane, cyclohexane, benzene, n-heptane, toluene, acetonitrile, chlorobenzene, naphthalene, n-hexane, and 1-naphthol. Further research is needed to improve the tolerance of *R. ruber* SD3 to compounds such as phenol. Summary of the Invention

[0003] The purpose of this invention is to provide an enhanced strain of R. ruber SD3 site-2 protease-like protein 2 gene, named Rhodococcus ruber s2plp2-E, deposited at the China Center for Type Culture Collection (CCTCCNO: M20251446), dated June 23, 2025, at the China Center for Type Culture Collection, Wuhan University.

[0004] A recombinant strain was obtained by enhancing the expression of the s2plp2 gene in a Rhodococcus SD3 site-2 protease-like protein 2 gene strain from the wild-type R. ruber SD3 strain. The s2plp2 gene sequence of the recombinant strain is shown in Table SEQ ID NO:1. The wild-type R. ruber SD3 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2012035, deposited on February 26, 2012, at the China Center for Type Culture Collection, Wuhan University.

[0005] A method for constructing a *Rhodotorula ruber* SD3 site-2 protease-like protein 2 gene-enhanced strain as described in claim 1, characterized by comprising: PCR amplification of the *s2plp2* gene of *R. ruber* SD3; constructing a recombinant plasmid pNV18-s2plp2 carrying the *s2plp2* gene; transforming the plasmid into *E. coli* Top 10 competent cells; culturing recombinant *E. coli* in large quantities and extracting the plasmid; electroporating the extracted plasmid into *R. ruber* SD3 competent cells to obtain the *R. ruber* SD3 s2plp2 gene-enhanced strain *R. ruber* s2plp2-E.

[0006] Preferably, the construction method includes the following specific steps:

[0007] (1) Take R. ruber SD3 seed culture for streaking culture, pick single colonies of R. ruber SD3 from the streaking culture for culture, extract R. ruber SD3 whole genome DNA, use R. ruber SD3 whole genome DNA as template, PCR amplify the target gene s2plp2 and purify the PCR product;

[0008] (2) The empty vector pNV18 was obtained by double digestion with EcoRI and HindIII to obtain the linearized vector pNV18, and the double digestion products were recovered by gel extraction.

[0009] (3) The purified linearized vector pNV18 was homologously recombinated with the target gene s2plp2, and the homologous recombination product was transformed into E. coli Top 10 competent cells;

[0010] (4) Select white monoclonal colonies for kanamycin antibiotic screening, and sequence the selected monoclonal colonies for verification; then, expand the sequenced monoclonal colonies and extract the recombinant plasmid pNV18-s2plp2.

[0011] (5) Prepare R. ruber SD3 competent cells and electroporate the recombinant plasmid pNV18-s2plp2 into R. ruber SD3 competent cells to obtain R. ruber s2plp2-E.

[0012] Preferably, in step (1), the method for extracting the whole genome DNA of R. ruber SD3 is as follows: take 3 mL of R. ruber SD3 bacterial culture in the logarithmic growth phase, centrifuge to remove the supernatant, add 500 μL of sterile deionized water to the precipitate to resuspend the bacterial precipitate, centrifuge and wash repeatedly; add 300 μL of sterile deionized water again to resuspend the precipitate, place it in a water bath and heat until boiling for 10 min, then immediately place it in a -20℃ freezer for 10 min; after thawing, centrifuge and take the supernatant for later use.

[0013] Preferably, in step (1), the PCR amplification of the target gene s2plp2 and the purification of the PCR product are as follows: First, PCR amplification primers for the gene s2plp2 are synthesized: the sequence of the forward primer is 5'-tatgaccatgattacgaattcATGCTGCGC GGATCGGTC-3'; the sequence of the reverse primer is 5'-acgacggccagtgccaagcttCTACTCCGGCCG GGGCTG-3', with the enzymatic restriction sites EcoRI and HindIII at the underlined positions, respectively. Then, using R. ruber SD3 genomic DNA as a template, the s2plp2 fragment is amplified and purified using a kit.

[0014] Preferably, in step (2), the method for obtaining the linearized vector pNV18 is as follows: Prepare the reaction system: add 30 μL of plasmid pNV18, add 2 μL each of restriction endonucleases EcoR I and Hind III, add 10 μL of double enzyme digestion buffer 10×M, and adjust the volume to 50 μL using ddH2O; place the PCR tube containing 50 μL of reaction system in a 37℃ water bath for 3 h, and detect the size and purity of the PCR product by 1% (m / v) agarose gel electrophoresis.

[0015] Preferably, in step (3), the method for transforming E. coli Top 10 competent cells with the homologous recombination product is as follows: Take 100 μL of E. coli Top 10 competent cells from a -80℃ freezer, thaw them on ice, add all the homologous recombination product after the reaction to 100 μL of E. coli Top 10 competent cells under aseptic conditions, shake gently, place on ice for 30 min, heat shock in a 42℃ water bath for 90 s, and then cool on ice for about 5 min; add 600 μL of LB liquid culture medium to the tube, mix well, and rejuvenate at 37℃ and 200 r / min for 45-60 min to restore the bacteria to normal growth state. Shake the above bacterial solution well and take 100 μL to spread on an LB solid plate containing 50 μg / mL kanamycin antibiotic, place it face up for half an hour, and after the bacterial solution is completely absorbed by the culture medium, invert the culture dish and incubate at 37℃ for 12-16 h.

[0016] Preferably, in step (5), the method for preparing R. ruber SD3 competent cells is as follows: In a clean bench, R. ruber SD3 seed culture is picked up with a 10 μL pipette tip and streaked onto an antibiotic-free LB agar plate, and then placed in a constant temperature incubator at 35°C for 48 h; a single colony from the above plate is picked up with a 10 μL pipette tip and transferred to 50 mL of antibiotic-free LB liquid medium, and cultured at 35°C and 200 r / min until OD. 595 The nm value is approximately 0.7. Take 100 mL of bacterial culture, centrifuge at 8,000 × g and 4 °C for 20 min, discard the supernatant, add 20 mL of 300 mM sucrose containing 10% (m / v) glycerol pre-cooled and sterilized at 4 °C to resuspend the bacterial precipitate, centrifuge at 8,000 × g and 4 °C for 20 min, discard the supernatant, repeat the washing of the precipitate 3 times, finally add 2 mL of 300 mM sucrose solution containing 10% (m / v) glycerol to the bacterial precipitate and gently pipette to resuspend the precipitate, aliquot 100 μL into each new sterile 1.5 mL centrifuge tube, and store at -80 °C.

[0017] Preferably, in step (5), the method for electroporating the recombinant plasmid pNV18-s2plp2 into R. ruber SD3 competent cells is as follows: Take 10 μL of recombinant plasmid pNV18-s2plp2 and add it to thaw on ice or to 100 μL of freshly prepared R. ruber SD3 competent cells. Gently tap the bottom of the 1.5 ml centrifuge tube with your finger to mix the cells and plasmid. Incubate on ice for 100 min. Set the electroporation conditions: voltage 1.5 KV, electroporation time 2.8 ms. Pre-cool the sterilized electroporation cup, add the mixture to it, and quickly place the electroporation cup into the electroporation tank. After electroporation twice, immediately aspirate the mixture and add it to a centrifuge tube containing 500 μL of antibiotic-free LB liquid medium. Place the centrifuge tube in a shaker and incubate at 35℃ and 200 r / min for 6 h to revive the cells. Take 100 μL of the revived bacterial solution and spread it on LB medium with a final kanamycin concentration of 50 μg / mL. Solid plates were incubated upside down at 35°C for 48 h. Multiple single colonies from the solid plates were picked and placed in 5 mL LB liquid medium with a final kanamycin concentration of 50 μg / mL and incubated for about 48 h. Seed culture was then retained.

[0018] Applications of the Rhodococcus SD3 site-2 protease-like protein 2 enhanced strain include: degradation of isooctane, cyclohexane, benzene, n-heptane, toluene, acetonitrile, chlorobenzene, naphthalene, n-hexane, and 1-naphthol contaminants.

[0019] The technical effects of this invention are: the expression level of the s2plp2 gene in R. ruber s2plp2-E is significantly enhanced by 3.35 times, and it exhibits stronger tolerance to phenol stress. Attached Figure Description

[0020] Figure 1 The construction of recombinant plasmid pNV18-s2plp2; Figure A. Double digestion of empty vector pNV18 with EcoRI and HindIII; Figure B. Agarose gel recovery of linearized vector pNV18; Figure C. PCR amplification of target gene s2plp2; Figure D. Agarose gel recovery of target gene s2plp2; Figure E. PCR amplification to verify positive clones of recombinant plasmid pNV18-s2plp2.

[0021] Figure 2 Figure A shows the PCR amplification and verification of the positive clone of the s2plp2 gene enhancement strain; Figure B shows the PCR amplification of the kanamycin resistance gene; Figure B shows the PCR amplification of the target gene and the fragment connected to the vector.

[0022] Figure 3 This study measured the expression level of the s2plp2 gene in R. ruber SD3 s2plp2-E.

[0023] Figure 4 The growth curves of wild-type R. ruber SD3 and R. ruber s2plp2-E under different concentrations of phenol stress are shown. Detailed Implementation

[0024] To better explain the present invention, a detailed description of specific embodiments is provided.

[0025] In one embodiment, the Rhodococcus ruber SD3 (R. ruberSD3) is the R. ruber SD3 described in the invention patent with authorization announcement number CN102604875B and invention title "Rhodococcus and its application in the degradation of phenol pollutants".

[0026] In a first aspect, the present invention provides a s2plp2 gene-enhanced strain of R. ruber SD3, which is a recombinant strain obtained by enhancing the expression of the s2plp2 gene in R. ruber SD3 from wild-type R. ruber SD3, the gene sequence of which is shown in Table SEQ ID NO:1.

[0027] Secondly, the present invention provides a method for constructing an enhanced strain of the s2plp2 gene of R. ruber SD3, comprising: PCR amplification of the s2plp2 gene of R. ruber SD3, constructing a recombinant plasmid pNV18-s2plp2 carrying the s2plp2 gene; transforming the plasmid into E. coli Top 10 competent cells, culturing recombinant E. coli in large quantities and extracting the plasmid, and electroporating the extracted plasmid into R. ruber SD3 competent cells to obtain an enhanced strain of the s2plp2 gene of R. ruber SD3, R. ruber s2plp2-E.

[0028] The method for constructing R. ruber s2plp2-E includes the following steps:

[0029] (1) Take R. ruber SD3 seed culture for streaking culture, pick single colonies of R. ruber SD3 from the streaking culture for culture, extract R. ruber SD3 whole genome DNA, use R. ruber SD3 whole genome DNA as template, PCR amplify the target gene s2plp2 and purify the PCR product;

[0030] (2) The empty vector pNV18 was obtained by double digestion with EcoRI and HindIII to obtain the linearized vector pNV18, and the double digestion products were recovered by gel extraction.

[0031] (3) The purified linearized vector pNV18 was homologously recombinated with the target gene s2plp2, and the homologous recombination product was transformed into E. coli Top 10 competent cells;

[0032] (4) Select white monoclonal colonies for kanamycin antibiotic screening, and then verify the selected monoclonal colonies by sequencing. Subsequently, the monoclonal colonies that have been successfully verified by sequencing are expanded and the recombinant plasmid pNV18-s2plp2 is extracted.

[0033] (5) Prepare R. ruber SD3 competent cells and electroporate the recombinant plasmid pNV18-s2plp2 into R. ruber SD3 competent cells to obtain R. ruber s2plp2-E;

[0034] In step (1), the method for extracting the whole genome DNA of R. ruber SD3 is as follows: Take 3 mL of R. ruber SD3 bacterial culture in the logarithmic growth phase, centrifuge and discard the supernatant. Add 500 μL of sterile deionized water to the precipitate to resuspend the bacterial precipitate, centrifuge at 12,000 r / min for 1 min, and repeat the washing 3 times. Add 300 μL of sterile deionized water again to resuspend the precipitate, place it in a water bath and heat until boiling for 10 min, then immediately place it in a -20℃ freezer for 10 min. After thawing, centrifuge at 12,000 r / min for 1 min, and collect the supernatant for later use.

[0035] In step (1), the preparation method for PCR amplification of the target gene s2plp2 and purification of the PCR product is as follows: First, synthesize PCR amplification primers for gene s2plp2: the sequence of the forward primer is 5'-tatgaccatgattac gaattc ATGCTGCGCGGATCGGTC-3'; the reverse primer sequence is 5'- acgacggccagtgcc aagctt CTACTCCGGCCG GGGCTG-3', where the underlined areas represent the EcoRI and HindIII restriction sites, respectively. Using R. ruber SD3 genomic DNA as a template, the s2plp2 fragment was amplified, and the PCR product was purified using an Omega DNA agarose gel extraction kit.

[0036] In step (2), the linearized vector pNV18 was prepared as follows: The reaction system was prepared by adding 30 μL of plasmid pNV18, 2 μL each of restriction endonucleases EcoRI and HindIII, and 10 μL of double digestion buffer 10 × M. The volume was then adjusted to 50 μL using ddH2O. The PCR tube containing 50 μL of the reaction system was placed in a 37℃ water bath for 3 h. The size and purity of the PCR products were detected by 1% (m / v) agarose gel electrophoresis. DNA agarose gel extraction was performed using an Omega DNA extraction kit.

[0037] In step (5), the preparation method of R. ruber SD3 competent cells is as follows: In a clean bench, R. ruber SD3 seed culture is picked up with a 10 μL pipette tip and streaked onto an antibiotic-free LB agar plate. The plate is then placed in a constant temperature incubator and cultured at 35℃ for 48 h. Single colonies from the above plate are picked up with a 10 μL pipette tip and transferred to 50 mL of antibiotic-free LB liquid medium. The culture is then carried out at 35℃ and 200 r / min until OD500. 595 The nm value is approximately 0.7. Take 100 mL of bacterial culture, centrifuge at 8,000 × g, 4 °C for 20 min, discard the supernatant, add 20 mL of 300 mM sucrose solution containing 10% (m / v) glycerol (pre-cooled and sterilized at 4 °C) to resuspend the bacterial pellet, centrifuge at 8,000 × g, 4 °C for 20 min, discard the supernatant, repeat the washing of the pellet 3 times, finally add 2 mL of 300 mM sucrose solution containing 10% (m / v) glycerol to the bacterial pellet and gently pipette to resuspend the pellet, aliquot 100 μL into new sterile 1.5 mL centrifuge tubes, and store at -80 °C. The bacterial pellet needs to be kept at a low temperature throughout the process.

[0038] In step (5), the method for electroporating the recombinant plasmid pNV18-s2plp2 into R. ruber SD3 competent cells is as follows: Take 10 μL of the recombinant plasmid pNV18-s2plp2 and add it to thawed cells on ice or freshly prepared 100 μL of R. ruber SD3 competent cells. Gently tap the bottom of the 1.5 ml centrifuge tube with your finger to mix the cells and plasmid. Incubate on ice for 100 min. Set the electroporation conditions: voltage 1.5 kV, electroporation time 2.8 ms. Pre-cool the sterilized electroporation cuvette, add the mixture to it, and quickly place the cuvette into the electroporation tank. After electroporation twice, immediately aspirate the mixture and add it to a centrifuge tube containing 500 μL of antibiotic-free LB liquid medium. Place the centrifuge tube in a shaker and incubate at 35°C and 200 r / min for 6 h to allow the cells to recover. Take 100 μL of the revived bacterial culture and spread it on LB agar plates with a final kanamycin concentration of 50 μg / mL. Incubate at 35°C upside down for 48 h. Pick multiple single colonies from the agar plates and place them in 5 mL of LB liquid medium with a final kanamycin concentration of 50 μg / mL. Incubate for approximately 48 h and retain the seed culture. Further verify the positive clones of R. ruber s2plp2-E by PCR.

[0039] Example 1

[0040] This embodiment describes the construction of R. ruber s2plp2-E. The specific experimental method is as follows:

[0041] (1) The linearized vector pNV18 was obtained by double digestion of the empty vector plasmid pNV18 with EcoRI and HindIII. The reaction system is shown in Table 1. PCR tubes containing 50 μL of the reaction system were placed in a 37℃ water bath for 3 h. The size and purity of the PCR products were detected by agarose gel electrophoresis at a concentration of 1% (m / v). Figure 1 As shown in Figure A, a distinct band is visible between 4,000 and 7,000 bp, matching the target band size of 4,411 bp, indicating successful linearization of the empty vector plasmid pNV18. DNA was extracted and recovered using the Omega DNA agarose gel extraction kit. Figure 1 As shown in Figure B, a distinct band can be seen between 4,000 and 7,000 bp, indicating successful recovery of the linearized vector pNV18.

[0042] Table 1. Double enzyme digestion reaction system

[0043] Table1 Double digestion reaction system

[0044] reagents Usage pNV18 30μL EcoR I 2 μL Hind III 2 μL 10 ×M buffer 10 μL <![CDATA[ddH2O]]> Add to 50 μL

[0045] (2) The target gene was analyzed, and primer fragments containing the homologous recombination ligation vector pNV18 were designed. Forward primer sequence: 5'-tatgaccatgattacgaattcATGCTGCGCGGATCGGTC-3', reverse primer sequence: 5'-acgacggccagtgccaagcttCTACTCCGGCCGGGGCTG-3'. Specific PCR parameters were as follows: 95℃ pre-denaturation for 5 min; followed by 95℃ denaturation for 1 min, 64℃ annealing for 30 s, and 72℃ extension for 55 s, repeated 34 times; then a final extension at 72℃ for 5 min, followed by storage at 16℃ for 10 min. The size and purity of the PCR products were detected using 1% (m / v) agarose gel electrophoresis. Figure 1 As shown in C, a distinct band can be observed in lanes 1-2, between 1,000-2,000 bp, matching the size of the target band at 1,173 bp, indicating successful PCR amplification of the target gene s2plp2. The DNA was recovered from the gel using the Omega DNA Agarose Gel Extraction Kit. Figure 1 As shown in D, in lane 1, a distinct band can be seen between 1,000 and 2,000 bp, which matches the size of the target band of 1,173 bp, indicating that the target gene s2plp2 has been successfully recovered. Figure 1 The middle lanes M are for DL2,000 marker and DL10,000 marker respectively.

[0046] (3) Homologous recombination was performed between the purified linearized vector pNV18 and the target gene s2plp2. The homologous recombination system is shown in Table 2. The PCR tube containing 10 μL of the reaction system was placed in a 37℃ water bath for 15 min.

[0047] Table 2 Homologous recombination reaction system

[0048] Table 2 Homologous recombination reaction system

[0049] reagents Usage Linearized carrier pNV18 3.5 μL s2plp2 1.5 μL 2 × Ezmax Universal CloneMix 5.0 μL

[0050] (4) Subsequently, the homologous recombination product needs to be transformed into E. coli Top 10 competent cells. Take 100 μL of E. coli Top 10 competent cells from a -80℃ freezer, thaw them on ice, and add all the homologous recombination product after the reaction to 100 μL of E. coli Top 10 competent cells under aseptic conditions. Shake gently and place on ice for 30 min. Heat shock in a 42℃ water bath for 90 s, and then cool on ice for about 5 min. Add 600 μL of LB liquid medium to the tube, mix well, and incubate at 37℃ and 200 r / min for 45-60 min to allow the bacteria to return to normal growth. Shake the above bacterial solution well and take 100 μL to spread on an LB solid plate containing 50 μg / mL kanamycin antibiotic. Place it face up for half an hour until the bacterial solution is completely absorbed by the medium. Then invert the culture dish and incubate at 37℃ for 12-16 h.

[0051] (5) Select white monoclonal colonies and add them to LB liquid medium with a final concentration of kanamycin antibiotic of 50 μg / mL. Incubate at 37℃ and 200 r / min. 595 The nm value was 0.8-1.0, and a portion of the bacterial culture was taken to retain the seed culture. Simultaneously, after culturing at 37℃ and 200 r / min for 12-16 h, plasmid extraction was performed using the SanPrep column-based plasmid mini-extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). Plasmid size and purity were detected by 1% (m / v) agarose gel electrophoresis. Positive clones of the recombinant plasmid pNV18-s2plp2 were verified by PCR amplification. Figure 1 As shown in Figure E, a distinct band between 1,000 and 2,000 bp is visible in lane 1, matching the target band size of 1,173 bp, indicating successful amplification of the target gene s2plp2 using plasmid number 1 as a template. Sample number 1 was then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to further identify positive clones. Analysis of the sequenced nucleotides confirmed the successful construction of the recombinant plasmid pNV18-s2plp2.

[0052] Example 2

[0053] This embodiment describes the construction of R. ruber s2plp2-E, and the specific experimental method is as follows:

[0054] (1) In a clean bench, use a 10 μL pipette tip to pick up the R. ruber SD3 seed culture and streak it onto an antibiotic-free LB agar plate. Incubate at 35°C for 2 days. Use a 10 μL pipette tip to pick up a single colony from the above plate and transfer it to 50 mL of antibiotic-free LB liquid medium. Incubate at 35°C and 200 r / min until OD reaches 0.50. 595 The nm value is approximately 0.7. Take 100 mL of bacterial culture, centrifuge at 8,000 × g, 4 °C for 20 min, discard the supernatant, add 20 mL of 300 mM sucrose solution containing 10% (v / v) glycerol (pre-cooled and sterilized at 4 °C) to resuspend the bacterial pellet, centrifuge at 8,000 × g, 4 °C for 20 min, discard the supernatant, repeat the washing of the pellet 3 times, finally add 2 mL of 300 mM sucrose solution containing 10% (v / v) glycerol to the bacterial pellet and gently pipette to resuspend the pellet, aliquot 100 μL into new sterile 1.5 mL centrifuge tubes, and store at -80 °C. The bacterial pellet needs to be kept at a low temperature throughout the process.

[0055] (2) Take 10 μL of recombinant plasmid pNV18-s2plp2 and add it to thawed on ice or freshly prepared 100 μL of R. ruberSD3 competent cells. Gently tap the bottom of the 1.5 ml centrifuge tube with your finger and incubate on ice for 100 min.

[0056] (3) Set the electroporation conditions: voltage 1.5 kV, electroporation time 2.8 ms. Pre-cool the sterilized electroporation cup, add the mixture into it, quickly place the electroporation cup into the electroporation tank, and after electroporation 2 times, immediately add the mixture into a centrifuge tube containing 500 μL LB liquid culture medium.

[0057] (4) The above centrifuge tubes were cultured at 35°C and 200 r / min for 6 h to revive the cells.

[0058] (5) Take 100 μL of the revived bacterial culture and spread it on an LB solid plate with a final kanamycin concentration of 50 μg / mL. Incubate at 35°C upside down for 48 h.

[0059] (6) Pick multiple single colonies from the solid plate and add them to 5 mL of LB liquid medium with a final kanamycin concentration of 50 μg / mL. Incubate at 35℃ and 200 r / min for 48 h, and retain the seed culture. Centrifuge 3 mL of the bacterial culture to remove the supernatant. Add 500 μL of sterile ddH2O to the precipitate to resuspend the bacterial pellet, centrifuge at 12,000 r / min for 1 min, and repeat the washing of the pellet 3 times. Add 300 μL of sterile ddH2O again to resuspend the bacterial pellet, boil at 100℃ for 15 min, and immediately freeze at -20℃ for 10 min. After thawing, centrifuge at 12,000 r / min for 2 min, and collect the whole genome supernatant for later use.

[0060] (7) PCR was performed using kanamycin resistance gene primers (forward primer: 5'-ATGATTGAACAAGATG G-3', reverse primer: 5'-TCAGAAGAACTCGTCAAGAA-3') to preliminarily verify the success of electroporation transformation. The specific PCR parameters were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 52℃ annealing for 30 s, 72℃ extension for 55 s, for a total of 34 cycles; 72℃ extension for 5 min; and 16℃ incubation for 10 min. Positive clones were verified by 1% (m / v) agarose gel electrophoresis. Figure 2 As shown in Figure A, lanes 1 and 2 represent the amplification of the kanamycin resistance gene using recombinant plasmid pNV18-s2plp2 and plasmid pNV18, respectively. In lanes 3-4 of the experimental group, the size of the PCR-amplified fragment matches the size of the kanamycin resistance gene, which is 795 bp.

[0061] (8) Design identification primers based on a fragment from the pNV18 vector plus a target fragment connected to the vector. For the recombinant plasmid pNV18-s2plp2, design the forward identification primer: 5'-TTAGGCACCCCAGGCTTTACAC-3', and the reverse identification primer: 5'-GGCAGCAGGTTGA A CACGG-3'. Further verify the enhanced positive clones by PCR. The specific PCR parameters are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 56℃ annealing for 30 s, 72℃ extension for 55 s, for a total of 34 cycles; 72℃ full extension for 5 min; 16℃ incubation for 10 min. Verify the positive clones by 1% (m / v) agarose gel electrophoresis. Figure 2 As shown in Figure B, lanes 1 and 2 represent the amplification of the target gene and vector-linked fragment using recombinant plasmid pNV18-s2plp2 and plasmid pNV18, respectively. In lanes 3-4 of the experimental group, the size of the PCR-amplified fragment matches the theoretical size of 591 bp. This indicates that the s2plp2 gene-enhanced strain was successfully constructed. Figure 2 The middle lane (M) is the DL2,000 marker.

[0062] Example 3

[0063] This embodiment verifies the relative expression level of s2plp2 mRNA in R. ruber s2plp2-E. The specific experimental method is as follows:

[0064] (1) R. ruber SD3 wild strain and R. ruber s2plp2-E were streaked on solid plates and activated by constant temperature incubation at 35℃ for 2 days. Single colonies were picked and inoculated into 50 mL of liquid culture medium without antibiotics and cultured at 35℃ and 200 r / min until saturation.

[0065] (2) At a ratio of 1:50, saturated R. ruber SD3 wild-type strain and R. ruber s2plp2-E bacterial culture were transferred to 5 mL of antibiotic-free LB liquid medium and cultured at 35℃ and 200 r / min until OD. 595 The nm value was approximately 0.5. The cells were cultured at 35℃ and 200 r / min for 2 days. 3 mL of OD was collected. 595 Bacterial suspensions with an nm value of approximately 0.5 were centrifuged to collect the bacterial cell precipitate. The precipitate was then washed twice with 0.85% (m / v) NaCl, and the precipitate was transferred to a 1.5 mL sterile cryovial. The precipitate was then treated with liquid nitrogen for 10–15 min and stored at -80°C. Three biological replicates were taken from each sample group.

[0066] (3) RNA was extracted from the treated bacterial precipitate using the Omega Bio-Tek Bacterial RNA Kit R6950. After extraction, 1% (m / v) agarose gel electrophoresis was performed, and the RNA concentration was detected using an ultra-micro nucleic acid detector. RNA samples can be stored at -80℃, and repeated freeze-thaw cycles should be avoided as much as possible. cDNA was synthesized using the PrimeScript FAST RT reagent Kit with gDNAEraser reverse transcription kit from Takara Biotech (Beijing) Co., Ltd. (first, follow the system in Table 3, react at 42℃ for 2 min, and use the final product as the RT reaction solution. Then, follow the system in Table 4, and the PCR instrument reaction program is 37℃ for 15 min; 85℃ for 5 s. cDNA samples can be stored at -20℃).

[0067] Table 3 Reaction system for removing genomic DNA

[0068] Table 3 Reaction System for Genomie DNA Removal

[0069] reagents Usage 8 × gDNA Eraser Premix 2.0 μL Total RNA 5.0 μL <![CDATA[RNase Free ddH2O]]> Add to 16.0 μL

[0070] Table 4 Reverse transcription reaction system

[0071] Table 4 Reverse transcription reaction system

[0072] reagents Usage RT reaction 16.0 μL 5 × RT Premix 4.0 μL Total 20.0 μL

[0073] (4) Based on the gene sequence of s2plp2, primers that meet the requirements for real-time PCR were designed. The primer sequences are shown in Table 5. After the primer specificity was tested, they were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0074] Table 5. Names, sequences, and lengths of qRT-PCR amplification primers

[0075] Table 5 The name, sequence and length of qRT-PCR amplificationprimers

[0076] Gene Forward sequence (5'-3') Reverse sequence(5'-3') PCR Products s2plp2 GCAGCAGGTTGAACACGG CTACGGAGTGGGGGTGGA 91bp 16S rRNA ACTGGGCGTAAAGAGYTCGT CGCATTTCACCGCTACAC 138bp

[0077] Using the qRT-PCR reaction kit from Takara Biotech (Beijing) Co., Ltd., the qRT-PCR reaction system was prepared according to Table 6 for gene expression level detection. The qRT-PCR instrument program was set as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing and extension for 60 s, with a cycle count of 40; the melting curve was automatically set by the machine. 2 -∆∆Ct qRT-PCR data were processed using relative quantification and bar charts were generated using Graphpad Prism 8 software.

[0078] Table 6 qRT-PCR reaction system

[0079] Table 6 qRT-PCR reaction system

[0080] reagents Usage TB Green Premix Ex TaqⅡ (TliRNaseH Plus) (2 ×) 5.0 μL PCR Forward Primer (10 μM) 0.2 μL PCR Reverse Primer (10 μM) 0.2 μL ROX Reference Dye or Dye Ⅱ(50X) 0.2 μL cDNA solution 2.0 μL <![CDATA[ddH2O]]> Add to 10.0 μL

[0081] like Figure 3 As shown, the relative mRNA expression level of the s2plp2 gene in R. ruber s2plp2-E was significantly increased by 3.35 times compared with the wild-type strain.

[0082] Example 4

[0083] This example describes the organic solvent resistance analysis of R. ruber s2plp2-E. The specific experimental method is as follows:

[0084] (1) Wild-type R. ruber SD3 and R. ruber s2plp2-E were streaked onto LB agar plates and incubated at 35°C in an inverted incubator for 2 days. The activated monoclonal strains were then inoculated into 50 mL of LB liquid medium and incubated at 35°C and 200 r / min until OD. 595 A nm value of approximately 1.0 indicates that it is a seed culture.

[0085] (2) At a ratio of 1:50, wild-type R. ruber SD3 and seed culture of R. ruber s2plp2-E were transferred to 50 mL of LB liquid medium, respectively. Phenol was added to a final concentration of 0, 0.02%, and 0.04% (m / v), respectively. The cultures were incubated at 35℃ and 200 r / min. Samples were taken at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 108 h, 132 h, and 156 h to detect the OD value. 595 The absorbance value at nm was used to obtain 3 biological replicates for each sample group.

[0086] (3) OD at 12 h 595 The absorbance at nm was used as a control, and the growth rate of the strains was calculated at different time points. Graphpad Prism 8 software was used for plotting.

[0087] The bacterial growth rates of wild-type R. ruber SD3 and R. ruber s2plp2-E were as follows: Figure 4As shown, the growth rate of wild-type R. ruber SD3 gradually increased with increasing phenol concentration at different time points before 60 h of culture. After 60 h, the growth rate of wild-type R. ruber SD3 under different phenol stresses tended to stabilize. Under 0.02% (m / v) phenol stress, the growth rate of wild-type strains was about 320% higher than that of the untreated group, and under 0.04% (m / v) phenol stress, the growth rate of wild-type strains was about 580% higher than that of the untreated group. Under no phenol stress, the growth rate of R. ruber s2plp2-E gradually increased over time until it gradually stabilized after 72 h of growth. As the phenol concentration increased, the bacterial growth rate also gradually increased, and the growth rate was significantly higher than that of the wild-type strain. Under 0.02% (m / v) phenol stress, the growth rate of *R. ruber* s2plp2-E was approximately 1,300% higher than that of the untreated group, and under 0.04% (m / v) phenol stress, the growth rate of the s2plp2 gene-enhanced strain was approximately 2,100% higher than that of the untreated group. These results indicate that the s2plp2 gene can enhance the phenol tolerance of *R. ruber* SD3.

[0088] Figure 3 and Figure 4 The s2plp2-E in the middle represents the enhanced strain of R. ruber SD3 s2plp2 gene.

[0089] The sequence listing of a strain of Rhodococcus rubers2plp2-E with an enhanced SD3 site-2 protease-like protein 2 gene is as follows:

[0090]

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Rhodococcus SD3-2 protease-like protein 2 gene enhanced, characterized in that, exist R.ruber Increased in wild-type SD3 s2plp2 The recombinant strain obtained by gene expression, wherein the recombinant strain s2plp2 The gene sequence is shown in SEQ ID NO:

1.

2. A method for constructing an enhanced strain of the Rhodococcus SD3 site-2 protease-like protein 2 gene of claim 1, characterized in that, include: PCR amplification R.ruber SD3 gene s2plp2 Constructing gene-carrying s2plp2 Recombinant plasmid pNV18- s2plp2 The plasmid was transformed into... E. coli Recombinant E. coli were cultured in large quantities in the Top 10 competent cells, and the plasmid was extracted. The extracted plasmid was then electroporated to... R.ruber In SD3 competent cells, we obtained R. ruber SD3s2plp2 gene-enhanced strain R. ruber s2plp2-E.

3. The method according to claim 2, characterized in that, The construction method includes the following specific steps: (1) Take R. ruber SD3 seed culture was streaked, and samples were picked from the streaked culture. R .ruber SD3 single colonies were cultured and extracted. R. ruber SD3 whole genome DNA, with R. ruber Using SD3 whole-genome DNA as a template, the target gene was amplified by PCR. s2plp2 The PCR products were then purified. (2) Adopt Eco RI and Hind III. Obtain the linearized vector pNV18 by double digestion of the empty vector plasmid pNV18, and then perform gel recovery of the double digestion products. (3) The purified linearized vector pNV18 was combined with the target gene s2plp2 Perform homologous recombination and transform the homologous recombination product. E. coli Top 10 competent cells; (4) Select white monoclonal colonies for kanamycin antibiotic screening, and then sequence the selected monoclonal colonies for verification. Subsequently, the single-clone colonies that were successfully sequenced were expanded and the recombinant plasmid pNV18- was extracted. s2plp2 ; (5) Preparation R. ruber SD3 competent cells, and the recombinant plasmid pNV18- s2plp2 Electric transfer to R. ruber In SD3 competent cells, we obtained R. ruber s2plp2-E.

4. The method according to claim 3, characterized in that, In step (1), the extraction R. ruber The method for obtaining SD3 whole genome DNA is as follows: Take 3 mL of DNA in the logarithmic growth phase... R. ruber Centrifuge the SD3 bacterial culture to remove the supernatant, add 500 μL of sterile deionized water to the precipitate to resuspend the bacterial precipitate, centrifuge and wash repeatedly; add 300 μL of sterile deionized water again to resuspend the precipitate, place it in a water bath and heat until boiling for 10 min, then immediately place it in a -20℃ freezer for 10 min; after thawing, centrifuge and collect the supernatant for later use.

5. The method according to claim 3, characterized in that, In step (1), the PCR amplification of the target gene s2plp2 The PCR product was purified as follows: PCR amplification primers for the s2plp2 gene were first synthesized: the sequence of the forward primer was 5'-tatgaccatgattacgaattcATGCTGCGC GGATCGGTC-3'; the sequence of the reverse primer was 5'-acgacggccagtgccaagcttCTACTCCGGCCG GGGCTG-3', with the enzymatic restriction sites represented by the horizontal lines. EcoR I and Hind III. Again R. ruber Using SD3 genomic DNA as a template, amplification s2plp2 The fragments were purified using a kit.

6. The method according to claim 3, characterized in that, In step (2), the method for obtaining the linearized vector pNV18 is as follows: Prepare the reaction system: add 30 μL of plasmid pNV18, restriction endonuclease... EcoR I and Hind Add 2 μL of each of the III enzymes, add 10 μL of the double digestion reaction buffer 10 × M, and bring the volume to 50 μL using ddH2O. Place the PCR tube containing 50 μL of the reaction system in a 37℃ water bath for 3 h, and detect the size and purity of the PCR products by 1% (m / v) agarose gel electrophoresis.

7. The method according to claim 3, characterized in that, In step (3), the homologous recombination product is transformed E. coli The method for obtaining the Top 10 competent cells is as follows: Take 100 μL from a -80℃ freezer. E. coli The top 10 competent cells were thawed on ice, and the entire homologous recombinant product from the reaction was added to 100 μL under aseptic conditions. E. coli In the Top10 competent cells, gently shake well and place on ice for 30 min; then heat shock in a 42℃ water bath for 90 s, followed by cooling on ice for about 5 min; add 600 μL of LB liquid medium to the tube, mix well, and incubate at 37℃ and 200 r / min for 45-60 min to allow the bacteria to return to normal growth. Shake well again and take 100 μL to spread on an LB solid plate containing 50 μg / mL kanamycin antibiotic, place face up for half an hour, and after the bacterial solution is completely absorbed by the medium, invert the culture dish and incubate at 37℃ for 12-16 h.

8. The method according to claim 3, characterized in that, In step (5), the preparation R. ruber The method for preparing SD3 competent cells is as follows: In a clean bench, use a 10 μL pipette tip to pick up... R. ruber SD3 seed culture was streaked onto antibiotic-free LB agar plates and incubated at 35°C for 48 h. Single colonies from the plates were then picked with a 10 μL pipette tip and transferred to 50 mL of antibiotic-free LB liquid medium, and incubated at 35°C and 200 rpm until OD500 was reached. 595 The nm value is approximately 0.

7. Take 100 mL of bacterial culture, centrifuge at 8,000 × g and 4 °C for 20 min, discard the supernatant, add 20 mL of 300 mM sucrose containing 10% (m / v) glycerol pre-cooled and sterilized at 4 °C to resuspend the bacterial precipitate, centrifuge at 8,000 × g and 4 °C for 20 min, discard the supernatant, repeat the washing of the precipitate 3 times, finally add 2 mL of 300 mM sucrose solution containing 10% (m / v) glycerol to the bacterial precipitate and gently pipette to resuspend the precipitate, aliquot 100 μL into each new sterile 1.5 mL centrifuge tube, and store at -80 °C.

9. The method according to claim 3, characterized in that, In step (5), the recombinant plasmid pNV18- s2plp2 Electric transfer to R. ruber The method for processing SD3 competent cells is as follows: Take 10 μL of recombinant plasmid pNV18- s2plp2 Add to ice to thaw or to freshly prepared 100 μL R. ruber In SD3 competent cells, gently tap the bottom of the 1.5 ml centrifuge tube with your finger to mix the cells and plasmids, and incubate on ice for 100 min. Set the electroporation conditions as follows: voltage 1.5 kV, electroporation time 2.8 ms. Pre-cool the sterilized electroporation cuvette, add the mixture to it, and quickly place the cuvette into the electroporation tank. After two electroporations, immediately aspirate the mixture and add it to a centrifuge tube containing 500 μL of antibiotic-free LB liquid medium. Place the centrifuge tube in a shaker and incubate at 35°C and 200 r / min for 6 h to revive the cells. Take 100 μL of the revived bacterial culture and spread it on an LB agar plate with a final kanamycin concentration of 50 μg / mL. Incubate upside down at 35°C for 48 h. Pick multiple single colonies from the agar plate and place them in 5 mL of LB liquid medium with a final kanamycin concentration of 50 μg / mL. Incubate for about 48 h and retain the seed culture.

10. The application of the Rhodococcus SD3 site-2 protease-like protein 2 enhanced strain according to claim 1, or the Rhodococcus SD3 site-2 protease-like protein 2 enhanced strain constructed according to claims 2-9, characterized in that, The applications include the degradation of isooctane, cyclohexane, benzene, n-heptane, toluene, acetonitrile, chlorobenzene, naphthalene, n-hexane, and 1-naphthol contaminants.

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