Shikimic acid dehydrogenase mutant from escherichia coli and method for producing shikimic acid

Abstract

The invention discloses a shikimic acid dehydrogenase mutant from escherichia coli and a method for producing shikimic acid, and belongs to the technical field of biology. The shikimic acid dehydrogenase mutant is obtained by mutating amino acids at sites 1, 12, 27, 40, 141, 153, 156 and 198 of SEQ ID NO: 1 into lysine and mutating amino acids at sites 2, 104, 161, 240 and 244 of SEQ ID NO: 1 into arginine. The shikimic acid dehydrogenase mutant provided by the invention can change the surface potential of wild type shikimic acid dehydrogenase, improve the optimum pH and optimum temperature of the reaction, reduce the oxidation reaction of 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 field of biotechnology, and particularly relates to a shikimate dehydrogenase mutant from Escherichia coli and a method for producing shikimic acid. 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, chemical formula 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 a natural organic compound with important medical and chemical applications. It is best known as a key precursor of the anti-influenza drug Tamiflu (oseltamivir). In the pharmaceutical industry, the extraction, synthesis and application of shikimic acid and its related derivatives have attracted much attention. The traditional star anise extraction and preparation method has no cost advantage. 3-dehydroshikimate dehydrogenase (3-dehydroshikimate dehydrogenase) is one of the key enzymes in the shikimic acid synthesis pathway. It catalyzes the reduction of 3-dehydroshikimate (DHS) to shikimic acid. This reaction is an indispensable step in the shikimic acid pathway and the core link in the biosynthesis of shikimic acid. Shikimic acid dehydrogenase is one of the key enzymes in the shikimic acid biosynthesis 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 accumulates in the engineering bacteria, its miscellaneous acids quinic acid and dehydroshikimic acid also accumulate accordingly, thus greatly reducing the crystallization efficiency, purity and quality of shikimic acid. It can be seen that reducing the content of the 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] The reaction temperature and pH have a great influence on shikimate dehydrogenase when it catalyzes the dehydrogenation of shikimate. At a suitable acidic pH, shikimate dehydrogenase performs a reduction reaction, and at an alkaline pH, shikimate dehydrogenase performs an oxidation reaction. At different temperatures, the enzyme activity also varies greatly.

[0006] In summary, we hope to find a more ideal shikimate dehydrogenase, which has a higher activity in catalyzing the reduction of 3-dehydroshikimic acid to shikimic acid and a lower activity in catalyzing the reverse conversion of shikimic acid to 3-dehydroshikimic acid under certain temperature and pH conditions. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a shikimate dehydrogenase mutant. 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 invention provides a shikimate dehydrogenase mutant. The shikimate dehydrogenase mutant is a protein obtained by mutating the first amino acid of SEQ ID NO:1 from methionine to lysine, the second amino acid from glutamic acid to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamic acid to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

[0010] 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 adding a shikimate dehydrogenase mutant to a reaction solution 1 containing the fermentation product to obtain a reaction solution 2, reacting the reaction solution 2 at 25-42° C. 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 pH of the reaction solution 2 being 6-7; the shikimate dehydrogenase mutant is SEQ ID A protein obtained by mutating the first amino acid of NO:1 from methionine to lysine, the second amino acid from glutamate to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamate to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

[0011] 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.

[0012] The present invention also provides a method for preparing shikimic acid or increasing the yield of shikimic acid, the method comprising the following steps:

[0013] 1) Cultivating Escherichia coli in a microbial liquid culture medium and collecting fermentation broth, wherein the Escherichia coli is HYY1458;

[0014] 2) reacting the shikimate dehydrogenase mutant with the fermentation broth to obtain a reaction product, wherein the shikimate content in the reaction product is higher than that in the fermentation broth, and / or the 3-dehydroshikimate content in the reaction product is lower than that in the fermentation broth; the shikimate dehydrogenase mutant is A protein obtained by mutating the first amino acid of NO:1 from methionine to lysine, the second amino acid from glutamate to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamate to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

[0015] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains the above-mentioned protein.

[0016] The above recombinant bacteria contains the coding gene of the above protein. The nucleotide sequence of the coding gene is SEQ ID NO:4.

[0017] The above-mentioned recombinant bacteria are obtained by introducing a nucleic acid molecule encoding the above-mentioned protein, or introducing an expression cassette containing the nucleic acid molecule, or introducing a recombinant vector containing the nucleic acid molecule into a recipient bacterium.

[0018] The present invention also provides use of the above-mentioned protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in preparing shikimic acid or increasing the yield of shikimic acid. The protein is a protein obtained by mutating the first amino acid of SEQ ID NO:1 from methionine to lysine, the second amino acid from glutamic acid to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamic acid to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

[0019] 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.

[0020] In the above application, the substance is a biological material, and the biological material is any one of the following:

[0021] B1), a nucleic acid molecule encoding the aforementioned protein;

[0022] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] 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).

[0025] In the above application, the expression vector used to prepare the recombinant vector is a PET-28a(+) vector.

[0026] Preferably, the expression vector also contains a gene KanR encoding kanamycin resistance protein with a His tag.

[0027] In the above application, the host cell of the recombinant bacteria is Escherichia coli, preferably BL21 (DE3).

[0028] The present invention provides an application of the recombinant bacteria, wherein the recombinant bacteria is used to convert dehydroshikimic acid into shikimic acid. The recombinant bacteria is frozen overnight at -20°C and broken, and the reaction substrate is shikimic acid fermentation liquid.

[0029] The present invention provides a liquid chromatography detection method for the recombinant bacteria. A 1 g / L concentration shikimic acid solution is prepared and diluted to 0.1, 0.2, 0.4, 0.6 and 0.8 g / L. A 1 g / L concentration 3-dehydroshikimic acid solution is also prepared and diluted to 0.1, 0.2, 0.4, 0.6 and 0.8 g / L. A high performance liquid chromatograph (HPLC) is used for detection to obtain the peak time and the peak areas corresponding to different concentrations of shikimic acid and 3-dehydroshikimic acid. 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 to obtain a linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used. The instrument was a Shimadzu high performance liquid chromatograph, and the chromatographic column used was a chromcore AQ C18 column; the mobile phase was 0.1% phosphoric acid; the flow rate was set at 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.

[0030] Among the above substances, the expression cassette containing nucleic acid molecules described in B3) refers to DNA that can express the above-mentioned proteins in host cells. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules of all regulatory sequences necessary for expressing the nucleic acid molecules of any of the above-mentioned proteins. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that has transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation of the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence attached to the amino terminus of the protein that can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory sequences are systems that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can amplify genes.

[0031] The use of the aforementioned biomaterials in preparing shikimic acid or increasing the yield of shikimic acid also falls within the protection scope of the present invention.

[0032] 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 by using the same. By mutating important amino acid sites in the wild-type shikimic acid dehydrogenase AroE of Escherichia coli, the surface potential of the shikimic acid dehydrogenase is changed, thereby changing the optimal reaction temperature and pH of the shikimic acid dehydrogenase, 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), the reaction of shikimic acid dehydrogenase catalyzing the reduction of 3-dehydroshikimic acid to shikimic acid can be strengthened, so that the content of the byproduct 3-dehydroshikimic acid in the shikimic acid fermentation liquid is significantly reduced, and the production cost of industrialization is greatly saved.

[0033] The project number of this invention is: Discovery of new anti-infection drug targets based on hosts and screening of new veterinary drugs 2022YFD1802104.

[0034] Deposit certificate

[0035] Bacteria species: Escherichia coli

[0036] Latin name: Escherichia coli

[0037] Strain ID: HYY1458

[0038] Collection institution: China Center for Type Culture Collection

[0039] Abbreviation of depository institution: CCTCC

[0040] Address: Wuhan University, Wuhan, China

[0041] Deposit date: December 23, 2022

[0042] Registration number of the Collection Center: CCTCC NO: M 20222036. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the PET28a(+) plasmid map.

[0044] Figure 2 This is the liquid phase detection spectrum of the fermentation broth.

[0045] Figure 3 PET28a(+)-AroE at pH 7 改造 Liquid phase detection spectrum of fermentation broth.

[0046] Figure 4PET28a(+)-AroE at 37°C 改造 Liquid phase detection spectrum of fermentation broth. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference.

[0050] Example 1: Construction of amino acid screening of shikimate dehydrogenase AroE mutants

[0051] By using Rosetta SuperCharge calculation to modify the surface potential of the protease, the surface electrostatic charge of AroE was changed, and AroE was obtained under the condition of increasing the optimal pH and temperature without affecting the efficiency of AroE in catalyzing the synthesis of shikimic acid. 改造 protein (SEQ ID NO: 2).

[0052] AroE 改造 The protein is a protein obtained by mutating the first amino acid of the amino acid sequence of AroE (SEQ ID NO: 1) from M to K, the second amino acid from E to R, the 12th amino acid from A to K, the 27th amino acid from L to K, the 40th amino acid from I to K, the 104th amino acid from V to R, the 141st amino acid from L to K, the 153rd amino acid from S to K, the 156th amino acid from E to K, the 161st amino acid from L to R, the 198th amino acid from A to K, the 240th amino acid from M to R, the 244th amino acid from Q to R, and keeping the other amino acid sequences unchanged.

[0053] Example 2: Construction of shikimate dehydrogenase AroE mutant

[0054] According to the coding sequence (SEQ ID NO: 2) of E. coli ECOLI E.Coil shikimate dehydrogenase AroE (NCBI GenBank ID: P15770.1) published by NCBI and AroE 改造 The coding sequence (SEQ ID NO: 4) was sent to Beijing Qingke Biotechnology Co., Ltd. Fuzhou Branch for gene synthesis, and then the DNA molecules with nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4 were respectively inserted into the nucleotides between the BamHI and XhoI enzyme recognition sites of the pET28a (+) plasmid to obtain pET28-a (+)-AroE and pET28-a (+)-AroE 改造 The mutant plasmid was transformed into Escherichia coli BL21 (ED3) competent cells to obtain the corresponding shikimate dehydrogenase mutant recombinant expression strain.

[0055] pET28-a(+)-AroE is a recombinant expression vector obtained by replacing the small fragment between BamHI and Xho I of pET28-a(+) plasmid with the DNA sequence shown in SEQ ID NO:2, while keeping other nucleotide sequences unchanged. The recombinant expression vector expresses a protein with an amino acid sequence of SEQ ID NO:1.

[0056] pET28a(+)-AroE 改造 The mutant plasmid is a recombinant expression vector 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.

[0057] Example 3, preparation of fermentation broth

[0058] 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.

[0059] 1. Primary seed culture of E. coli HYY008 and E. coli HYY1458

[0060] (1) Inoculate a single clone of Escherichia coli HYY1458 into LB slant medium and culture at 33°C (30°C±3°C in actual application) for 24 h (24 h±2 h in actual application);

[0061] (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 (a 5 L seed shake flask has a liquid volume of 1 L) containing a primary seed shake flask culture medium at an inoculum amount of 2% (volume percentage) (in practical applications, 2% ± 0.5% is acceptable), and cultured at 30° C. (in practical applications, 30° C. ± 3° C. is acceptable) for 16 h (in practical applications, 16 h ± 2 h is acceptable) to obtain a primary seed solution.

[0062] 2. Secondary seed liquid culture of E. coli HYY008 and E. coli HYY1458

[0063] The primary seed solution prepared by the above method is inoculated into a fermentation tank containing secondary seed culture medium at a transplanting amount of 5% (volume percentage) (5%±1% is acceptable in practical applications), and cultured at 30°C (30°C±3°C is acceptable in practical applications) for 12h (12h±2h is acceptable in practical applications), the dissolved oxygen concentration can be 40% (40%-80% is acceptable in practical applications), and a secondary seed solution with an OD610 of approximately 3 (3±0.5 is acceptable in practical applications) is obtained.

[0064] 3. Fermentation of shikimic acid by E. coli HYY008 and E. coli HYY1458

[0065] The secondary seed liquid prepared by the above method is inoculated into a 50L fermentation tank filled with fermentation medium at a transplanting amount of 15% (volume percentage) (15%±2% can be used in actual applications), and cultured at 30℃±2℃ for 24±2h, the dissolved oxygen concentration can be 15%-20%, and when OD610 is about 80, the temperature is raised to 38℃±2℃ and cultured for 16±2h, the dissolved oxygen concentration can be 30%-40%, and when the glucose content during the fermentation process is lower than 2g / L, 600g / L of glucose aqueous solution is automatically added to the fermentation tank to keep the glucose content of the fermentation system between 0.2g / L-2g / L.

[0066] 4. Detection of Shikimic Acid Content in the Fermentation Broth

[0067] 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).

[0068] 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 in the supernatant of the fermentation broth of Escherichia coli HYY1458 is 64±0.5 g / L.

[0069] Example 4: Activity test of oxalate dehydrogenase AroE mutant

[0070] TB liquid culture medium formula: 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.

[0071] (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.

[0072] (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 shikimate dehydrogenase proteins PET28a(+)-AroE and PET28a(+)-AroE 改造 .

[0073] (3) Determination of 3-dehydroshikimic acid and shikimic acid content by HPLC

[0074] Source of shikimic acid standard: Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0075] Source of 3-dehydroshikimic acid standard: Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0076] The experiment was repeated three times, and the specific operations for each repetition were 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 at 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.

[0077] ② Establishment of standard curve: Prepare 1g / concentration shikimic acid solution and dilute it to 0.1, 0.2, 0.4, 0.6 and 0.8g / L. At the same time, prepare 1g / L concentration 3-dehydroshikimic acid solution and dilute it to 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, draw the standard curve with the concentration of shikimic acid and 3-dehydroshikimic acid solution as the horizontal coordinate and the peak area as the vertical coordinate, and obtain the linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used.

[0078] ③ Reaction system 1: 1 mL fermentation broth (prepared in Example 3), 1 mL NADPH, 0.1 mL shikimate dehydrogenase (Ni-NAT column eluate) and 7.9 mL 0.1 M potassium phosphate buffer were used to prepare the enzyme activity assay reaction system. The reaction conditions were: pH 6, 7, 8, 9, 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 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.

[0079] ④ Reaction system 2: 1 mL fermentation broth (prepared in Example 3), 1 mL NADPH, 0.1 mL shikimate dehydrogenase (Ni-NAT column eluate) and 7.9 mL 0.1 M potassium phosphate buffer were used to prepare the enzyme activity assay reaction system. The reaction conditions were: pH 7, temperature: 25°C, 30°C, 32°C, 37°C, 40°C in a water bath for 5 h to obtain a reaction solution. 100 μL of the above reaction solution was mixed with 100 uL 2M sulfuric acid solution (the rest was distilled water), centrifuged at 1200 rpm for 5 min, and the supernatant was retained by 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.

[0080] (4) Data processing

[0081] GraphPad Prism 8 statistical software was used to process the data. The data of reaction system 1 are shown in Table 1, and the data of reaction system 2 are shown in Table 2. The experimental results are expressed as mean values, and P < 0.01 (**) indicates a very significant difference.

[0082] It can be seen that compared with the unreacted supernatant of the fermentation broth of E. coli HYY1458, the content of shikimic acid in reaction system 1 and reaction system 2 increased significantly, and the content of 3-dehydroshikimic acid decreased significantly. 改造The removal effect of 3-dehydroshikimic acid in the supernatant of the fermentation broth of Escherichia coli HYY1458 is much better than that of protein AroE.

[0083] Table 1. Catalytic results of shikimate dehydrogenase at different pH

[0084]

[0085] Table 2. Catalytic results of shikimate dehydrogenase at different temperatures

[0086]

[0087] SEQ ID NO:1

[0088] METYAVFGNPIAHSKSPFIHQQFAQQLNIEHPYGRVLAPINDFINTLNAFFSAGGKGANVTVPFKEEAFARADELTERAALAGAVNTLMRLEDGRLLGDNTDGVGLLSDLERLSFIRPGLRILLIGAGGASRGVLL PLLSLDCAVTITNRTVSRAEELAKLFAHTGSIQALSMDELEGHEFDLIINATSSGISGDIPAIPSSLIHPGIYCYDMFYQKGKTPFLAWCEQRGSKRNADGLGMLVAQAAHAFLLWHGVLPDVEPVIKQLQEELSA.

[0089] SEQ ID NO:2

[0090] 5'-ATGGAAACCTATGCGGTGTTCGGTAATCCAATCGCACACTCTAAATCTCCGTTCATCCACCAGCAGTTCGCTC AGCAGCTGAACATCGAACATCCGTATGGTCGTGTACTGGCGCCAATCAACGACTTCATCAACACTCTGAACGCTTTCTTCTCTGCGGGTGGTAAAGGTGCTAACGTGACCGTTCCGTTCAAAGAAGAAGCGTTCGCTCGTGCGGATGAACTGACCGAACGTGCTGCTCTGGCAGGTGCTGTTAACACTCTGATGCGTCTGGAAGATGGTCGTTTGCTGGGTGACAACACCGATGGTGTAGGTCTGCTGTCTGACCTGGAACGTCTGTCTTTCATCCGTCCGGGTTTGCGTATCTTGCTGATCGGTGCAGGTGGTGCATCTCGTGGTGTTCTTCTGCCGCTGTTGTCTCTGGATTGCGCTGTTACCATCACCAACCGCACCGTGTCTCGTGCTGAAGAACTGGCGAAACTGTTCGCGCACACCGGTTCCATCCAGGCTCTGTCTATGGACGAACTGGAAGGTCACGAGTTCGACCTGATCATCAACGCGACCTCTTCTGGTATCTCTGGTGACATCCCGGCAATTCCGTCTAGCCTGATCCACCCAGGTATCTACTGCTACGACATGTTCTACCAGAAAGGTAAGACTCCATTCTTGGCATGGTGCGAACAGCGTGGTTCTAAACGTAACGCGGACGGCCTGGGTATGCTGGTTGCACAGGCAGCTCACGCGTTTCTGCTGTGGCACGGTGTTCTGCCGGACGTTGAACCGGTTATCAAACAGCTGCAAGAAGAACTGTCTGCA-3'。

[0091] SEQ ID NO:3

[0092] KRTYAVFGNPIKHSKSPFIHQQFAQQKNIEHPYGRVLAPKNDFINTLNAFFSAGGKGANVTVPFKEEAFARADELTERAALAGAVNTLMRLEDGRLLGDNTDGRGLLSDLERLSFIRPGLRILLIGAGGASRGVLLPLLSKDCAVTITNRTVKRAKELAKRFAHTGSIQALSMDELEGHEFDLIINATSSGISGDIPKIPSSLIHPGIYCYDMFYQKGKTPFLAWCEQRGSKRNADGLGRLVARAAHAFLLWHGVLPDVEPVIKQLQEELS。

[0093] SEQ ID NO:4

[0094] 5'-AAACGTACCTACGCAGTGTTCGGTAATCCGATCAAACACTCCAAGAGTCCGTTCATCCACCAGCAGTTCGCAC AGCAGAAGAACATCGAACACCCATACGGTCGTGTACTGGCACCGAAGAACGACTTCATCAACACTCTGAACGCATTCTTCTCTGCAGGTGGTAAAGGTGCTAACGTTACCGTTCCGTTCAAAGAAGAAGCATTCGCTCGTGCAGACGAACTGACCGAACGTGCTGCACTGGCAGGTGCTGTTAACACCTTGATGCGTCTGGAAGATGGTCGTCTGCTGGGTGACAACACTGACGGTCGTGGTTTGCTGTCTGACCTGGAACGTCTGTCTTTCATCCGTCCAGGTCTGCGTATTCTGCTGATCGGTGCTGGTGGTGCTTCTCGTGGTGTGCTTCTGCCGCTGCTGTCCAAAGACTGCGCTGTTACCATCACCAACCGTACCGTTAAACGTGCGAAAGAACTGGCTAAACGTTTCGCTCATACCGGTTCTATCCAGGCACTGTCTATGGACGAACTGGAAGGTCACGAGTTCGACCTGATCATCAACGCTACCAGCTCTGGTATCTCTGGTGACATCCCGAAGATCCCATCTTCTCTGATCCATCCGGGTATCTACTGCTACGACATGTTCTACCAGAAAGGTAAGACTCCGTTTCTGGCTTGGTGCGAACAGCGTGGTTCTAAACGTAACGCGGATGGTCTGGGTCGTCTGGTTGCTCGTGCTGCACACGCTTTCTTGCTGTGGCACGGTGTACTGCCAGACGTTGAACCGGTTATCAAACAGCTGCAAGAAGAACTGTCT-3'。

[0095] 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 shikimate dehydrogenase mutant, characterized in that: The shikimate dehydrogenase mutant is a protein obtained by mutating the first amino acid of SEQ ID NO:1 from methionine to lysine, the second amino acid from glutamic acid to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamic acid to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

2. 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 adding a shikimate dehydrogenase mutant to a reaction solution 1 containing the fermentation product to obtain a reaction solution 2, reacting the reaction solution 2 at 25-42° C. to increase the content of 3-dehydroshikimic acid and / or the content of shikimic acid in the fermentation product, wherein the pH of the reaction solution 2 is 6-7; the shikimate dehydrogenase mutant is SEQ ID A protein obtained by mutating the first amino acid of NO:1 from methionine to lysine, the second amino acid from glutamate to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamate to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

3. 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 shikimate dehydrogenase mutant with the fermentation broth to obtain a reaction product, wherein the shikimate content in the reaction product is higher than that in the fermentation broth, and / or the 3-dehydroshikimate content in the reaction product is lower than that in the fermentation broth; the shikimate dehydrogenase mutant is A protein obtained by mutating the first amino acid of NO:1 from methionine to lysine, the second amino acid from glutamate to arginine, the 12th amino acid from alanine to lysine, the 27th amino acid from leucine to lysine, the 40th amino acid from isoleucine to lysine, the 104th amino acid from valine to arginine, the 141st amino acid from leucine to lysine, the 153rd amino acid from serine to lysine, the 156th amino acid from glutamate to lysine, the 161st amino acid from leucine to arginine, the 198th amino acid from alanine to lysine, the 240th amino acid from methionine to arginine, the 244th amino acid from glutamine to arginine, and keeping other amino acid sequences unchanged.

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

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

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria are obtained by introducing a nucleic acid molecule encoding the protein of claim 1, or an expression cassette containing the nucleic acid molecule, or a recombinant vector containing the nucleic acid molecule into a recipient bacterium.

7. Use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in preparing shikimic acid or increasing the yield of shikimic acid, wherein the protein is a protein obtained by mutating the amino acid at position 1 of SEQ ID NO:1 from methionine to lysine, the amino acid at position 2 from glutamic acid to arginine, the amino acid at position 12 from alanine to lysine, the amino acid at position 27 from leucine to lysine, the amino acid at position 40 from isoleucine to lysine, the amino acid at position 104 from valine to arginine, the amino acid at position 141 from leucine to lysine, the amino acid at position 153 from serine to lysine, the amino acid at position 156 from glutamic acid to lysine, the amino acid at position 161 from leucine to arginine, the amino acid at position 198 from alanine to lysine, the amino acid at position 240 from methionine to arginine, the amino acid at position 244 from glutamine to arginine, and keeping other amino acid sequences unchanged.

8. The use according to claim 7, characterized in that: The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein in claim 7 or the content or activity of the protein.

9. The use according to claim 8, 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 7; 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. Use of the biomaterial according to claim 9 in preparing shikimic acid or increasing the yield of shikimic acid.

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