Method for preparing shikimic acid or increasing yield of shikimic acid

Abstract

The invention discloses a method for preparing shikimic acid or increasing the yield of shikimic acid, and belongs to the technical field of biology. The method comprises the following steps: reacting protein with a fermentation product to increase the content of 3-dehydroshikimic acid in the fermentation product and / or increase the content of shikimic acid in the fermentation product; the protein is obtained by mutating the 19th amino acid of SEQ ID No.1 from glutamic acid to serine and mutating the 102nd amino acid of SEQ ID No.1 from threonine to serine and keeping other amino acid residues of SEQ ID No.1 unchanged. The mutant can change the substrate binding specificity to 3-dehydroshikimic acid, so that the affinity to the substrate dehydroshikimic acid is enhanced, the oxidation reaction to the shikimic acid is reduced, the reduction reaction for generating the shikimic acid from the 3-dehydroshikimic acid is increased, and finally the yield of the shikimic acid is increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing shikimic acid or increasing the yield of shikimic acid. Background Art

[0002] Shikimic acid is a single-configuration chiral compound extracted from the natural raw material star anise. It is the raw material for the synthesis of many alkaloids, indole derivatives, aromatic amino acids and chiral drugs, and is widely used in the food and chemical industries. Although shikimic acid has been a research object for a long time, it was only after the global spread of the avian influenza epidemic that Roche produced the shikimic acid derivative "Tamiflu", and shikimic acid really became a hot spot for research and development. The avian influenza virus poses a serious threat to human health, and Tamiflu is currently the only effective specific drug for treating avian influenza. As an important raw material for the anti-avian influenza drug Tamiflu, shikimic acid has become the object of development and research by many scholars. With the continuous and in-depth exploration of shikimic acid, the application of shikimic acid is not only limited to chemical raw materials, but can also be used to transform and produce other chemical substances and preparations, and its application is becoming more and more extensive.

[0003] In addition to being widely used as a spice, shikimic acid has pharmacological functions such as anti-thrombosis, thereby inhibiting arterial and venous thrombosis and cerebral thrombosis. It can also play a good anti-inflammatory, antiviral and anti-tumor role. In addition, shikimic acid also has a good analgesic effect and is an important intermediate in modern chemical and pharmaceutical synthesis.

[0004] Compared with plant extraction and chemical synthesis of shikimic acid, the use of microbial fermentation synthesis to produce shikimic acid has become a new trend. An obvious problem with existing microbial fermentation strains is that the fermentation broth contains impurities such as 3-dehydroshikimic acid, which affects the quality of the fermentation broth and the extraction process. Therefore, how to improve the yield and purity of shikimic acid to promote its research and application in chemical, pharmaceutical and other related fields has become the key to the production of shikimic acid by microbial fermentation.

[0005] In the biological metabolism process, shikimic acid metabolism only exists in plants, fungi and microorganisms, and is a critical metabolic pathway. Shikimic acid dehydrogenase catalyzes the fourth step of the metabolic pathway, using reduced coenzyme II (NADPH) to catalyze 3-dehydroshikimic acid (DHS) to produce shikimic acid. During the catalytic process, the enzyme is also subject to feedback inhibition by shikimic acid in the fermentation broth, which increases the accumulation of 3-dehydroshikimic acid, affects the quality of the fermentation broth and increases the extraction cost.

[0006] In summary, we hope to find a new shikimate dehydrogenase, which has a high activity in catalyzing the reduction of 3-dehydroshikimate to shikimate under certain conditions, so that it has a high 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 a new shikimate dehydrogenase. 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:

[0009] The present invention also provides a method for reducing the content of 3-dehydroshikimic acid in a fermentation product containing 3-dehydroshikimic acid and / or increasing the content of shikimic acid in the fermentation product, the method comprising reacting a protein with the fermentation product to increase the content of 3-dehydroshikimic acid in the fermentation product and / or increasing the content of shikimic acid in the fermentation product; the protein is a protein obtained by mutating the 19th amino acid of SEQ ID No.1 from glutamic acid to serine and the 102nd amino acid from threonine to serine, while keeping other amino acid residues of SEQ ID No.1 unchanged.

[0010] In the above method, the fermentation product is a fermentation broth obtained by culturing Escherichia coli in a microbial culture medium, the fermentation broth contains 3-dehydroshikimic acid, and the Escherichia coli is specifically HYY1458.

[0011] The present invention also provides a method for preparing shikimic acid or increasing the yield of shikimic acid, the method comprising the following steps:

[0012] 1) Cultivating Escherichia coli in a microbial liquid culture medium and collecting fermentation broth, wherein the Escherichia coli is HYY1458;

[0013] 2) reacting the protein with the fermentation broth to obtain a reaction product, wherein the content of shikimic acid in the reaction product is higher than that in the fermentation broth, and / or the content of 3-dehydroshikimic acid in the reaction product is lower than that in the fermentation broth; the protein is a mutant protein obtained by mutating the amino acid at position 19 of SEQ ID No.1 from glutamic acid to serine and the amino acid at position 102 from threonine to serine, while keeping other amino acid residues of SEQ ID No.1 unchanged.

[0014] In the above method, the reaction is carried out at 32° C. for 5 hours. The fermentation broth also contains shikimic acid.

[0015] The present invention provides a method for preparing shikimic acid or increasing the yield of shikimic acid, comprising preparing shikimic acid or increasing the yield of shikimic acid by using a protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein; the protein is a protein in which the 19th amino acid of SEQ ID No.1 is mutated from glutamic acid to serine, the 102nd amino acid is mutated from threonine to serine, and the other amino acid sequences remain unchanged.

[0016] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains the above-mentioned protein.

[0017] The above-mentioned recombinant bacteria contain the coding gene of the above-mentioned protein. The nucleotide sequence of the coding gene is SEQ ID No.4.

[0018] The aforementioned proteins and biological materials also fall within the protection scope of the present invention.

[0019] The present invention also provides an application, namely, the application of the aforementioned 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.

[0020] 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.

[0021] In the above application, the substance is a biological material, and the biological material is any one of the following:

[0022] B1), a nucleic acid molecule encoding the aforementioned protein;

[0023] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0024] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0025] 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).

[0026] In the above application, the nucleotide sequence of the protein is SEQ ID No.4.

[0027] In the above application, the expression vector uses PET-28a(+) as a vector and also contains a gene KanR encoding a kanamycin resistance protein with a His tag.

[0028] In the above application, the microorganism is Escherichia coli BL21 (ED3) competent cells.

[0029] The present invention also provides an application, namely, the application of the aforementioned biological material in preparing shikimic acid or increasing the yield of shikimic acid.

[0030] The advantage of the present invention is that the mutant of shikimate dehydrogenase AroE provided by the present invention changes its substrate binding specificity for 3-dehydroshikimic acid by mutating the amino acid site therein, thereby enhancing the affinity for the substrate dehydroshikimic acid, reducing the oxidation reaction of shikimic acid, and improving the reduction reaction of shikimic acid generated from 3-dehydroshikimic acid, thereby ultimately increasing the shikimic acid yield.

[0031] The project number of this invention is: Discovery of new anti-infection drug targets based on hosts and screening of new veterinary drugs 2022YFD1802104.

[0032] Deposit certificate

[0033] Bacteria species: Escherichia coli

[0034] Latin name: Escherichia coli

[0035] Strain ID: HYY1458

[0036] Collection institution: China Center for Type Culture Collection

[0037] Abbreviation of depository institution: CCTCC

[0038] Address: Wuhan University, Wuhan, China

[0039] Date of deposit: December 23, 2022

[0040] Registration number of the Collection Center: CCTCC NO: M 20222036. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The shikimate dehydrogenase protein of Amphritea atlantica was purified by NI column and identified by SDS-PAGE.

[0042] Figure 2 This is the PET-28a+ plasmid map.

[0043] Figure 3 This is the liquid phase detection spectrum of shikimic acid in the fermentation broth at 210nm.

[0044] Figure 4 This is the liquid phase detection spectrum of dehydroshikimic acid in the fermentation broth at 254nm.

[0045] Figure 5 This is the spectrum of shikimic acid detected by AroE liquid phase at 210nm.

[0046] Figure 6 This is the spectrum of dehydroshikimic acid detected by AroE liquid phase at 254nm.

[0047] Figure 7 AroE at 210nm E19S / T22S Liquid chromatography detection of shikimic acid spectrum.

[0048] Figure 8 AroE at 254nm E19S / T22S Liquid chromatography detection of shikimic acid spectrum. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] Example 1. Construction of shikimate dehydrogenase AroE mutant

[0052] The Amphritea atlantica shikimate dehydrogenase AroE (NCBI GenBank ID: UTW04290.1) (nucleotide sequence is SEQ ID No. 2) and AroE E19S / T22S The nucleotide sequence (nucleotide sequence is SEQ ID No. 4) replaced the nucleotides between the BamHI and XhoI restriction enzyme recognition sites of pET28-a(+) to obtain pET28-a(+)-AroE and pET28-a(+)-AroE E19S / T22S The mutant plasmid was transformed into Escherichia coli BL21 (ED3) competent cells to obtain the corresponding shikimate dehydrogenase mutant recombinant expression strain.

[0053] 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.

[0054] pET28-a(+)-AroE E19S / T22SThe recombinant expression vector is obtained by replacing the small fragment between BamHI and XhoI 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.

[0055] Example 2: Activity test of shikimate dehydrogenase AroE mutant

[0056] 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.

[0057] (1) Strain culture: The mutant recombinant strain constructed in Example 2 was inoculated into LB medium and cultured at 37°C with shaking overnight. 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.

[0058] (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 ultrahigh 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 pretreated 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. E19S / T22S The mutant proteins were stained with Coomassie Brilliant Blue.

[0059] (3) Determination of 3-dehydroshikimic acid and shikimic acid content by HPLC

[0060] 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.

[0061] Source of 3-dehydroshikimic acid standard: Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number 05616.

[0062] The experiment was repeated three times, and the specific operation of each repetition was as follows:

[0063] ① HPLC detection instrument and method parameters: The instrument is Shimadzu HPLC, the chromatographic column is chromcore AQ C18 column; the mobile phase is 0.1% phosphoric acid; the flow rate is set at 0.8 mL / min; the detection time is 20 min; the detector is an ultraviolet detector, the detection wavelength is 210 nm, and the column temperature is 25°C. The elution time of shikimic acid is 5 min, and the elution time of 3-dehydroshikimic acid is 5.7 min.

[0064] 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 the standard solution with a pipette, and then dilute to 1000uL with ultrapure water, then the sample concentration is 0.1, 0.2, 0.4, 0.6 and 0.8g / L. Use high performance liquid chromatography (HPLC) to detect, obtain the peak time and the peak area corresponding to different concentrations of shikimic acid and 3-dehydroshikimic acid, and use the concentration of shikimic acid and 3-dehydroshikimic acid solution as the horizontal coordinate and the peak area as the vertical coordinate to draw a standard curve to obtain a linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used.

[0065] 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 reaction was carried out in a water bath at 32° C. for 5 h to obtain a reaction solution. 100 μL of the above 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. After the above supernatant was diluted 100 times, it was put on the machine to detect the content of shikimate and 3-dehydroshikimate according to the parameters shown in ① above.

[0066] (4) Data processing

[0067] 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.

[0068] Mutant AroE E19S / T22S Coomassie brilliant blue staining results Figure 1 The results of HPLC determination of 3-dehydroshikimic acid and shikimic acid content are shown in Table 1. Figure 5-Figure 8 As shown. It can be seen that compared with the wild-type mutant, AroE E19S / T22S The reduction of shikimate dehydrogenase was improved, indicating that the mutant AroE E19S / T22S The affinity for the substrate dehydroshikimic acid is enhanced.

[0069] Table 13 - Dehydroshikimic acid and shikimic acid content

[0070] Mutants Shikimic acid content (g / L) 3-Dehydroshikimic acid(g / L) Significance analysis Before reaction 87.812 13.0 After AroE reaction 91.849 10.026 / <![CDATA[AroE E19S / T22S After reaction]]> 93.324 9.055 P＜0.0002

[0071] Note: Significance refers to the use of AroE E19S / T22S There was a significant difference in the content of shikimic acid in the fermentation broth after treating the fermentation broth and after treating the fermentation broth with AroE.

[0072] Example 3, preparation of fermentation broth

[0073] 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.

[0074] 1. Primary seed culture of Escherichia coli HYY1458

[0075] (1) Inoculate a single clone of Escherichia coli HYY1458 into LB slant medium and culture at 33°C for 24 h;

[0076] (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.

[0077] 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.

[0078] 2. Secondary seed liquid culture of Escherichia coli HYY1458

[0079] 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.

[0080] 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.

[0081] 3. Shikimic acid production by E. coli HYY1458 fermentation

[0082] 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.

[0083] 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.

[0084] 4. Detection of Shikimic Acid Content in the Fermentation Broth

[0085] 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).

[0086] The HPLC determination conditions are as follows: the instrument is Shimadzu high performance liquid chromatograph, the chromatographic column is chromcore AQC18 column; the mobile phase is 0.1% phosphoric acid; the flow rate is set at 0.8mL / min; the detection time is 20min; the detector is an ultraviolet detector, the detection wavelength is 210nm, and the column temperature is 25°C. The shikimic acid peak time is 5min, the 3-dehydroshikimic acid peak time is 5.7min, and the shikimic acid concentration of the fermentation supernatant of Escherichia coli HYY1458 is 87.812g / L ( Figure 3 ) and 13g / L( Figure 4 ).

[0087] SEQ ID No.1

[0088] MNKNFLSNLTGSFAMPAMENPTVAMVEAAYQHHQLDWRYINCEVSPELLKDAVRGARAMGWAGFNCSIPHKVAVIEHLDGLGQSAMIIGAVNTVVRNARGEYIGENTDGRGFVTALNEVTDPQGAQVILFGAGGAARAVAVEM AFAGAAHITVVNRSRERGEKLTRLICKKTPATATFAIWDGDFRLPADTDIVINATSIGLYPNIDQQLDINWETLSSDMVVADGIHNPPMTPLLRTAQQKGCKCVNGQQMLVHQGAIGIHYWTGIEVDTRVMYQALREIFNPE.

[0089] SEQ ID No.2

[0090] 5'-GGATCCATGAACAAGAACTTTCTGTCCAACCTGACTGGTAGCTTCGCAATGCCAGCGATGGAGAATCCAACTG TAGCGATGGTTGAAGCGGCTTACCAGCACCATCAGCTGGACTGGCGTTACATCAACTGCGAAGTTAGTCCAGAACTGCTGAAAGACGCAGTTCGTGGTGCGCGTGCAATGGGTTGGGCAGGTTTCAACTGCTCTATCCCGCACAAAGTTGCGGTTATCGA ACACCTGGACGGTCTGGGTCAGTCTGCAATGATCATCGGTGCAGTTAACACCGTTGTTCGTAACGCTCGTGGTGAATACATCGGTGAGAACACCGACGGTCGTGGTTTCGTTACCGCACTGAACGAAGTTACCGATCCACAGGGTGCGCAGGTTATTCTGTTCGGTGCTGGTGGTGCAGCTCGTGCTGTAGCAGTGGAAATGGCGTTCGCTGGTGCAGCGCACATCACCGTTGTTAACCGTTCTCGTGAACGTGGTGAGAAACTGACTCGTCTGATCTGCAAGAAAACTCCAGCAACCGCAACCTTCGCGATCTGGGACGGTGATTTCCGTCTGCCAGCAGACACCGACATCGTTATCAACGCGACCTCTATCGGTCTGTATCCGAACATCGACCAGCAGCTGGACATCAACTGGGAAACTCTCTCTTCTGACATGGTTGTTGCAGACGGTATCCACAATCCGCCGATGACTCCGCTGTTGCGTACCGCTCAGCAGAAAGGTTGCAAATGCGTTAACGGTCAGCAGATGCTGGTTCACCAGGGTGCAATCGGTATCCATTACTGGACTGGTATCGAAGTTGACACTCGTGTTATGTACCAGGCACTGCGTGAACTCGAG-3'。

[0091] SEQ ID No.3(AroE E19S / T22S )

[0092] MNKNFLSNLTGSFAMPAMSNPSVAMVEAAYQHHQLDWRYINCEVSPELLKDAVRGARAMGWAGFNCSIPHKVAVIEHLDGLGQSAMIIGAVNTVVRNARGEYIGENTDGRGFVTALNEVTDPQGAQVILFGAGGAARAVAVEMAFAGAAHITVVNRSRERGEKLTRLICKKTPATATFAIWDGDFRLPADTDIVINATSIGLYPNIDQQLDINWETLSSDMVVADGIHNPPMTPLLRTAQQKGCKCVNGQQMLVHQGAIGIHYWTGIEVDTRVMYQALREIFNPE。

[0093] SEQ ID No.4(AroE E19S / T22S )

[0094] 5'-GGATCCATGAACAAGAACTTTCTGTCCAACCTGACTGGTAGCTTCGCAATGCCAGCGATGAGCAATCCAAGCG TAGCGATGGTTGAAGCGGCTTACCAGCACCATCAGCTGGACTGGCGTTACATCAACTGCGAAGTTAGTCCAGAACTGCTGAAAGACGCAGTTCGTGGTGCGCGTGCAATGGGTTGGGCAGGTTTCAACTGCTCTATCCCGCACAAAGTTGCGGTTATCGAACACCTGGACGGTCTGGGTCAGTCTGCAATGATCATCGGTGCAGTTAACACCGTTGTTCGTAACGCTCGTGGTGAATACATCGGTGAGAACACCGACGGTCGTGGTTTCGTTACCGCACTGAACGAAGTTACCGATCCACAGGGTGCGCAGGTTATTCTGTTCGGTGCTGGTGGTGCAGCTCGTGCTGTAGCAGTGGAAATGGCGTTCGCTGGTGCAGCGCACATCACCGTTGTTAACCGTTCTCGTGAACGTGGTGAGAAACTGACTCGTCTGATCTGCAAGAAAACTCCAGCAACCGCAACCTTCGCGATCTGGGACGGTGATTTCCGTCTGCCAGCAGACACCGACATCGTTATCAACGCGACCTCTATCGGTCTGTATCCGAACATCGACCAGCAGCTGGACATCAACTGGGAAACTCTCTCTTCTGACATGGTTGTTGCAGACGGTATCCACAATCCGCCGATGACTCCGCTGTTGCGTACCGCTCAGCAGAAAGGTTGCAAATGCGTTAACGGTCAGCAGATGCTGGTTCACCAGGGTGCAATCGGTATCCATTACTGGACTGGTATCGAAGTTGACACTCGTGTTATGTACCAGGCACTGCGTGAACTCGAG-3'.

[0095] Nucleotide sequence of pET28-a(+):

[0096]

[0097] 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 method for reducing the content of 3-dehydroshikimic acid in a fermentation product containing 3-dehydroshikimic acid and / or increasing the content of shikimic acid in the fermentation product, characterized in that: The method comprises reacting a protein with the fermentation product to increase the content of 3-dehydroshikimic acid in the fermentation product and / or to increase the content of shikimic acid in the fermentation product; the protein is a protein obtained by mutating the 19th amino acid of SEQ ID No.1 from glutamic acid to serine and the 102nd amino acid from threonine to serine, while keeping other amino acid residues of SEQ ID No.1 unchanged.

2. A method for preparing shikimic acid or increasing the yield of shikimic acid, characterized in that: The method comprises the following steps: 1) Cultivating Escherichia coli in a microbial liquid culture medium and collecting fermentation broth, wherein the Escherichia coli is HYY1458; 2) reacting the protein with the fermentation broth to obtain a reaction product, wherein the content of shikimic acid in the reaction product is higher than that in the fermentation broth, and / or the content of 3-dehydroshikimic acid in the reaction product is lower than that in the fermentation broth; the protein is a mutant protein obtained by mutating the amino acid at position 19 of SEQ ID No.1 from glutamic acid to serine and the amino acid at position 102 from threonine to serine, while keeping other amino acid residues of SEQ ID No.1 unchanged.

3. A recombinant bacterium, characterized in that: The recombinant bacteria contains the protein according to claim 1.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria contain the coding gene of the above-mentioned protein.

5. 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.

6. The use according to claim 5, 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.

7. The use according to claim 6, characterized in that: The substance is a biological material, and the biological material is any one 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).

8. The use according to claim 7, characterized in that: The nucleotide sequence of the protein is SEQ ID No.

4.

9. The protein of claim 1 or the biomaterial of claim 7.

10. Use of the biomaterial according to claim 7 in preparing shikimic acid or increasing the yield of shikimic acid.

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