A method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida

Abstract

The invention discloses a method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida. The method comprises the following steps: taking a cym operon induced by 4-isopropylbenzoic acid (cumate) in a metabolic pathway of Pseudomonas putida F1 as a basis, replacing an original promoter for expression and an expression target gene, and combining multiple repressor sites to obtain a modified cym operon, thereby improving the expression intensity of a fluorescent protein. The invention determines the effective response range of an inducer of the induction system in Pseudomonas putida, and simultaneously screens and obtains a multiple repressor site induction system with the best response.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida. Background Art

[0002] Pseudomonas putida is a saprophytic soil bacillus with a rich nutritional background. It is widely found in soil, water, the surface of plants and animals, and various protein-rich substances. Pseudomonas putida has a complex enzyme background, multiple metabolic pathways, and can utilize a variety of carbon sources. Therefore, it is often used in environmental pollution control and organic waste recycling, and it shows high robustness and tolerance to extreme environmental conditions such as pH and solvents. As a Gram-negative bacterium, Pseudomonas putida also has the characteristics of low culture cost, short growth cycle, safety and harmlessness.

[0003] During the growth of prokaryotes, they often need to synthesize different types and quantities of proteins according to changes in environmental conditions to adapt themselves to them. Therefore, there are many mechanisms for regulating gene expression and protein synthesis in prokaryotes, such as the lac operon induced by lactose or isopropylthiogalactoside (IPTG) and the Tet resistance operon induced by tetracycline. These classic regulatory models can also be effectively applied in Pseudomonas putida. However, as a host bacterium with the potential for metabolic engineering, Pseudomonas putida needs to develop more available inducible expression systems to expand its range of expressing exogenous proteins.

[0004] Choi et al. used the pNEW vector and T5 promoter to construct a 4-isopropylbenzoic acid induction system in E. coli, and obtained good response effects in various E. coli. The induction system constructed by Seo et al. can be used in Bacillus subtilis and Bacillus megaterium, and the response time is measured. Mullick et al. combined CymR with VP16 to construct a reverse activation model and successfully applied it in mammalian cells. However, due to the endogenous metabolic pathway for the decomposition of 4-isopropylbenzoic acid in its source, Pseudomonas putida F1, this induction system cannot be used in its source strain at present. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for transforming an operon induced by 4-isopropylbenzoic acid.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] Based on the 4-isopropylbenzoic acid-induced cym operon in the metabolic pathway of Pseudomonas Putida F1, the original expression promoter and the target gene for expression are replaced, and the modified cym operon is constructed by combining the repression site;

[0010] Wherein, the cym operon sequence induced by 4-isopropylbenzoic acid is shown in SEQ ID NO: 23;

[0011] The modified cym operon was applied to expression in Pseudomonas putida, and the expression system was able to effectively respond to the addition of 4-isopropylbenzoic acid in Pseudomonas putida.

[0012] As a preferred embodiment of the method for constructing the high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the modified cym operon includes a gene encoding a repressor protein, a repressor site, an exogenously introduced promoter, and a green fluorescent protein.

[0013] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the replacement of the original expression promoter of the cym operon and the expression of the target gene include:

[0014] Using plasmid pBBR1-pTrc as a template, primers 1 and 2 were designed to amplify the resistance gene Kan, replicon pBBR1 oriV, and pBBR1 Rep by PCR to obtain fragment 1;

[0015] Using the purified genome of Pseudomonas putida F1 as a template, primers 3 and 4 were designed to amplify the repressor protein gene cymR by PCR to obtain fragment 2;

[0016] Primers 5 and 6 were designed to amplify the repressor site CuO1 and promoter of the cym operon by PCR to obtain fragment 3;

[0017] Primers 7 and 8 were designed to amplify the green fluorescent protein gene eGFP by PCR to obtain fragment 4;

[0018] Fragment 1, fragment 3, and fragment 4 were subjected to Gibson assembly at a molar ratio of 1:2:2, cultured and extracted to obtain the pBBR1-lacI-CuO1-eGFP plasmid;

[0019] Using plasmid pBBR1-lacI-CuO1-eGFP as a template, primers 9 and 10 were designed to amplify the lacI gene part (5949 bp) by PCR to obtain fragment 5;

[0020] Fragment 5 was subjected to Gibson assembly, cultured and extracted to obtain pBBR1-CuO1-eGFP plasmid, the nucleotide sequence of which is shown in SEQ ID NO: 13;

[0021] Using plasmid pBBR1-CuO1-eGFP as a template, the plasmid was double-digested by restriction sites XbaI and HindIII. The restriction enzyme system is shown in Table 3. The product was recovered by gel recovery to obtain linearized vector fragment 6;

[0022] Fragment 2 and fragment 6 were subjected to Gibson assembly at a molar ratio of 1:4, cultured and extracted to obtain pBBR1-cymR-CuO1-eGFP plasmid, the nucleotide sequence of which is shown in SEQ ID NO:14;

[0023] Using pBBR1-CuO1-eGFP and pBBR1-cymR-CuO1-eGFP plasmids as templates, primers 11 and 12 were designed to amplify the two plasmid vectors by PCR to obtain fragments 7 and 8;

[0024] Fragment 7 and fragment 8 were assembled by Gibson to obtain pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP plasmids respectively;

[0025] Wherein, the primer 1 is pBBR1vector-F, and the nucleotide sequence is shown in SEQ ID NO: 1;

[0026] Primer 2 is pBBR1vector-R, the nucleotide sequence of which is shown in SEQ ID NO:2;

[0027] Primer 3 is cymR-F, the nucleotide sequence of which is shown in SEQ ID NO:3;

[0028] Primer 4 is cymR-R, the nucleotide sequence of which is shown in SEQ ID NO:4;

[0029] Primer 5 is CuO1-F, the nucleotide sequence of which is shown in SEQ ID NO:5;

[0030] Primer 6 is CuO1-R, the nucleotide sequence of which is shown in SEQ ID NO:6;

[0031] Primer 7 is eGFP-F, the nucleotide sequence of which is shown in SEQ ID NO:7;

[0032] Primer 8 is eGFP-R, the nucleotide sequence of which is shown in SEQ ID NO:8;

[0033] Primer 9 is dlacI-F, the nucleotide sequence of which is shown in SEQ ID NO:9;

[0034] Primer 10 is dlacI-R, the nucleotide sequence of which is shown in SEQ ID NO:10;

[0035] Primer 11 is tac-F, and the nucleotide sequence is shown in SEQ ID NO: 11;

[0036] Primer 12 is tac-R, and the nucleotide sequence is shown in SEQ ID NO:12.

[0037] As a preferred embodiment of the method for constructing the high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, wherein: the method combines multiple repressor sites, including:

[0038] Primers 15, 16, 17, and 18 were designed to add repressor sites at the corresponding positions to obtain fragments 9 and 10;

[0039] Primers 21 and 22 were designed to linearize and amplify plasmids pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP to obtain fragments 11 and 12;

[0040] Fragment 9 and fragment 11, fragment 9 and fragment 12, fragment 10 and fragment 11, and fragment 10 and fragment 12 are respectively subjected to Gibson assembly to obtain a transformed cym operon plasmid;

[0041] Wherein, the primer 15 is 2CuO1-F, and the nucleotide sequence is shown in SEQ ID NO: 15;

[0042] Primer 16 is 2CuO1-R, and the nucleotide sequence is shown in SEQ ID NO: 16;

[0043] Primer 17 is 3CuO1-F, the nucleotide sequence of which is shown in SEQ ID NO: 17;

[0044] Primer 18 is 3CuO1-R, and the nucleotide sequence is shown in SEQ ID NO: 18;

[0045] The nucleotide sequence of fragment 9 is shown in SEQ ID NO: 19;

[0046] The nucleotide sequence of fragment 10 is shown in SEQ ID NO:20.

[0047] The primer 21 is CuO1-vector-F, and the nucleotide sequence is shown in SEQ ID NO: 21;

[0048] Primer 22 is CuO1-vector-R, and the nucleotide sequence is shown in SEQ ID NO:22.

[0049] As a preferred scheme for the construction method of the high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida described in the present invention, the modified cym operon plasmid includes pBB R1-Ptac-2CuO1-eGFP, pBBR1-cymR-Ptac-2CuO1-eGFP, pBBR1-Ptac-3CuO1-eGF P, and pBBR1-cymR-Ptac-3CuO1-eGFP.

[0050] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the modified cym operon can be applied to Pseudomonas putida KT2440.

[0051] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the modified cym operon has inducing activity and can be induced to express by 4-isopropylbenzoic acid in Pseudomonas putida KT2440.

[0052] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the induction concentration of 4-isopropylbenzoic acid is 50-800 mg / L.

[0053] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the modified cym operon reaches the highest fluorescent protein expression response intensity at a concentration of 400 mg / L 4-isopropylbenzoic acid inducer.

[0054] As a preferred embodiment of the method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida of the present invention, the cym operon plasmid that achieves the highest fluorescent protein expression response intensity is pBBR1-cymR-Ptac-2CuO1-eGFP.

[0055] Beneficial effects of the present invention:

[0056] The invention is based on the cym operon induced by 4-isopropylbenzoic acid (cumate) in the metabolic pathway of Pseudomonas putida F1, replaces its original expression promoter and expression target gene, and obtains the modified cym operon by combining multiple repressor sites, so that the expression intensity of fluorescent protein is improved; the invention determines the effective response range of the inducer of the induction system in Pseudomonas putida, and simultaneously screens and obtains the multiple repressor site induction system with the best response. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0058] Figure 1 The figure is a schematic diagram of the cym operon transformation method and the inducible expression principle of the present invention.

[0059] Figure 2 This is a diagram showing the detection and comparison of the fluorescent protein expression response intensities of the original expression promoter and Ptac in Example 2 of the present invention.

[0060] Figure 3 OD of different concentrations of cumate inducer added to multiple repressor sites in Pseudomonas putida KT2440 in Example 2 of the present invention 600 Value comparison chart.

[0061] Figure 4 Fluorescence / OD of adding different concentrations of cumate inducer to multiple repressor sites in Pseudomonas putida KT2440 in Example 2 of the present invention 600 Comparison chart of values ​​and response levels. DETAILED DESCRIPTION

[0062] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0063] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0064] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0065] The primers described in the present invention were all synthesized by Genewise Co., Ltd.

[0066] The PCR reaction conditions in the present invention are:

[0067] Pre-denaturation at 98℃ for 3min

[0068] Denaturation at 98°C for 15 s–annealing at 55°C for 10 s–extension at 72°C for 56 s (40 cycles)

[0069] Keep warm at 72°C for 5 minutes

[0070] The reaction system is shown in Table 1:

[0071] Table 1 PCR reaction system

[0072]

[0073]

[0074] The Gibson assembly system in the present invention is shown in Table 2:

[0075] Table 2 Assembly system

[0076]

[0077] The enzyme digestion system of the present invention is shown in Table 3:

[0078] Table 3 Enzyme digestion system

[0079]

[0080] Example 1

[0081] This example provides a method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida, see Figure 1 , is a schematic diagram of the principle of transforming the cym operon in the present invention, Figure 1 A is the arrangement of genes and expression elements of the cym operon on the genome of Pseudomonas putida F1. On this basis, the original expression promoter was replaced by the tac promoter and the target gene was replaced by eGFP to construct the Figure 1 The expression system shown in B, the specific steps are as follows:

[0082] Using plasmid pBBR1-pTrc as a template, primers were designed to amplify the resistance gene Kan, replicon pBBR1oriV, and pBBR1 Rep by PCR to obtain fragment 1 with a sequence length of 4722 bp. The primers were pBBR1vector-F and pBBR1vector-R, and their nucleotide sequences were shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0083] Using the purified genome of Pseudomonas putida F1 as a template, primers were designed to amplify the repressor protein gene cymR by PCR to obtain a fragment 2 with a sequence length of 1195 bp, the primers were cymR-F and cymR-R, and the nucleotide sequences thereof were shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0084] The cym operon repression site CuO1 and the promoter were amplified to obtain a fragment 3 with a sequence length of 256 bp, the primers were CuO1-F and CuO1-R, and the nucleotide sequences were as shown in SEQ ID NO:5 and SEQ ID NO:6; the green fluorescent protein gene eGFP was amplified to obtain a fragment 4 with a sequence length of 767 bp, the primers were eGFP-F and eGFP-R, and the nucleotide sequences were as shown in SEQ ID NO:7 and SEQ ID NO:8, and the above primers were synthesized at GENEWIZ.

[0085] The PCR amplified product was subjected to agarose gel electrophoresis, and the agarose gel band with the correct molecular weight was recovered and the concentration was determined before use;

[0086] Fragment 1, fragment 3 and fragment 4 were Gibson assembled at a molar ratio of 1:2:2. The assembly system is shown in Table 2. The reaction temperature was 50°C and the reaction time was 1h. The ligation product was transformed into Escherichia coli JM109, coated with kanamycin LB plates, and cultured at 37°C overnight to obtain transformants. A single colony of the transformant was picked and cultured at 37°C overnight in a kanamycin-resistant LB liquid medium to extract the plasmid, thereby obtaining the pBBR1-lacI-CuO1-eGFP plasmid.

[0087] Using plasmid pBBR1-lacI-CuO1-eGFP as a template, primers were designed to obtain fragment 5 with a sequence length of 5949 bp by PCR amplification except for the lacI gene part, and the primers were dlacI-F and dlacI-R, and the nucleotide sequences thereof were shown in SEQ ID NO:9 and SEQ ID NO:10. Fragment 5 was subjected to Gibson assembly. The plasmid was extracted to obtain pBBR1-CuO1-eGFP, and the nucleotide sequence thereof was shown in SEQ ID NO:13. Using plasmid pBBR1-CuO1-eGFP as a template, the plasmid was double-digested by restriction sites XbaI and HindIII, and the restriction system was shown in Table 3. The product was recovered by gel to obtain linearized vector fragment 6. Fragment 2 and fragment 6 were subjected to Gibson assembly at a molar ratio of 1:4, and the plasmid was extracted to obtain pBBR1-cymR-CuO1-eGFP, and the nucleotide sequence thereof was shown in SEQ ID NO:14.

[0088] Using pBBR1-CuO1-eGFP and pBBR1-cymR-CuO1-eGFP plasmids as templates, primers were designed to amplify two plasmid vectors, fragment 7 and fragment 8, by PCR, the primers were tac-F and tac-R, and their nucleotide sequences were shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The primers were synthesized at GENEWISE.

[0089] Fragment 7 and fragment 8 were Gibson assembled with ddH2O at a volume ratio of 1:1 to obtain pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP plasmids.

[0090] Primers were designed to add the repressor site CuO1 at the corresponding position, the primers were 2CuO1-F, 2CuO1-R, 3CuO1-F, 3CuO1-R, the nucleotide sequences were shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, respectively, to obtain fragment 9 and fragment 10, the sequences were SEQ ID NO: 19 and SEQ ID NO: 20;

[0091] Primers were designed to linearize and amplify plasmids pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP, the primers were CuO1-vector-F and CuO1-vector-R, the nucleotide sequences were shown in SEQ ID NO:21 and SEQ ID NO:22, respectively, to obtain fragments 11 and 12;

[0092] Fragment 9 and fragment 11 were Gibson assembled at a molar ratio of 4:1, fragment 9 and fragment 12 were Gibson assembled at a molar ratio of 4:1, fragment 10 and fragment 11 were Gibson assembled at a molar ratio of 4:1, and fragment 10 and fragment 12 were Gibson assembled at a molar ratio of 4:1, the reaction temperature was 50°C, and the reaction time was 1h. The ligation products were transformed into Escherichia coli JM109, coated with kanamycin LB plates, and cultured at 37°C overnight to obtain transformants. Single colonies of transformants were picked, cultured at 37°C overnight in kanamycin-resistant LB liquid culture medium, and plasmids were extracted to obtain the modified operon plasmids pBBR1-Ptac-2CuO1-eGFP, pBBR1-cymR-Ptac-2CuO1-eGFP, pBBR1-Ptac-3CuO1-eGFP, and pBBR1-cymR-Ptac-3CuO1-eGFP.

[0093] Example 2

[0094] See also Figure 1 to Figure 4 In order to verify the technical effect of the present invention, this example tests the response degree of the plasmid constructed in Example 1 in Pseudomonas putida KT2440.

[0095] Plasmids pBBR1-CuO1-eGFP, pBBR1-cymR-CuO1-eGFP, pBBR1-Ptac-CuO1-eGFP, pBBR1-cymR-Ptac-CuO1-eGFP, pBBR1-Ptac-2CuO1-eGFP, pBBR1-cymR-Ptac-2CuO1-eGFP, pBBR1-Ptac-3CuO1-eGFP, and pBBR1-cymR-Ptac-3CuO1-eGFP were transformed into Pseudomonas putida KT2440 strain to obtain strain 1, strain 2, strain 3, strain 4, strain 5, strain 6, strain 7, and strain 8, and the steps are as follows:

[0096] Take 2 μL of plasmid pBBR1-CuO1-eGFP, pBBR1-cymR-CuO1-eGFP, pBBR1-Ptac-CuO1-eGFP, pBBR1-cymR-Ptac-CuO1-eGFP, pBBR1-Ptac-2CuO1-eGFP, pBBR1-cymR-Ptac-2CuO1-eGFP, pBBR1-Ptac-3CuO1-eGFP, and pBBR1-cymR-Ptac-3CuO1-eGFP respectively, add them to 80 μL Pseudomonas putida KT2440 competent cells, and incubate on ice for 5 min;

[0097] Transfer the mixture of plasmid and competent cells to a pre-cooled electroporation cuvette with an inner diameter of 1 mm, and quickly transform the plasmid at 1.2 kV electroporation conditions;

[0098] Immediately add 500 μL LB liquid medium into the electroporation cup to rinse the electroporation liquid, transfer it to a 1.5 mL EP tube, and place it in a 30°C shaker to recover the culture for 2 h;

[0099] Strain 1, strain 2, strain 3, strain 4, strain 5, strain 6, strain 7 and strain 8 were spread on 50 μg / mL kanamycin-resistant LB solid medium and cultured in a 30° C. incubator for 16 h.

[0100] 1. Detection and comparison of the expression response intensity of fluorescent protein from the original expression promoter and Ptac

[0101] Strain 1, strain 2, strain 3, and strain 4 were inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured at 30°C overnight;

[0102] After incubation at 30°C for 3 h, the OD 600 When it reached 0.6, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L of 4-isopropylbenzoic acid cumate were added, respectively, and the cells were grown at 30 °C for another 24 h;

[0103] Take 50 μL of the culture medium and dilute it 4-fold with H2O for OD 600 The eGFP fluorescence was measured by Spectramax M2 with 485 nm excitation and 528 nm emission. Figure 2 As shown, Figure 2 A is the OD of different concentrations of cuma te inducer added when the inducible expression system uses the original promoter or Ptac 600 Value comparison, Figure 2 B is the fluorescence / OD of the inducible expression system using the original promoter or Ptac with different concentrations of cumate inducer added 600 The results showed that the bacterial growth state was better after the expression promoter was replaced with Pta c than before the replacement, and the expression intensity of green fluorescent protein was greatly improved after the replacement.

[0104] 2. Detection and comparison of fluorescent protein expression response intensity between systems with multiple repression sites

[0105] Strain 3, strain 4, strain 5, strain 6, strain 7, and strain 8 were inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured at 30°C overnight;

[0106] After incubation at 30°C for 3 h, the OD 600 When the pH value reached 0.6, strain 3, strain 4, strain 5, strain 6, strain 7, and strain 8 were added with 20 mg / L, 50 mg / L, 100 mg / L, 400 mg / L, and 800 mg / L of 4-isopropylbenzoic acid cumate, respectively, and continued to grow at 30°C for 24 h;

[0107] Take 50 μL of the culture medium and dilute it 4-fold with H2O for OD 600 The eGFP fluorescence was measured by Spectramax M2 with 485 nm excitation and 528 nm emission. Figure 3 , Figure 4 shown.

[0108] Figure 3 OD of different concentrations of cumate inducer added to multiple repressor sites in Pseudomonas putida KT2440 600 The results showed that the growth of the bacteria was not greatly affected by the increase in the concentration of the inducer.

[0109] Figure 4 A is the fluorescence / OD after adding different concentrations of cumate inducer to multiple repressor sites in Pseudomonas putida KT2440 600 Value comparison, Figure 4 B is the response degree of the multi-repression site induction system to different concentrations of cumate inducer in Pseudomonas putida KT2440. The results show that all three induction systems can respond in the inducer concentration range of 0-800 mg / L, and the dual repression site, i.e. pBBR1-cymR-Ptac-2CuO1-eGFP, reaches the highest fluorescent protein expression intensity at a 400 mg / L 4-isopropylbenzoic acid inducer concentration.

[0110] The invention is based on the cym operon induced by 4-isopropylbenzoic acid (cumate) in the metabolic pathway of Pseudomonas putida F1, replaces its original expression promoter and expression target gene, and obtains the modified cym operon by combining multiple repressor sites, so that the expression intensity of fluorescent protein is improved; the invention determines the effective response range of the inducer of the induction system in Pseudomonas putida, and simultaneously screens and obtains the multiple repressor site induction system with the best response.

[0111] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida, characterized in that: include, Pseudomonas putida F1 ( Pseudomonas Putida F1 )The 4-isopropylbenzoic acid-induced cym operon in the metabolic pathway is used as the basis to replace the original expression promoter and the expression target gene, and the modified cym operon is constructed by combining the repression site; The replacing of the original expression promoter and the expression of the target gene comprises: Using plasmid pBBR1-pTrc as a template, primers 1 and 2 were designed to amplify the resistance gene Kan, replicon pBBR1oriV, and pBBR1 Rep by PCR to obtain fragment 1; Using the purified genome of Pseudomonas putida F1 as a template, primers 3 and 4 were designed to amplify the repressor protein gene cymR by PCR to obtain fragment 2; Primers 5 and 6 were designed to amplify the repression site CuO1 and promoter of the cym operon by PCR to obtain fragment 3; Primers 7 and 8 were designed to amplify the green fluorescent protein gene eGFP by PCR to obtain fragment 4; Fragments 1, 3, and 4 were subjected to Gibson assembly and cultured and extracted to obtain the pBBR1-lacI-CuO1-eGFP plasmid; Using plasmid pBBR1-lacI-CuO1-eGFP as a template, primers 9 and 10 were designed to amplify the lacI gene portion by PCR to obtain fragment 5; Fragment 5 was subjected to Gibson assembly, cultured and extracted to obtain pBBR1-CuO1-eGFP plasmid, the nucleotide sequence of which is shown in SEQ ID NO: 13; Using plasmid pBBR1-CuO1-eGFP as a template, the plasmid was double-digested through the restriction sites XbaI and HindIII, and the product was recovered by gel recovery to obtain the linearized vector fragment 6; Fragment 2 and fragment 6 were subjected to Gibson assembly, cultured and extracted to obtain pBBR1-cymR-CuO1-eGFP plasmid, the nucleotide sequence of which is shown in SEQ ID NO: 14; Using pBBR1-CuO1-eGFP and pBBR1-cymR-CuO1-eGFP plasmids as templates, primers 11 and 12 were designed to amplify the two plasmid vectors by PCR to obtain fragments 7 and 8; Fragment 7 and fragment 8 are respectively subjected to Gibson assembly to obtain pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP plasmids, wherein the modified operon plasmid comprises pBBR1-cymR-Ptac-CuO1-eGFP plasmid; Wherein, the primer 1 is pBBR1vector-F, and the nucleotide sequence is shown in SEQ ID NO: 1; Primer 2 is pBBR1vector-R, the nucleotide sequence of which is shown in SEQ ID NO:2; Primer 3 is cymR-F, the nucleotide sequence of which is shown in SEQ ID NO:3; Primer 4 is cymR-R, the nucleotide sequence of which is shown in SEQ ID NO:4; Primer 5 is CuO1-F, the nucleotide sequence of which is shown in SEQ ID NO:5; Primer 6 is CuO1-R, the nucleotide sequence of which is shown in SEQ ID NO:6; Primer 7 is eGFP-F, the nucleotide sequence of which is shown in SEQ ID NO:7; Primer 8 is eGFP-R, the nucleotide sequence of which is shown in SEQ ID NO:8; Primer 9 is dlacI-F, the nucleotide sequence of which is shown in SEQ ID NO:9; Primer 10 is dlacI-R, the nucleotide sequence of which is shown in SEQ ID NO:10; Primer 11 is tac-F, and the nucleotide sequence is shown in SEQ ID NO: 11; Primer 12 is tac-R, and the nucleotide sequence is shown in SEQ ID NO: 12; The modified cym operon includes the repressor protein encoding gene cymR, the repressor site CuO1, the exogenously introduced promoter tac, and the green fluorescent protein eGFP; Wherein, the repression site CuO1 in the cym operon sequence induced by 4-isopropylbenzoic acid is shown in SEQ ID NO: 23; The modified cym operon plasmid was transferred into Pseudomonas putida KT2440 as a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida; The expression system can effectively respond to the addition of 4-isopropylbenzoic acid in Pseudomonas putida, the 4-isopropylbenzoic acid induction concentration is 50-800 mg / L, and the modified cym operon reaches the highest fluorescent protein expression response intensity at a 400 mg / L 4-isopropylbenzoic acid inducer concentration.

2. The method for constructing a high-intensity expression system induced by 4-isopropylbenzoic acid in Pseudomonas putida according to claim 1, characterized in that: By combining repressor sites, include, Primers 15, 16, 17, and 18 were designed to add repressor sites at the corresponding positions to obtain fragments 9 and 10; Primers 21 and 22 were designed to linearize and amplify plasmids pBBR1-Ptac-CuO1-eGFP and pBBR1-cymR-Ptac-CuO1-eGFP to obtain fragments 11 and 12; Fragment 9 and fragment 11, fragment 9 and fragment 12, fragment 10 and fragment 11, and fragment 10 and fragment 12 are respectively subjected to Gibson assembly to obtain a transformed cym operon plasmid; The modified cym operon plasmids include pBBR1-cymR-Ptac-2CuO1-eGFP and pBBR1-cymR-Ptac-3CuO1-eGFP; Wherein, the primer 15 is 2CuO1-F, and the nucleotide sequence is shown in SEQ ID NO: 15; Primer 16 is 2CuO1-R, and the nucleotide sequence is shown in SEQ ID NO: 16; Primer 17 is 3CuO1-F, the nucleotide sequence of which is shown in SEQ ID NO: 17; Primer 18 is 3CuO1-R, and the nucleotide sequence is shown in SEQ ID NO: 18; The nucleotide sequence of fragment 9 is shown in SEQ ID NO: 19; The nucleotide sequence of fragment 10 is shown in SEQ ID NO:

20.

3. The primer 21 is CuO1-vector-F, and the nucleotide sequence is shown in SEQ ID NO: 21; Primer 22 is CuO1-vector-R, and the nucleotide sequence is shown in SEQ ID NO:22.

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