Tac promoter mutant, recombinant plasmid, recombinant bacteria and application

Abstract

This invention discloses tac Promoter mutants, recombinant plasmids, recombinant bacteria and their applications tac Promoter mutant, named M tac 1~M tac 4. The nucleotide sequences of these sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 9, respectively. This invention... tac Promoter mutants can effectively increase the functional expression level of the target protein in E. coli, with the optimal mutant regulating the expression level of the functional protein as follows: tac The promoter is 1.53 times that of the T7 promoter and 4.92 times that of the T7 promoter. Under the same reaction conditions, the present invention... tac The target protein expressed by the promoter mutant exhibits superior PET degradation performance, with the total release of PET degradation products increasing by approximately 25% compared to the T7 promoter system. This invention illustrates the present invention. tac Promoter mutants can maintain good PET degradation activity while increasing the functional expression level of the target protein.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and genetic engineering, specifically to... tac Promoter mutants, recombinant plasmids, recombinant bacteria and their applications. Background Technology

[0002] Plastics are widely used in various industrial sectors, and the resulting waste pollution has become an increasingly serious global environmental problem. Polyethylene terephthalate (PET), in particular, is widely used in packaging materials and industrial products due to its excellent mechanical properties and chemical stability. However, PET has a degradation cycle of hundreds of years in the natural environment. Traditional treatment methods such as landfilling and incineration are not only inefficient in resource utilization but also generate toxic byproducts, failing to meet the requirements of green and sustainable development.

[0003] Bio-enzymatic catalysis of PET depolymerization technology has advantages such as mild reaction conditions, low energy consumption, and environmental friendliness, and is considered an important approach to realizing the resource utilization of PET waste. In 2020, a French team engineered leaf-branch compost cutinase (LCC) to obtain the PET hydrolase ICCG. This hydrolase has excellent thermal stability and catalytic performance, and can achieve efficient degradation of high-solids-content PET in a short time, showing good prospects for industrial application. However, the expression level of this enzyme in heterologous expression systems is still limited, which has become a key factor restricting its large-scale application.

[0004] Escherichia coli, with its clear genetic background, rapid growth, and ease of operation, is widely used for the industrial production of recombinant proteins. Current technologies typically employ expression systems based on the strong T7 promoter to achieve heterologous expression of PET hydrolases. However, excessively high transcriptional intensity can lead to an overburdened translation, causing misfolding of the target protein and the formation of inclusion bodies, thereby reducing the proportion of soluble protein and increasing subsequent purification costs. Therefore, to improve the yield of PET hydrolases (ICCG) in heterologous expression systems, promoter design and optimization are necessary. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide... tac Promoter mutant.

[0006] A second objective of this invention is to provide recombinant plasmids.

[0007] A third objective of the present invention is to provide recombinant bacteria containing the above-mentioned recombinant plasmid.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned recombinant bacteria in the preparation of PET hydrolase.

[0009] The technical solution of this invention is summarized as follows: tac Promoter mutant, named M tac 1~M tac 4, the M tac 1~M tac The nucleotide sequences of 4 are shown in SEQ ID NO. 6 to SEQ ID NO. 9, respectively.

[0010] Recombinant plasmid, the recombinant plasmid comprising the above-mentioned... tac The promoter mutant and the gene encoding the PET hydrolase ICCG, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0011] The plasmid backbone of the recombinant plasmid is pET26b(+).

[0012] Recombinant bacteria containing the above-mentioned recombinant plasmids.

[0013] The host of the recombinant bacteria is Escherichia coli BL21(DE3).

[0014] The application of the above-mentioned recombinant bacteria in the preparation of PET hydrolase.

[0015] Beneficial effects (1) This invention, through the application of... tac The spacing sequence between the -35 and -10 regions of the promoter was designed and optimized in a targeted manner, thereby enabling the regulation of promoter expression intensity. (2) The present invention provides tac Promoter mutants can effectively increase the functional expression level of the target protein in E. coli, with the optimal mutant regulating the functional expression level of the target protein as follows: tac The boot subsystem is 1.53 times that of the T7 boot subsystem, and 4.92 times that of the T7 boot subsystem. (3) Under the same reaction conditions, the present invention is used. tac The target protein expressed by the promoter mutant exhibits superior PET degradation performance, with the total release of PET degradation products increasing by approximately 25% compared to the T7 promoter system. This illustrates the invention. tac Promoter mutants can improve the functional expression of the target protein while maintaining good PET degradation activity, and have good potential for industrial applications. Attached Figure Description

[0016] Figure 1 For recombinant plasmid pET26b(+)- tac A schematic diagram of the construction of -ICCG-EGFP.

[0017] Figure 2 Fluorescence assays showing the expression levels of the target protein under different promoter regulation.

[0018] Figure 3 SDS-PAGE gel images of target proteins expressed under different promoter regulation.

[0019] Figure 4 A graph showing the functional expression levels of target proteins regulated by different promoters.

[0020] Figure 5 A graph showing the PET degradation activity of target proteins regulated by different promoters.

[0021] in, Figures 2-5 In the middle: 1 is the T7 starter, 2 is... tac Promoter, 3 is M tac 1 promoter, 4 for M tac 2 promoters, 5 is M tac 3 promoters, 6 for M tac 4. Promoter. Detailed Implementation

[0022] To make the technical solution of the present invention clearer, the present invention will be further described below with reference to embodiments. The following descriptions are only some preferred embodiments of the present invention and are not intended to limit the present invention in other ways. Any modifications or equivalent changes made to the embodiments based on the technical essence of the present invention without departing from the scope of the present invention are within the protection scope of the present invention.

[0023] The Escherichia coli DH5α and BL21(DE3) competent cells used in this invention, and the expression vector pET-26b(+) used, can all be obtained commercially.

[0024] Experimental materials LB liquid medium: Weigh 10 g peptone, 10 g NaCl, and 5 g yeast extract, dissolve in double-distilled water (ddH2O), and bring the volume to 1 L. Sterilize at 121℃ for 20 min.

[0025] When preparing LB solid medium, add 1.5% (w / v) of agar powder to LB liquid medium.

[0026] 50×TAE buffer: Weigh 242 g Tris, 59.2 mL glacial acetic acid, and 100 mL 0.5 M EDTA, dissolve in ddH2O, adjust pH to 8.0, and bring the volume to 1 L.

[0027] 1 M isopropyl-β-D-thiogalactoside (IPTG): Weigh 11.9 g IPTG, dissolve in sterile ddH2O, and bring the volume to 50 mL.

[0028] 50 mg / mL kanamycin: Weigh 2.5 g of kanamycin, dissolve it in sterile ddH2O, and bring the volume to 50 mL.

[0029] 100 mM potassium phosphate buffer: Weigh 16.284 g K2HPO4 and 0.888 g KH2PO4, dissolve them in 900 mL ddH2O, adjust the pH to 8.0, and bring the volume to 1 L.

[0030] The high-fidelity enzyme used in this invention was purchased from YEASEN; the restriction endonuclease, seamless cloning enzyme, and Dpn I enzyme were purchased from GenStar.

[0031] The DNA gel recovery kit and plasmid miniprep kit used in this invention were purchased from Sangon Biotech.

[0032] The PET film used in this invention was purchased from Goodfellow.

[0033] tac Promoter mutant, named M tac 1~M tac 4, whose nucleotide sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 9 respectively.

[0034] The tac Promoter mutants have the following characteristics: (1) Origin tac promoter; (2) Retain tac Conservative sequences in regions -35 and -10 of the promoter; (3) Introduce nucleotide mutations in the spacer region between the -35 region and the -10 region; (4) The length of the interval is 16 bp.

[0035] Example 1 Recombinant plasmid pET26b(+)- tac Preparation of -ICCG-EGFP.

[0036] The recombinant plasmid pET26b(+)-ICCG-EGFP was constructed by ligating the ICCG-EGFP fusion gene into the pET-26b(+) expression vector. The ICCG-EGFP fusion gene consists of a gene encoding the PET hydrolase ICCG (SEQ ID NO. 1), a linker peptide (SEQ ID NO. 2), and a gene encoding enhanced green fluorescent protein EGFP (SEQ ID NO. 3). The gene encoding enhanced green fluorescent protein EGFP is linked to the 3' end of the gene encoding the PET hydrolase ICCG via the linker peptide.

[0037] The ICCG-EGFP fusion gene is the target gene in this invention, and its expression product, the ICCG-EGFP fusion protein, is the target protein.

[0038] The codon-optimized fusion gene from *E. coli* was cloned into the expression vector pET-26b(+). Nde I and Xho I restriction endonuclease sites were used to obtain the recombinant plasmid pET26b(+)-ICCG-EGFP, wherein the fusion gene is expressed under the regulation of the T7 promoter on the expression vector.

[0039] The T7 promoter sequence (SEQ ID NO. 4) in the recombinant plasmid pET26b(+)-ICCG-EGFP was replaced with the tac promoter sequence (SEQ ID NO. 5) to construct the recombinant plasmid pET26b(+)-tac-ICCG-EGFP containing the tac promoter. Its structure is shown below. Figure 1 As shown.

[0040] The preparation method of recombinant plasmid pET26b(+)-tac-ICCG-EGFP is as follows: Following the instructions of the EZ-HiFi Seamless Cloning Kit (GenStar), pET26b(+)-ICCG-EGFP was used as a template. Forward primer F1 (SEQ ID NO. 10) and reverse primer R1 (SEQ ID NO. 11) were designed for PCR amplification. The PCR system is shown in Table 1. Table 1 PCR system

[0041] The PCR reaction conditions were: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 56℃ annealing for 5 s, 72℃ extension for 1 min, with 33 cycles of denaturation, annealing and extension steps, and finally 72℃ extension for 2 min to end the reaction. The circular vector template in the PCR product was digested with Dpn I enzyme. Electrophoresis was performed on a 1% agarose gel, followed by gel extraction using a gel extraction kit to obtain the linearized vector. The linearized vector was mixed with a seamless cloning enzyme, and circular vectors were obtained through homologous recombination. The seamless cloning system is shown in Table 2. Table 2 Seamless Cloning System

[0042] React at 50℃ for 30 min, then add 10 μL of the reaction product directly to 100 μL of the reaction mixture. E. coli In DH5α competent cells, the mixture was gently mixed, incubated on ice for 30 min, then heated at 42°C for 60 s, followed by incubation on ice for 2 min. 800 μl of LB liquid medium was added, and the mixture was incubated at 37°C with shaking for 60 min. The bacterial culture was then evenly spread onto LB solid medium containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single clones were picked and sequenced to confirm their correctness, yielding the recombinant plasmid pET26b(+)-. tac -ICCG-EGFP.

[0043] In this invention, EGFP is used as a reporter protein to characterize the expression level of the ICCG gene. Under the conditions of this invention, the expression system fused with EGFP can reflect the differences in the expression level of the target protein caused by different promoters. In practical applications, the EGFP tag can be retained, replaced, or removed as needed.

[0044] Example 2 tac Targeted design and optimization of promoter mutants.

[0045] The recombinant plasmid pET26b(+)- constructed in Example 1 tac Based on -ICCG-EGFP, construct tac A promoter random mutation library was used to obtain the correspondence data between promoter sequences and expression intensity, and a promoter expression intensity prediction model was established by combining a deep learning model containing convolutional neural networks and Transformer structures.

[0046] Based on the promoter expression intensity prediction model, a genetic algorithm is used to... tac Promoter sequences were optimized and screened, and the obtained candidate mutation sequences were experimentally verified, ultimately yielding four mutations. tac Promoter mutant, named M tac 1~M tac 4, whose nucleotide sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 9 respectively.

[0047] Example 3 Recombinant plasmid pET26b(+)-M tac Preparation of -ICCG-EGFP.

[0048] Obtained in Example 2 tac The promoter mutant sequences (SEQ ID NO. 6 to SEQ ID NO. 9) replaced the recombinant plasmid pET26b(+)- tac -ICCG-EGFP tac The promoter sequence (SEQ ID NO. 5) was used to prepare the recombinant plasmid pET26b(+)-M. tac -ICCG-EGFP, the specific steps are as follows: pET26b(+)- tac Using ICCG-EGFP as a template, PCR amplification was performed using primers SEQ ID NO. 12 and SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19, respectively. PCR amplification, seamless cloning, transformation and screening were performed according to Example 1. Four recombinant plasmids were obtained: pET26b(+)-M tac 1-ICCG-EGFP, pET26b(+)-M tac 2-ICCG-EGFP, pET26b(+)-M tac 3-ICCG-EGFP and pET26b(+)-M tac 4-ICCG-EGFP.

[0049] Example 4 Recombinant strain BL21(DE3) / pET26b(+)-M tac Preparation of -ICCG-EGFP The four recombinant plasmids obtained in Example 3 were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB solid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C to obtain recombinant bacteria BL21(DE3) / pET26b(+)-M. tac 1-ICCG-EGFP, BL21(DE3) / pET26b(+)-M tac 2-ICCG-EGFP, BL21(DE3) / pET26b(+)-M tac 3-ICCG-EGFP and BL21(DE3) / pET26b(+)-M tac 4-ICCG-EGFP.

[0050] The control strain BL21(DE3) / pET26b(+)-ICCG-EGFP was constructed according to the above method.

[0051] The control strain BL21(DE3) / pET26b(+) was constructed according to the above method. tac -ICCG-EGFP.

[0052] Example 5 Fluorescence intensity determination of each recombinant strain and control strain.

[0053] The recombinant bacteria and control bacteria obtained in Example 4 were inoculated onto LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Single colonies were picked and cultured in 96-well plates. After the cells entered the logarithmic growth phase, IPTG was added to a final concentration of 1 mM, and expression was induced at 25°C for 12 h. After induction, the fluorescence intensity (excitation wavelength 488 nm, emission wavelength 509 nm) and OD of the bacterial culture were measured using a multi-functional microplate reader. 600 Value, in Fluorescence / OD 600 Characterize protein expression levels.

[0054] Fluorescence measurement results (see) Figure 2 The results showed that the fluorescence intensity of the four recombinant bacteria was higher than that of the two control bacteria: recombinant bacteria BL21(DE3) / pET26b(+)-M tac 1-ICCG-EGFP, BL21(DE3) / pET26b(+)-M tac 2-ICCG-EGFP, BL21(DE3) / pET26b(+)-M tac 3-ICCG-EGFP and BL21(DE3) / pET26b(+)-M tac The fluorescence intensity of 4-ICCG-EGFP relative to the control strain BL21(DE3) / pET26b(+)-ICCG-EGFP was 1.57, 1.57, 1.58, and 1.65 times, respectively; relative to the control strain BL21(DE3) / pET26b(+)-ICCG-EGFP, it was also 1.57, 1.57, 1.58, and 1.65 times higher. tac The concentrations of -ICCG-EGFP were 1.2, 1.21, 1.22, and 1.27 times, respectively.

[0055] Example 6 Induction of expression and purification of target protein in recombinant and control strains.

[0056] (1) Induced expression Each recombinant bacterial strain and each control bacterial strain were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. The overnight culture was then inoculated into fresh LB liquid medium containing kanamycin at a ratio of 1:100 (v / v) and cultured until OD500 was reached. 600 The initial concentration was 0.2–0.4, followed by the addition of IPTG to a final concentration of 0.5 mM, and expression was induced at 25°C for 8 h.

[0057] (2) Protein purification After induction, bacterial cells were collected by centrifugation at 4000 rpm for 15 min at 4°C and resuspended in lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 5% glycerol, pH 7.5). The resuspended cells were lysed using a high-pressure cell disruptor, and the resulting lysis buffer was centrifuged at 18000 rpm for 50 min, and the supernatant was collected. The supernatant was incubated with nickel affinity chromatography media at 4°C for 2 h, followed by washing and elution steps: Non-specifically bound proteins were removed using a washing buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 5% glycerol, pH 7.5). The target protein was eluted using elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, 5% glycerol, pH 7.5).

[0058] The eluted target protein was detected by SDS-PAGE gel electrophoresis (see [link]). Figure 3 Protein yield (mg / L) was collected based on molecular weight and normalized to cell density. -1 ·OD 600 -1 To evaluate the expression levels of functional proteins.

[0059] Results of functional protein expression assay (see) Figure 4 The results showed that all four recombinant bacteria were superior to the control bacteria: recombinant bacteria BL21(DE3) / pET26b(+)-M tac 1-ICCG-EGFP, pET26b(+)-M tac 2-ICCG-EGFP, pET26b(+)-M tac 3-ICCG-EGFP and pET26b(+)-M tacThe functional protein expression levels of 4-ICCG-EGFP were 3.92, 4.15, 4.54, and 4.92 times higher than those of the control strain BL21(DE3) / pET26b(+)-ICCG-EGFP, respectively; and higher than those of the control strain BL21(DE3) / pET26b(+)-ICCG-EGFP. tac The values ​​of -ICCG-EGFP were 1.22, 1.29, 1.41, and 1.53 times, respectively.

[0060] In summary, the fluorescence intensity results of Example 5 and the protein purification results of this example together demonstrate that, in this invention, through the analysis of... tac The interval sequence between the -35 and -10 regions of the promoter was obtained through optimization. tac The promoter mutants (SEQ ID NO. 6 to SEQ ID NO. 9) can effectively increase the functional expression level of the target protein.

[0061] Example 7 Experiments on the degradation of PET by the target protein expressed by recombinant bacteria.

[0062] (1) PET degradation reaction A commercially available PET membrane (6 mm diameter disc, approximately 8 mg in mass) was selected as the substrate and placed in a 600 μL reaction system. The reaction system included 100 mM potassium phosphate buffer (pH 8.0) to a final concentration of 500 nM of the target protein.

[0063] The above reaction system was incubated in a 72℃ constant temperature reaction apparatus for 18 h to carry out the PET degradation reaction. After the reaction was completed, an equal volume of methanol was added to the reaction system to terminate the enzymatic reaction. After thorough mixing and centrifugation, the supernatant was used for subsequent product analysis.

[0064] (2) UPLC quantitative analysis of PET depolymerization products.

[0065] The reaction products were quantitatively analyzed using ultra-high performance liquid chromatography (UPLC), including terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). The UPLC detection methods are shown in Table 3. Table 3 UPLC Detection Methods

[0066] Experimental results (see) Figure 5 This indicates that the invention described herein... tac The target protein expressed by the promoter mutant exhibits superior PET degradation performance, with the total release of PET degradation products increasing by approximately 25% compared to the T7 promoter system.

Claims

1. tac Promoter mutant, named M tac 1~M tac 4, characterized in that, The M tac 1~M tac The nucleotide sequences of 4 are shown in SEQ ID NO. 6 to SEQ ID NO. 9, respectively.

2. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the one described in claim 1. tac The promoter mutant and the gene encoding the PET hydrolase ICCG, the nucleotide sequence of which is shown in SEQ ID NO.

1.

3. The recombinant plasmid according to claim 2, characterized in that, The plasmid backbone is pET26b(+).

4. Recombinant bacteria containing the recombinant plasmid of claim 2 or 3.

5. The recombinant bacteria according to claim 4, characterized in that, The host of the recombinant bacteria is Escherichia coli BL21(DE3).

6. The use of the recombinant bacteria according to claim 4 or 5 in the preparation of PET hydrolase.

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