Method for improving soluble expression of non-ribosome peptide synthetase in escherichia coli

Abstract

The invention discloses a method for improving soluble expression of non-ribosome peptide synthetase in escherichia coli. According to the method, a polyion oligopeptide label is connected to the N end or the C end of the non-ribosome peptide synthetase; the method comprises the following steps: adding a polyion oligopeptide tag (D) 5 at the N / C end of ebony in a fusion protein SGE formed by non-ribosomal peptide synthetase ebony for producing decarboxylated carnosine and phosphokinase Sfp through a connecting peptide GSG, or replacing a GSG sequence in an original SGE sequence with the polyion oligopeptide tag (D) 5 to construct new plasmids, and transforming the obtained three new plasmids in recombinant escherichia coli to obtain the decarboxylated carnosine-producing non-ribosomal peptide synthetase ebony-phosphokinase Sfp. The solubility of the non-ribosome peptide synthetase in escherichia coli is greatly improved, and the addition of the polyion oligopeptide tag (D) 5 at the N end is more beneficial to reducing the formation of inclusion bodies and improving the yield of decarboxylated carnosine.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering and relates to a method for improving the soluble expression of non-ribosomal peptide synthetase in Escherichia coli. (II) Background technology

[0002] Soluble expression has always been an issue that cannot be ignored in the process of efficient protein production. However, the prokaryotic system lacks complex post-translational protein modification mechanisms, and the reducing environment in the bacterial cytoplasm is not conducive to the formation of disulfide bonds. Therefore, heterologous expression of target proteins in prokaryotes is more likely to form inclusion bodies.

[0003] Methods to reduce inclusion body formation include: selecting low-copy vectors, choosing appropriate hosts, co-expressing with other molecules and adding chemical chaperones, and changing expression conditions such as lowering the induction temperature or reducing the concentration of inducers. However, the best solution so far is still soluble tags, which consist of proteins or peptides connected to the target protein, which help to achieve natural folding, thereby producing appropriate functional activity, or achieving higher levels of poorly expressed proteins.

[0004] At present, decarboxylated carnosine is mainly produced by chemical methods, but chemical methods are prone to produce by-products, complex operations, high production costs and easy to pollute the environment, which does not meet the requirements of sustainable development. Biosynthesis is a more environmentally friendly method, which has been widely used in industrial production using whole cells as biocatalysts. However, when non-ribosomal peptide synthetase is heterologously expressed in Escherichia coli, it is a gene from eukaryotic organisms and the protein is large, so it is very easy to form inclusion bodies, which greatly limits the application prospects of non-ribosomal peptide synthetase SGE.

[0005] Therefore, it is urgent to find a method to improve the soluble expression of heterologous proteins in Escherichia coli, reduce the formation of inclusion bodies, and increase the yield of decarboxylated carnosine. (III) Summary of the invention

[0006] The present invention aims to provide a method for improving the soluble expression of non-ribosomal peptide synthetase in Escherichia coli. By introducing a polyion short peptide tag (D) 5, the soluble expression of non-ribosomal peptide synthetase in Escherichia coli is effectively improved, the formation of inclusion bodies is reduced, and the yield of decarboxylated carnosine is increased.

[0007] The technical solution adopted by the present invention is:

[0008] In the first aspect, the present invention provides a method for improving the soluble expression of a non-ribosomal peptide synthetase in Escherichia coli using a polyion short peptide tag (D) 5, wherein the method comprises connecting a polyion short peptide tag (D) 5 to the N-terminus or C-terminus of the non-ribosomal peptide synthetase; the polyion short peptide tag (D) 5 consists of 5 aspartic acids and has an amino acid sequence of: DDDDD.

[0009] In a second aspect, the present invention provides a method for increasing the yield of decarboxylated carnosine synthetase synthesized by non-ribosomal peptide synthetase by utilizing polyion short peptide tag (D) 5. The method comprises connecting the polyion short peptide tag (D) 5 to the N-terminus or C-terminus of the non-ribosomal peptide synthetase of the fusion protein of the decarboxylated carnosine-producing phosphotransferase and the non-ribosomal peptide synthetase, or replacing the connecting peptide between the non-ribosomal peptide synthetase and the phosphotransferase with the polyion short peptide tag (D) 5; the polyion short peptide tag (D) 5 consists of 5 aspartic acids, and the amino acid sequence is: DDDDD.

[0010] Furthermore, the non-ribosomal peptide synthetase is D. melanogaster non-ribosomal peptide synthetase Ebony, and the phosphotransferase is B. subtilis phosphotransferase Sfp.

[0011] Furthermore, the fusion protein of the phosphotransferase and non-ribosomal peptide synthetase producing decarboxylated carnosine is obtained by operably connecting the non-ribosomal peptide synthetase Ebony and the phosphotransferase Sfp via a connecting peptide GSG.

[0012] Furthermore, the method for synthesizing decarboxylated carnosine by the non-ribosomal peptide synthetase is as follows: whole cells of recombinant genetic engineering bacteria expressing a fusion protein of a non-ribosomal peptide synthetase and a phosphotransferase containing a polyion short peptide tag (D) 5 are induced to express as catalysts, histamine and β-alanine are used as substrates, and a reaction system is constructed with ATP and MgCl2 as cofactors; a whole-cell reaction is carried out in a 50 mM sodium phosphate buffer solution at pH 7 at 25° C., the oscillation reaction speed is 800 r / min, and the reaction time is not less than 48 h; in the reaction system, the final concentration of the whole cells is 12.5 g / L, the final concentration of the histamine is 20 mM, the final concentration of the β-alanine is 10 mM, the final concentration of the ATP is 2.5 mM, and the final concentration of the MgCl2 is 10 mM.

[0013] Furthermore, the recombinant genetically engineered bacteria is constructed as follows: the polyion short peptide tag (D) 5 is respectively connected to the N-terminus or C-terminus of ebony in the non-ribosomal peptide synthetase and phosphotransferase fusion protein plasmid pTrc-p15A-Sfp-GSG-Ebony by homologous recombination, or the GSG connecting peptide is replaced to construct recombinant plasmids pTrc-p15A-Sfp-GSG-DDDDD-Ebony, pTrc-p15A-Sfp-GSG-Ebony-DDDDD, and pTrc-p15A-Sfp-DDDDD-Ebony, and the recombinant plasmid is introduced into Escherichia coli to obtain a recombinant genetically engineered bacteria with improved decarboxylated carnosine production.

[0014] In a third aspect, the present invention provides a recombinant plasmid containing a polyion short peptide tag (D) 5, wherein the vector of the recombinant plasmid is pTrc-p15A-Sfp-GSG-Ebony; the recombinant plasmid is obtained by inserting the polyion short peptide tag (D) 5 into the N-terminus or C-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony, or by replacing the GSG connecting peptide.

[0015] Further, the recombinant plasmid is constructed as follows:

[0016] (1) Using the pTrc-p15A-Sfp-GSG-Ebony plasmid as a template, the following primers were used for inverse PCR amplification, and the nucleotide sequence shown in SEQ ID NO: 1 was inserted into the N-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony. The PCR product was purified and recovered to obtain the linearized vector pTrc-p15A-Sfp-GSG-DDDDD-Ebony;

[0017] NE-Asp5-F:GGTGATGATGATGATGATatgggcagcttaccgca;

[0018] NE-Asp5-R: ATCATCATCATCATCACCGCTACCTAAAAGCTCTTCG;

[0019] The linearized vector is transferred into E. coli W3110 competent cells, screened on a culture medium containing kanamycin, single clones are selected for sequencing verification, and plasmids are extracted to obtain the recombinant plasmid.

[0020] (2) Using the pTrc-p15A-Sfp-GSG-Ebony plasmid as a template, the following primers were used for inverse PCR amplification to insert the nucleotide sequence shown in SEQ ID NO: 1 into the C-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony, and the PCR product was purified and recovered to obtain the linearized vector pTrc-p15A-Sfp-GSG-Ebony-DDDDD;

[0021] EC-Asp5-F:GATGATGATGATGATtaaGGCTGTTTGGCGGA;

[0022] EC-Asp5-R:ATCATCATCATCATCtttaccaacttctttccaatg;

[0023] The linearized vector is transferred into E. coli W3110 competent cells, screened on a culture medium containing kanamycin, single clones are selected for sequencing verification, and plasmids are extracted to obtain the recombinant plasmid.

[0024] (3) Using the pTrc-p15A-Sfp-GSG-Ebony plasmid as a template, reverse PCR amplification was performed with the following primers to replace the GSG sequence in pTrc-p15A-Sfp-GSG-Ebony with the nucleotide sequence shown in SEQ ID NO: 1, and the PCR product was purified and recovered to obtain the linearized vector pTrc-p15A-Sfp-DDDDD-Ebony:

[0025] S-Asp5-EF:TTAGATGATGATGATGATatgggcagcttaccgca;

[0026] S-Asp5-ER:catATCATCATCATCATCTAAAAGCTCTTCGTACGAGACCA;

[0027] The linearized vector is transferred into E. coli W3110 competent cells, screened on a culture medium containing kanamycin, single clones are selected for sequencing verification, and plasmids are extracted to obtain the recombinant plasmid.

[0028] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium obtained by transformation of the recombinant plasmid. The recombinant genetically engineered bacterium is constructed according to the following method: the recombinant plasmid is transferred into E. coli W3110 competent cells by a heat shock method, and the transformation product is evenly spread on an LB solid culture medium containing 100 μg / mL kanamycin, and cultured overnight at 37°C. A single clone is picked for sequencing verification to obtain the recombinant genetically engineered bacterium.

[0029] In the fifth aspect, the present invention provides a polyion short peptide tag (D) 5 for improving the soluble expression of non-ribosomal peptide synthetase SGE in Escherichia coli. The polyion short peptide tag (D) 5 is composed of 5 aspartic acids, the amino acid sequence is shown as DDDDD, and the encoding gene nucleotide sequence is shown as GATGATGATGATGAT.

[0030] Compared with the prior art, the beneficial effects of the present invention are mainly embodied in that: the present invention adds a polyion short peptide tag (D) 5 to the N / C terminus of ebony in a fusion protein SGE composed of a non-ribosomal peptide synthetase ebony producing decarboxylated carnosine and a phosphokinase Sfp with a connecting peptide GSG, or replaces the GSG sequence in the original SGE sequence with the polyion short peptide tag (D) 5 to construct a new plasmid, and the obtained three new plasmids are transformed in recombinant Escherichia coli, so that the solubility of the non-ribosomal peptide synthetase in Escherichia coli is greatly improved, wherein the addition of the polyion short peptide tag (D) 5 to the N terminus is more helpful to reduce the formation of inclusion bodies and increase the yield of decarboxylated carnosine. (IV) Description of the drawings

[0031] Figure 1 , Agarose gel electrophoresis of the fusion protein obtained by PCR amplification, M represents Marker; (1) represents SGED5, lanes 1-3 represent parallel samples; (2) represents SGD5E, lanes 1-5 represent parallel samples; (3) represents SD5E, lanes 1-4 represent parallel samples.

[0032] Figure 2 , SDS-PAGE electrophoresis of bacterial solution, supernatant after disruption, and precipitate of WSGE, WSGD5E, WSGED5, and WSD5E engineered bacteria before and after induction expression, M represents Marker;

[0033] WSGE: 1 represents the bacterial solution before WSGE induction; 2 represents the egg bacterial solution after WSGE induction; 3 represents the supernatant after WSGE was broken; 4 represents the precipitate after WSGE was broken;

[0034] WSGD5E: 1 represents the bacterial solution before WSGD5E induction; 2 represents the bacterial solution after WSGD5E induction; 3 represents the supernatant after WSGD5E was broken; 4 represents the precipitate after WSGD5E was broken;

[0035] WSD5E: 1 represents the bacterial solution after WSD5E induction; 2 represents the supernatant after WSD5E was broken; 3 represents the precipitate after WSD5E was broken;

[0036] WSGED5: 4 represents the bacterial liquid after WSGED5 induction; 5 represents the supernatant after WSGED5 was broken; 6 represents the precipitate after WSGED5 was broken.

[0037] Figure 3 , Decarboxylated carnosine production catalyzed by whole cells of different strains (error bars represent the standard deviation of three biological replicates). a: WSGE-E indicates strains with ELP tags, WSGE-S indicates strains with SUMO tags, and WSGE-E indicates strains with SUMO+ELP tags. (V) Specific implementation methods

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] Unless otherwise specified, the materials and reagents used in the examples can be obtained from conventional sales channels; the experimental methods used in the examples are conventional methods unless otherwise specified.

[0040] The plasmid pTrc-p15A-Sfp-GSG-Ebony used in the embodiments of the present invention was constructed using the method of patent application CN114350692A, and the publication date was April 15, 2022.

[0041] The LB liquid medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent was deionized water, and the pH value was natural;

[0042] LB solid medium composition: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 20g / L, solvent is deionized water, pH value is natural.

[0043] The composition of 50mM sodium phosphate buffer, pH=7 is: 0.2mol / L Na2HPO4, 0.2mol / L NaH2PO4.

[0044] Example 1: Construction of recombinant expression plasmid

[0045] 1. Construction of pTrc-p15A-Sfp-GSG-DDDDD-Ebony plasmid

[0046] Insert the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) into the N-terminus of ebony in the plasmid pTrc-p15A-Sfp-GSG-Ebony to construct the plasmid pTrc-p15A-Sfp-GSG-DDDDD-Ebony. The specific operation is as follows:

[0047] (1) Using pTrc-p15A-Sfp-GSG-Ebony plasmid (referred to as P15A-SGE plasmid) as a template and the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) as the homology arm to design primers, reverse PCR amplification was performed using primers NE-Asp5-F and NE-Asp5-R in Table 1, and the PCR product was purified and recovered to obtain a linearized vector.

[0048] PCR amplification system (50 μL): 1 μL of 100 ng / μL P15A-SGE plasmid as template, 1 μL each of 10 μM primers NE-Asp5-F and NE-Asp5-R, 25 μL 2xPhanta Max Buffer, 1 μL dNTP Mix, 0.5 μL PhantaMax Super-Fidelity DNA Polymerase, and 20.5 μL ultrapure water.

[0049] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by temperature cycling at 95°C for 30 sec; 59°C for 30 sec; 72°C for 4 min; a total of 36 cycles at 72°C, final extension for 10 min, and a final termination temperature of 16°C.

[0050] The above PCR product (denoted as SGD5E) was subjected to agarose gel electrophoresis experiment. Figure 1 (2). As can be seen from the figure, the PCR product contains mixed bands, and gel cutting and recovery are required (DNA gel recovery kit purchased from Qingke Company). Cut off the target DNA band and try to cut off the gel without the target DNA band. The smaller the gel volume, the better. Put the gel containing the target DNA band into a 2mL centrifuge tube, add an appropriate amount of Buffer GL (just enough to cover the target band), and place it in a 65℃ water bath for 4 to 6 minutes until the gel is completely melted. Then activate the silica gel membrane: place the adsorption column in the collection tube, add 250μL Buffer BL, centrifuge at 12000rpm for 1min, and discard the waste liquid. Transfer the solution to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, and put the adsorption column back into the empty collection tube. Then add 700μL Buffer W2 to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, repeat twice, centrifuge at 12000rpm for 2min, finally take out the adsorption column, place it in a clean 1.5mL centrifuge tube, add 35-50μL Eluent (preheating Eluent at 60-65℃ has a better effect) to the middle part of the adsorption membrane, centrifuge at 12000rpm for 2min, and the resulting supernatant is the purified PCR linear product.

[0051] (2) Recombinant plasmid

[0052] The purified PCR linear product was digested with endonuclease Dpn I at 37°C for 1 h to remove the template DNA, and then directly transferred into E. coli W3110 competent cells by heat shock method. The transformation product was evenly spread on LB agar plates containing 100 μg / mL kanamycin resistance (Kan) and cultured at 37°C overnight to obtain transformants containing the recombinant expression plasmid pTrc-p15A-Sfp-GSG-DDDDD-Ebony.

[0053] 2. Construction of pTrc-p15A-Sfp-GSG-Ebony-DDDDD plasmid

[0054] Insert the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) into the C-terminus of ebony in the plasmid pTrc-p15A-Sfp-GSG-Ebony to construct the pTrc-p15A-Sfp-GSG-Ebony-DDDDD plasmid. The specific operation is as follows:

[0055] Using the pTrc-p15A-Sfp-GSG-Ebony plasmid as a template and the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) as the homology arm to design primers, reverse PCR amplification was performed using primers EC-Asp5-F and EC-Asp5-R in Table 1, and the PCR product was purified and recovered using the method in step 1 to obtain a linearized vector.

[0056] PCR amplification system (50 μL): 1 μL of 100 ng / μL P15A-SGE plasmid as template, 1 μL of 10 μM primer EC-Asp5-F and primer EC-Asp5-R, 25 μL 2xPhanta Max Buffer, 1 μL dNTP Mix, 0.5 μL PhantaMax Super-Fidelity DNA Polymerase, and 20.5 μL ultrapure water.

[0057] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 8 min, followed by temperature cycling at 95°C for 30 sec; 58°C for 30 sec; 72°C for 4 min 10 s; a total of 37 cycles at 72°C, final extension for 12 min, and a final termination temperature of 16°C.

[0058] The above PCR product (denoted as SGED5) was subjected to agarose gel electrophoresis experiment. Figure 1(1). As can be seen from the figure, there are bands of the same size as the target band. The plasmid PCR product of the reverse amplification was digested with endonuclease Dpn I at 37°C for 1h to remove the template DNA. After purification with a purification kit, it was directly transferred into E. coli W3110 competent cells by heat shock method. The transformation product was evenly spread on LB agar plates containing 100μg / mL Kan, and cultured at 37°C overnight to obtain transformants containing the recombinant expression plasmid pTrc-p15A-Sfp-GSG-Ebony-DDDDDD.

[0059] 3. Construction of pTrc-p15A-Sfp-DDDDD-Ebony plasmid

[0060] The GSG sequence in the plasmid pTrc-p15A-Sfp-GSG-Ebony was replaced by the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) to construct the pTrc-p15A-Sfp-DDDDD-Ebony plasmid. The specific operations are as follows:

[0061] Using the pTrc-p15A-Sfp-GSG-Ebony plasmid as a template and the polyion short peptide tag encoding gene sequence (GATGATGATGATGAT) as the homology arm to design primers, reverse PCR amplification was performed using the primers S-Asp5-EF and S-Asp5-ER in Table 1, and the PCR product was purified and recovered using the method in step 1 to obtain a linearized vector.

[0062] PCR amplification system (50 μL): 1 μL of 100 ng / μL P15A-SGE plasmid as template, 1 μL each of 10 μM primers S-Asp5-EF and S-Asp5-ER, 25 μL 2xPhanta Max Buffer, 1 μL dNTP Mix, 0.5 μL PhantaMax Super-Fidelity DNA Polymerase, and 20.5 μL ultrapure water.

[0063] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 8 min, followed by temperature cycling at 95°C for 30 sec; 58°C for 30 sec; 72°C for 4 min; a total of 35 cycles at 72°C, final extension for 15 min, and the final termination temperature was 16°C.

[0064] The above PCR product (denoted as SD5E) was subjected to agarose gel electrophoresis experiment. Figure 1(3). As can be seen from the figure, there are bands of the same size as the target band. The plasmid PCR product of the reverse amplification was digested with endonuclease Dpn I at 37°C for 1h to remove the template DNA. After purification with a purification kit, it was directly transferred into E. coli W3110 competent cells by heat shock method. The transformation product was evenly spread on LB agar plates containing 100 μg / mL kanamycin resistance (Kan), and cultured at 37°C overnight to obtain transformants containing the recombinant expression plasmid pTrc-p15A-Sfp-DDDDD-Ebony.

[0065] Table 1 Construction of recombinant plasmids and related primers for verification

[0066]

[0067] Example 2: Construction of recombinant genetically engineered bacteria

[0068] The transformants obtained in Example 1 were subjected to the following operations:

[0069] (1) Sequencing verification: Use a sterilized pipette tip to select 5-8 transformants on the plate for colony PCR (primers used are shown in Table 2). The correct bands were inoculated into LB liquid medium containing 100 μg / mL kanamycin resistance, and cultured at 37°C, 180 r / min, and shaking for 12 h to obtain seed solution, sequence and maintain the bacteria. The constructed strains are shown in Table 3.

[0070] Table 2 Colony PCR primers required for transformants in Example 1

[0071]

[0072] Table 3 Strains constructed in Example 2

[0073]

[0074] (2) Induced expression of recombinant plasmid: Under sterile conditions, 3 mL of seed solution (2% inoculum) was taken from the LB liquid medium in step (1) and transferred to 150 mL of LB medium (containing Kana at a final concentration of 500 μg / mL), and cultured at 37°C, 200 rpm, and shaken for about 1.5 h until the OD 600 The concentration of IPTG was 0.6-0.8, and the final concentration of IPTG was 0.2 mM. The cells were cultured in a shaking incubator at 16°C for 20 h to induce expression.

[0075] (3) SDS-PAGE protein electrophoresis verification: Take 1mL of the induced bacterial solution, centrifuge at 12000rpm for 2min, and discard the supernatant. Resuspend the precipitate with 500μL PB buffer, ultrasonically disrupt the bacterial suspension for 3min, centrifuge at 12000rpm for 5min, and keep the supernatant. Take 30μL each of the induced bacterial solution, the disrupted supernatant, and the precipitate, then add 10μL of protein loading buffer to the bottom, boil in boiling water for 10min to mix evenly. Take 10μL of the mixed solution and 5μL of Blue PlusⅡProtein Marker and load them into the wells of the SDS-PAGE precast gel. Perform gel electrophoresis separation and analysis at a constant voltage of 180V for 45min. After the electrophoresis, stain for 10min and decolorize for 10min. According to the relative mobility of the protein sample to be tested, the molecular weight of the corresponding protein band is obtained from the protein standard. The results are shown in the attached. Figure 2 .

[0076] Figure 2 The results showed that the fusion tag D5 was connected to the N-terminus and C-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony by homologous recombination, and the replacement of the GSG connecting peptide could increase the soluble protein expression of non-ribosomal peptide synthetase SGE in Escherichia coli.

[0077] Example 3: Whole-cell catalytic reaction

[0078] The bacterial solution induced in Example 2 was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded to collect the bacteria; 22.5 mg of the collected bacteria, 1.6 mg of β-alanine, 1.7 mg of magnesium chloride, 2.5 mg of ATP, and 4 mg of histamine (histamine was added after 30 min of reaction) were dissolved in 50 mM sodium phosphate buffer solution at pH = 7 to form a 2 mL reaction system, and the reaction was carried out in a constant temperature oscillator at 25°C, the oscillation reaction speed was 800 r / min, and the reaction time was 60 h.

[0079] Samples were taken at 12, 24, 36, 48, and 60 hours, with each sample being 50 μL. First, 200 μL of acetonitrile was added, and the mixture was centrifuged at 12000 rpm for 10 minutes after shaking. 200 μL of the supernatant was taken, and 200 μL of NaOH-saturated NaHCO3 buffer (pH = 10.12) and 100 μL of DNS (dansyl chloride, 8 mg / mL) were added to mix, and the derivatization reaction was carried out at 60°C for 40 minutes, and then extracted with ethyl acetate for 3 times. The content of decarboxylated carnosine generated by the reaction was then detected by high performance liquid chromatography (HPLC). HPLC detection method: the chromatographic column was Welchrom C18 (4.6 mm × 250 mm), the ultraviolet detection wavelength was 246 nm, the flow rate was 1 mL / min, the injection volume was 10 μL, the column temperature was 35°C, the mobile phase was 68% acetonitrile, and 32% ammonium acetate was used for liquid phase detection.

[0080] from Figure 3 As can be seen in Figure b, after adding the D5 polyion short peptide fusion tag, the yield in each time period was higher than that of the initial strain. At 48 h, the yield of strain WSGD5E reached 1.95 times that of the initial strain.

[0081] The same method was used to replace D5 in WSGED5 with ELP, SUMO, and ELP-SUMO, respectively. Other operations were the same. The results are shown in Figure 3 In a, it can be seen that other soluble tags ELP and SUMO have poor effects on protein soluble expression. The decarboxylated carnosine production of the recombinant expression strain WSGE-ES constructed with the soluble tag ELP+SUMO is 2.89mM. When using the soluble tags ELP and SUMO, the production of the recombinant expression strain WSGE-E / WSGE-S is lower than that of the WSGE-ES strain, which are 2.39mM and 2.59mM, respectively. The catalytic efficiency of all strains continued to increase within 48h, but the increase was small.

[0082] ELP: (VPGXG) 5

[0083] SUMO:

[0084] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0085] ELP-SUMO:

[0086] VPGSGVPGSGVPGSGVPGSGVPGSGGSGGSGMSDSEVNQEAKPEVKPEVKPETHIN

[0087] LKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0088] The above embodiments are only used to help understand the method of the present invention and its core concept. It should be noted that, for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications may be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the soluble expression of non-ribosomal peptide synthetase in Escherichia coli using polyion short peptide tags, characterized in that: The method comprises connecting a polyion short peptide tag to the N-terminus or the C-terminus of the non-ribosomal peptide synthetase; the polyion short peptide tag is composed of 5 aspartic acids.

2. A method for increasing the yield of decarboxylated carnosine synthetase by using polyion short peptide tags, characterized in that: The method comprises connecting a polyion short peptide tag to the N-terminus or C-terminus of a non-ribosomal peptide synthetase of a fusion protein of a phosphotransferase producing decarboxylated carnosine and a non-ribosomal peptide synthetase, or changing the connecting peptide between the non-ribosomal peptide synthetase and the phosphotransferase to a polyion short peptide tag; the polyion short peptide tag is composed of 5 aspartic acids.

3. The method according to claim 2, characterized in that The non-ribosomal peptide synthetase is Drosophilamelanogaster non-ribosomal peptide synthetase Ebony, and the phosphotransferase is Bacillus subtilis phosphotransferase Sfp.

4. The method according to claim 2, characterized in that The method comprises the following steps: using whole cells of recombinant genetic engineering bacteria expressing a non-ribosomal peptide synthetase and a phosphotransferase fusion protein containing a polyion short peptide tag and induced expression as a catalyst, using histamine and beta-alanine as substrates, and using ATP and MgCl2 as cofactors to construct a reaction system, carrying out whole-cell reaction in a 50 mM sodium phosphate buffer solution at pH 7 at 25° C., with an oscillating reaction speed of 800 r / min and a reaction time of not less than 48 hours, to obtain a reaction solution containing decarboxylated carnosine.

5. The method according to claim 4, characterized in that In the reaction system, the final concentration of the whole cells is 12.5 g / L, the final concentration of the histamine is 20 mM, the final concentration of the β-alanine is 10 mM, the final concentration of the ATP is 2.5 mM, and the final concentration of the MgCl2 is 10 mM.

6. The method according to claim 4, characterized in that The recombinant genetic engineering bacteria is constructed by the following method: a polyion short peptide tag is respectively connected to the N-terminus or the C-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony in a homologous recombination manner, or a GSG connecting peptide is replaced to construct recombinant plasmids pTrc-p15A-Sfp-GSG-DDDDD-Ebony, pTrc-p15A-Sfp-GSG-Ebony-DDDDD, and pTrc-p15A-Sfp-DDDDD-Ebony, and the recombinant plasmids are introduced into Escherichia coli to obtain recombinant genetic engineering bacteria with improved decarboxylated carnosine production.

7. A recombinant plasmid containing a polyion short peptide tag, characterized in that: The vector of the recombinant plasmid is pTrc-p15A-Sfp-GSG-Ebony; the recombinant plasmid is obtained by inserting the polyion short peptide tag into the N-terminus or C-terminus of ebony in pTrc-p15A-Sfp-GSG-Ebony, or replacing the GSG connecting peptide.

8. A polyionic short peptide tag for improving the soluble expression of non-ribosomal peptide synthetase in Escherichia coli, wherein the polyionic short peptide tag consists of 5 aspartic acids.

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