Method for producing shikimic acid by utilizing shikimic acid dehydrogenase mutant

Abstract

The invention discloses a method for producing shikimic acid by utilizing a shikimic acid dehydrogenase mutant, and belongs to the technical field of gene engineering. According to the method, the shikimic acid is prepared by fermenting a recombinant bacterium containing mutated shikimic acid dehydrogenase AroE or a biological material; the mutated shikimic acid dehydrogenase AroE has any one or more of the following mutations: the 61 amino acid of SEQ ID No.1 mutates from arginine to tryptophan, the 102 amino acid of SEQ ID No.1 mutates from alanine to aspartic acid, and the 241 amino acid of SEQ ID No.1 mutates from leucine to isoleucine. The mutant provided by the invention can enhance the affinity to the substrate dehydrogenation shikimic acid, reduce the oxidation reaction to shikimic acid, finally improve the yield of shikimic acid, reduce the accumulation of byproducts, improve the quality of shikimic acid fermentation liquor and greatly save the industrial production cost.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a method for producing shikimic acid by utilizing a shikimic acid dehydrogenase mutant. Background Art

[0002] Shikimic acid (SA), chemical name (3R,4S,5R)-(-)-3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid, has a structural feature of containing a six-carbon ring alcohol, three chiral carbons and a carboxylic acid functional group, and a chemical formula of C7H 10 O5. Shikimic acid is easily soluble in water with a solubility of 180g / L, and is difficult to dissolve in chloroform, benzene and petroleum ether. The melting point is 185℃-191℃, and the absorption peak is around 210nm. The optical rotation is -180°, and the smell is pungent and sour.

[0003] Shikimic acid is the key synthetic precursor of the anti-influenza drug oseltamivir, and its monomer itself also has certain anti-influenza effects. As people pay more and more attention to influenza viruses, the prevention and symptomatic treatment of influenza drugs have become quite popular, and the use of oseltamivir has increased significantly, followed by a surge in the use of shikimic acid. With the large-scale production of biosynthetic shikimic acid technology, the traditional star anise extraction and preparation method has no cost advantage. Shikimic acid dehydrogenase is one of the key enzymes in the shikimic acid biosynthesis pathway, catalyzing the fourth step reaction in the shikimic acid pathway. Shikimic acid dehydrogenase uses reduced coenzyme II (NADPH) to catalyze 3-dehydroshikimic acid (DHS) to generate shikimic acid.

[0004] 3-Dehydroshikimic acid has a crucial impact on the quality of the fermentation liquid in the production of shikimic acid by microbial engineering bacteria. As the target shikimic acid is accumulated by the engineering bacteria, its miscellaneous acids quinic acid and dehydroshikimic acid also accumulate accordingly. Studies have shown that in the process of fermentation and production of shikimic acid, every 27g of shikimic acid is accompanied by the production of 4.4g of 3-dehydroshikimic acid. Therefore, the crystallization efficiency, purity and quality of shikimic acid are greatly reduced. It can be seen that reducing the content of byproduct 3-dehydroshikimic acid in the fermentation product is an urgent problem to be solved for the large-scale production of shikimic acid by microbial fermentation.

[0005] Existing shikimate dehydrogenase is also subject to feedback inhibition by shikimate. When the product shikimate accumulates to a high concentration in the fermentation system, shikimate dehydrogenase is bound by the high concentration of shikimate and cannot bind to the forward reaction substrate 3-dehydroshikimate to play a normal catalytic function, thereby limiting the production of shikimate. Therefore, improving the binding specificity of shikimate dehydrogenase to 3-dehydroshikimate is expected to reduce the occurrence of this product feedback inhibition.

[0006] In summary, it is hoped to find a more ideal shikimate dehydrogenase, which has a higher activity in catalyzing the reduction of 3-dehydroshikimate to shikimate and a lower activity in catalyzing the reverse reaction of shikimate to 3-dehydroshikimate under certain conditions, and has a higher binding specificity for 3-dehydroshikimate, thereby increasing the accumulation of shikimate and reducing the content of impurities in the product. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an enzyme that can increase the accumulation of shikimic acid and reduce the content of impurities in the product. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a protein, wherein the protein is any one of the following: A1) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine (R) to tryptophan (W), and the amino acid at position 102 is mutated from alanine (A) to aspartic acid (D), while the other amino acid sequences remain unchanged; A2) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine (R) to tryptophan (W), the amino acid at position 102 is mutated from alanine (A) to aspartic acid (D), and the amino acid at position 241 is mutated from leucine (L) to isoleucine (I), while the other amino acid sequences remain unchanged; A3) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine (R) to tryptophan (W), while the other amino acid sequences remain unchanged; A4) a protein in which the amino acid at position 102 of SEQ ID No. 1 is mutated from alanine (A) to aspartic acid (D), while the other amino acid sequences remain unchanged; A5) A protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine (R) to tryptophan (W), and the amino acid at position 241 is mutated from leucine (L) to isoleucine (I), while the other amino acid sequences remain unchanged; A6) A protein in which the amino acid at position 241 of SEQ ID No. 1 is mutated from leucine (L) to isoleucine (I), while the other amino acid sequences remain unchanged.

[0009] Among the above proteins, the nucleotide sequence of the protein A1) is SEQ ID No.10, the nucleotide sequence of the protein A2) is SEQ ID No.14, the nucleotide sequence of the protein A3) is SEQ ID No.4, the nucleotide sequence of the protein A4) is SEQ ID No.6, the nucleotide sequence of the protein A5) is SEQ ID No.12, and the nucleotide sequence of the protein A6) is SEQ ID No.8.

[0010] The present invention also provides the use of the above-mentioned protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in preparing shikimic acid or increasing the yield of shikimic acid.

[0011] In the above application, the regulation is to upregulate, enhance or increase the expression of the gene encoding the aforementioned protein or the content or activity of the protein.

[0012] In the above application, the substance is any one of the following: B1), a nucleic acid molecule encoding the aforementioned protein; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0013] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains any one of the aforementioned proteins.

[0014] In the above, the recombinant bacteria contains a gene encoding any of the aforementioned proteins.

[0015] In the above, the recombinant bacteria include bacteria.

[0016] The present invention also provides the use of a biological material related to any one of the aforementioned proteins in preparing shikimic acid or increasing the yield of shikimic acid, wherein the biological material is any one of the following: B1), a nucleic acid molecule encoding the aforementioned protein; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0017] The present invention also provides a method for preparing shikimic acid or increasing the yield of shikimic acid, comprising using the aforementioned recombinant bacteria for fermentation or the aforementioned biological materials to prepare shikimic acid.

[0018] The present invention is to solve the problems of low conversion rate of 3-dehydroshikimic acid to shikimic acid in existing Escherichia coli shikimic acid engineering strains, reduced shikimic acid crystallization efficiency and poor fermentation liquid quality, and provide a shikimic acid dehydrogenase AroE mutant and a method for increasing the shikimic acid content in a fermentation liquid using the same. By mutating important amino acid sites in the wild-type shikimic acid dehydrogenase AroE of Corynebacterium glutamicum, its substrate binding specificity for 3-dehydroshikimic acid is changed, thereby increasing the reduction reaction of shikimic acid from 3-dehydroshikimic acid and reducing the content of the byproduct 3-dehydroshikimic acid. By overexpressing the shikimic acid dehydrogenase mutant in the host Escherichia coli BL21 (DE3), it can be used to strengthen the catalytic reduction reaction of 3-dehydroshikimic acid to shikimic acid by the shikimic acid dehydrogenase of Corynebacterium glutamicum, and significantly reduce the content of the byproduct 3-dehydroshikimic acid in the shikimic acid fermentation liquid. In general, the mutant of the shikimate dehydrogenase AroE of Corynebacterium glutamicum of the present invention utilizes the mutant to enhance the affinity for the substrate dehydrogenated shikimic acid, reduce the oxidation reaction to shikimic acid, and ultimately increase the shikimic acid yield, reduce the accumulation of by-products, improve the quality of shikimic acid fermentation broth, and greatly save the industrial production cost.

[0019] The project number of this invention is: Discovery of new anti-infection drug targets based on hosts and screening of new veterinary drugs 2022YFD1802104. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The shikimate dehydrogenase protein of Corynebacterium glutamicum was purified by NI column and identified by SDS-PAGE.

[0021] Figure 2 This is the pET28-a(+) plasmid map.

[0022] Figure 3 This is the liquid phase detection spectrum of the fermentation broth.

[0023] Figure 4 This is the liquid phase detection spectrum of AroE.

[0024] Figure 5 AroE R61W Liquid phase detection spectrum.

[0025] Figure 6 AroE A102D Liquid phase detection spectrum.

[0026] Figure 7 AroE L241I Liquid phase detection spectrum.

[0027] Figure 8 AroE R61W、A102D Liquid phase detection spectrum.

[0028] Fig. 9 AroE R61W、L241I Liquid phase detection spectrum.

[0029] Fig.10 AroE R61W、A102D、L241I Liquid phase detection spectrum.

[0030] Deposit certificate Bacteria species: Escherichia coli Latin name: Escherichia coli Strain ID: HYY1458 Collection institution: China Center for Type Culture Collection Abbreviation of depository institution: CCTCC Address: Wuhan University, Wuhan, China Deposit date: December 23, 2022 Registration number of the Collection Center: CCTCC NO: M 20222036. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Example 1: Construction of amino acid screening of shikimate dehydrogenase AroE mutants In order to improve the efficiency of AroE in catalyzing the synthesis of shikimic acid and reduce the reverse synthesis of 3-dehydroshikimic acid, it is feasible to change the binding energy of AroE for shikimic acid and 3-dehydroshikimic acid: that is, to enhance the affinity of AroE for 3-dehydroshikimic acid while reducing the affinity of AroE for shikimic acid. By establishing a docking model of the key enzyme AroE with the product shikimic acid and the substrate 3-dehydroshikimic acid, amino acid residues within 5Å were selected as initial candidate residues. Subsequently, these amino acid residues were mutated to the other three amino acid residues, and then the transition state binding energy of each virtual mutant with the substrate and product was calculated. According to the binding energy algorithm in Discovery studio (DS) 2019 software, the increase in the affinity of the protein-ligand complex is manifested as a negative binding energy value (0, stable mutation), and conversely, the decrease in the affinity of the protein-ligand complex is manifested as a positive binding energy value (>0, destabilizing mutation). The desired target mutant should have an increased or unchanged affinity for 3-dehydroshikimic acid and a decreased affinity for shikimic acid. On this basis, a total of 6 mutant strains were screened, including protein AroE R61W (amino acid sequence is SEQ ID No.3), AroE A102D (amino acid sequence is SEQ ID No.5), AroE L241I (amino acid sequence is SEQ ID No.7), AroE R61W、A102D (amino acid sequence is SEQ ID No.9), AroE R61W、L241I (amino acid sequence is SEQ ID No.11), AroE R61W、A102D、L241I (The amino acid sequence is SEQ ID No. 13). Their binding energies with shikimic acid and 3-dehydroshikimic acid are shown in Table 1 below.

[0034] Table 1 Binding energy of mutant strains with shikimic acid and 3-dehydroshikimic acid

[0035] Example 2: Construction of shikimate dehydrogenase AroE mutant AroE R61W (nucleotide sequence is SEQ ID No.4), AroE A102D (nucleotide sequence is SEQ ID No.6), AroE L241I (nucleotide sequence is SEQ ID No.8), AroE R61W、A102D (nucleotide sequence is SEQ ID No.10), AroE R61W、L241I (nucleotide sequence is SEQ ID No.12), AroE R61W、A102D、L241IThe nucleotide sequence of (nucleotide sequence is SEQ ID No.14) replaced the nucleotides between the BamHI and HindIII restriction enzyme recognition sites of pET28-a(+) to obtain pET28-a(+)-AroE and pET28-a(+)-AroE R61W , pET28-a(+)-AroE A102N , pET28-a(+)-AroE L241I , pET28-a(+)-AroE R61W、A102D , pET28-a(+)-AroE R61W、L241I , pET28-a(+)-AroE R61W、A102D、L241I The mutant plasmid was transformed into Escherichia coli BL21 (ED3) competent cells to obtain the corresponding shikimate dehydrogenase mutant recombinant expression strain.

[0036] pET28-a(+)-AroE is a recombinant expression vector obtained by replacing the pET28-a(+) plasmid with the DNA sequence shown in SEQ ID No.2 and keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.1.

[0037] pET28-a(+)-AroE R61W The recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.4, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.3.

[0038] pET28-a(+)-AroE A102N The recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.6, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.5.

[0039] pET28-a(+)-AroE L241I The recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.8, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.7.

[0040] pET28-a(+)-AroE R61W、A102DThe recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.10, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.9.

[0041] pET28-a(+)-AroE R61W、L241I The recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.12, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.11.

[0042] pET28-a(+)-AroE R61W、A102D、L241I The recombinant expression vector is obtained by replacing the small fragment between BamHI and HindIII of pET28-a(+) with the DNA sequence shown in SEQ ID No.14, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID No.13.

[0043] Example 3, preparation of fermentation broth The Escherichia coli HYY1458 in the following embodiments is recorded in the Chinese patent document with application number "CN202310501621.7" and titled "A temperature-controlled high-yield shikimic acid recombinant Escherichia coli and its application", with strain number YP097158, and has been deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCC NO: M 20222036. It is hereinafter referred to as Escherichia coli HYY1458.

[0044] 1. Primary seed culture of Escherichia coli HYY1458 (1) Inoculate a single clone of Escherichia coli HYY1458 into LB slant medium and culture at 33°C for 24 h; (2) The E. coli HYY1458 slant prepared in step (1) was resuspended with 10 mL of sterile water, and then inoculated into a seed shake flask containing primary seed shake flask culture medium (5L seed shake flask liquid volume is 1L) at an inoculum amount of 2% (volume percentage), and cultured at 30° C. for 16 h to obtain a primary seed solution.

[0045] The first-level seed shaking flask culture medium is composed of: 10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glycerol, and the balance is water, pH 6.8-7.2, high-pressure sterilization at 121° C. for 20 min, cooling to 60° C., and adding ampicillin to make the concentration of ampicillin in the system 50 mg / L.

[0046] 2. Secondary seed liquid culture of Escherichia coli HYY1458 The first-level seed solution prepared by the above method was transplanted into a fermenter containing a second-level seed culture medium at a transplant volume of 5% (volume percentage), and cultured at 30°C for 12 h with a dissolved oxygen concentration of 40%. The OD 610nm It is the secondary seed liquid of 3.

[0047] The secondary seed culture medium is composed of: 15 g / L peptone, 2.5 g / L yeast powder, 0.5 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 1 g / L glycerol, 6 g / L glucose; the rest is water; pH 7.0-7.2, high pressure sterilization at 121°C for 20 min, cooling to 60°C, adding ampicillin, and making the concentration of ampicillin in the system 50 mg / L.

[0048] 3. Shikimic acid production by E. coli HYY1458 fermentation The secondary seed solution prepared by the above method was transplanted into a 50L fermentation tank filled with fermentation medium at a transplant volume of 15% (volume percentage), and cultured at 30℃±2℃ for 24±2h, with a dissolved oxygen concentration of 15%-20%, and an OD 610nm When the temperature is about 80, the temperature is raised to 38℃±2℃ and cultured for 16±2h. The dissolved oxygen concentration is 30%-40%. When the glucose content in the fermentation process is lower than 2g / L, 600g / L of glucose aqueous solution is automatically added to the fermenter to keep the glucose content of the fermentation system between 0.2g / L-2g / L.

[0049] The composition of the fermentation medium is: glucose 20g / L, yeast extract 5g / L, peptone 2g / L, ammonium sulfate 5g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 2g / L, magnesium sulfate 0.02g / L, ferrous sulfate 0.01g / L, calcium chloride 0.015g / L, sodium citrate 0.005g / L, manganese chloride 0.05g / L, niacin 0.1mg / L, cobalt chloride 0.2mg / L, vitamin B1 0.01mg / L, the balance is water, pH 6.8-7.2.

[0050] 4. Detection of Shikimic Acid Content in the Fermentation Broth Detection method of shikimic acid: take an appropriate amount of the fermentation broth in step 3, centrifuge at 12000 rpm for 3 minutes, take the supernatant and dilute it with deionized water to an appropriate multiple, and detect the shikimic acid production by high performance liquid chromatography (HPLC).

[0051] The HPLC determination conditions are as follows: the chromatographic column is a Carbomix H-NP10 column; the mobile phase is a 0.25 mM sulfuric acid solution; the flow rate is 0.5 mL / min; the column temperature is 55°C; the detection wavelength is 214 nm; the injection volume is 5 μL, the peak at 5.5 min is shikimic acid, and the shikimic acid concentration of the supernatant of the fermentation broth of Escherichia coli HYY1458 is 86.98±0.1 g / L.

[0052] Example 4. Activity test of shikimate dehydrogenase AroE mutant The composition of TB liquid culture medium is: peptone 12 g / L, yeast 24 g / L, glycerol 5 g / L, dipotassium hydrogen phosphate trihydrate 16.43 g / L, potassium dihydrogen phosphate 2.31 g / L, and the balance is deionized water.

[0053] (1) Strain culture: The mutant recombinant strain constructed in Example 2 was inoculated into LB medium and cultured at 37°C overnight with shaking. The next day, it was inoculated into TB liquid medium. When the bacterial solution concentration reached 0D 610nm When the pH value was 4-6, IPTG was added at a final concentration of 0.1 mM for induction, and the bacteria were collected after induction culture at 25°C for 24 h.

[0054] (2) Bacterial lysis: Add sterile water to the collected bacteria to resuspend the bacteria and centrifuge at 4°C to remove the supernatant. Repeat twice. Add lysis buffer (100mM PBS buffer, pH 6.5) to fully suspend the bacteria. Disrupt the bacteria at low temperature and ultra-high pressure, and collect the supernatant by centrifugation at 4°C. Use Ni-NTA to purify the mutant protein in the above supernatant. The specific operation is as follows: The supernatant obtained by ultrasonic centrifugation is separated and purified by a pre-treated Ni column. Repeat the column twice, elute with Ni-NTA-50\NTA-100\NTA-200\NTA-500, collect the eluate, and obtain the mutant protein AroE of shikimate dehydrogenase. R61W 、AroE A102D 、AroE L241I and AroE R61W、A102D、L241I The mutant proteins were stained with Coomassie Brilliant Blue.

[0055] (3) Determination of 3-dehydroshikimic acid and shikimic acid content by HPLC Source of shikimic acid standard: Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Website: https: / / www.sigmaaldrich.cn / CN / zh?utm_campaign=SIAL%20Branded%20-%20China&utm_medium=cpc&utm_source=so&utm_content=Sial&utm_term=%E5%A5%A5%E5%BE%B7%E9%87%8C%E5%A5%87%E8%A5%BF%E6%A0%BC%E7%8E%9B), product number S5375.

[0056] 3-Dehydroshikimic acid standard source: Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number 05616.

[0057] The experiment was repeated three times, and the specific operation of each repetition was as follows: ① HPLC detection instrument and method parameters: the instrument was Shimadzu HPLC, and the chromatographic column was chromcore AQ C18 column; the mobile phase was 0.1% phosphoric acid; the flow rate was set to 0.8 mL / min; the detection time was 20 min; the detector was an ultraviolet detector, the detection wavelength was 210 nm, and the column temperature was 25 °C. The elution time of shikimic acid was changed to 5 min, and the elution time of 3-dehydroshikimic acid was 5.7 min. ②Standard curve establishment: Use ultrapure water to prepare 1g / concentration shikimic acid solution, dissolve 10mg of shikimic acid standard in 10ml volumetric flask with 5ml ultrapure water, and then dilute to 10mL with ultrapure water. Use a pipette to take 100uL, 200uL, 400uL, 600uL, 800uL of standard solution, and then dilute to 1000uL with ultrapure water. The sample concentrations are 0.1, 0.2, 0.4, 0.6 and 0.8g / L. At the same time, use ultrapure water to prepare 1g / L concentration 3-dehydroshikimic acid solution, dissolve 10mg of 3-dehydroshikimic acid standard in 10ml volumetric flask with 5ml ultrapure water, and then dilute to 10mL with ultrapure water. Take 100uL, 200uL, 400uL, 600uL, 800uL of standard solution with a pipette, and dilute to 1000uL with ultrapure water, then the sample concentration is 0.1, 0.2, 0.4, 0.6 and 0.8g / L. Detection is performed using a high performance liquid chromatograph (HPLC), and the peak areas corresponding to the peak times and different concentrations of shikimic acid and 3-dehydroshikimic acid are obtained. The concentrations of shikimic acid and 3-dehydroshikimic acid solutions are used as the abscissa, and the peak areas are used as the ordinate to draw a standard curve and obtain a linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used.

[0058] ③ Sample treatment: 1 mL of fermentation broth (prepared in Example 3), 1 mL of NADPH, 0.1 mL of shikimate dehydrogenase (Ni-NAT column eluate) and 7.9 mL of 0.1 M potassium phosphate buffer were used to prepare an enzyme activity assay reaction system (pH 6.5), and the mixture was reacted in a water bath at 32°C for 5 h to obtain a reaction solution. 100 μL of the aforementioned reaction solution was mixed with 100 uL of 2M sulfuric acid solution (the rest was distilled water), centrifuged at 1200 rpm for 5 min, and the supernatant was retained after centrifugation. The aforementioned supernatant was diluted 100 times and then placed on a machine to detect the content of shikimate and 3-dehydroshikimate according to the parameters shown in ① above.

[0059] (4) Data processing GraphPad Prism 8 statistical software was used to process the data. The experimental results were expressed as mean values, and P < 0.01 (**) indicated a very significant difference.

[0060] Mutant AroE R61W、A102D Coomassie brilliant blue staining results Figure 1 Shown (others not shown), Figure 4-10 As shown in Table 2, it can be seen that compared with wild-type bacteria, mutant AroE R61W、A102D The reducibility of shikimate dehydrogenase was greatly improved, indicating that the mutant AroE R61W、A102D The affinity for the substrate dehydroshikimic acid is enhanced.

[0061] Table 2 3-dehydroshikimic acid and shikimic acid content

[0062] Nucleotide sequence of pET28-a(+):

[0063] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protein, characterized in that The protein is any one of the following: A1) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine to tryptophan, and the amino acid at position 102 is mutated from alanine to aspartic acid, while the other amino acid sequences remain unchanged; A2) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine to tryptophan, the amino acid at position 102 is mutated from alanine to aspartic acid, and the amino acid at position 241 is mutated from leucine to isoleucine, while the other amino acid sequences remain unchanged; A3) a protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine to tryptophan, while the other amino acid sequences remain unchanged; A4) a protein in which the amino acid at position 102 of SEQ ID No. 1 is mutated from alanine to aspartic acid, while the other amino acid sequences remain unchanged; A5) A protein in which the amino acid at position 61 of SEQ ID No. 1 is mutated from arginine to tryptophan, and the amino acid at position 241 is mutated from leucine to isoleucine, while the other amino acid sequences remain unchanged; A6) A protein in which the amino acid at position 241 of SEQ ID No. 1 is mutated from leucine to isoleucine, while the other amino acid sequences remain unchanged.

2. The protein according to claim 1, characterized in that The nucleotide sequence of the protein A1) is SEQ ID No.10, the nucleotide sequence of the protein A2) is SEQ ID No.14, the nucleotide sequence of the protein A3) is SEQ ID No.4, the nucleotide sequence of the protein A4) is SEQ ID No.6, the nucleotide sequence of the protein A5) is SEQ ID No.12, and the nucleotide sequence of the protein A6) is SEQ ID No.

8.

3. Use of the protein as claimed in claim 1 or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in preparing shikimic acid or increasing the yield of shikimic acid.

4. The use according to claim 3, characterized in that: The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein in claim 1 or the content or activity of the protein.

5. The use according to claim 3, characterized in that: The substance is any of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

6. A recombinant bacterium, characterized in that: The recombinant bacterium contains the protein according to any one of claim 1.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacterium contains a gene encoding the protein according to any one of claim 1.

8. The recombinant bacterium according to claim 6 or 7, characterized in that The recombinant bacteria include bacteria.

9. Use of a biomaterial related to the protein according to any one of claims 1 in preparing shikimic acid or increasing the yield of shikimic acid, characterized in that: The biological material is any of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

10. A method for preparing shikimic acid or increasing the yield of shikimic acid, comprising fermenting shikimic acid using the recombinant bacteria described in any one of claims 6 to 8 or using the biological material described in claim 9.

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