Arog mutant and use thereof

Abstract

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Description

An aroG mutant and its application Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to an isozyme aroG mutant of 3-deoxy-D-arabinopimelic acid-7-phosphate synthase and applications thereof. Background Art

[0002] In Escherichia coli, 3-deoxy-D-arabinopimelic acid-7-phosphate synthase (DAHP synthase, EC 4.1.2.15) catalyzes the first step in the aromatic amino acid biosynthesis pathway, catalyzing the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form DAHP and phosphate [Ger YM, Chen SL, Chiang HJ, Shiuan DA single Ser-180 mutation desensitizes feedback inhibition of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase in Escherichia coli. J Biochem. 1994; 116(5): 986-990.]. DAHP synthase includes three isozymes, aroG, aroH, and aroF. Among them, the feedback inhibitor of aroG is phenylalanine, the feedback inhibitor of aroH is tryptophan, and the feedback inhibitor of aroF is tyrosine. The three isozymes, aroG, aroH and aroF, account for 80%, 1% and 20% of the total activity of DAHP synthase, respectively [Kikuchi Y, Tsujimoto K, Kurahashi O. Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli. Appl Environ Microbiol. 1997; 63(2): 761-762.].

[0003] As the industrial scale of amino acid fermentation has gradually expanded, the mechanisms that alleviate feedback inhibition by DAHP synthase have been extensively studied. Early work yielded two ideas about the mechanism of feedback inhibition: substitution of Asn8 with Lys8 and deletion of the first seven N-terminal amino acids of aroF resulted in desensitization of Tyr-sensitive DAHP synthase, suggesting that the inhibitor binding site may be located in the N- or C-terminal domain. At the same time, sites near the inhibitor binding pocket, such as Asp146Asn, Pro150Leu, Ser180Phe, and Phe209Ser, can significantly alleviate the feedback inhibition of aroG, indicating that phenylalanine induces changes in the internal conformation of the enzyme by binding to the feedback inhibition site [Xu J, Hu C, Shen S, Wang W, Jiang P, Huang W. Requirement of the N-terminus for dimer formation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate synthase AroG of Escherichia coli. J Basic Microbiol. 2004; 44(5): 400-406.].

[0004] Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a strategy that utilizes protein rational design, screens the amino acid sites around the inhibitor Phe binding pocket in aroG synthetase through computer simulation calculation and inhibitor binding activity pocket analysis, and thus obtains key sites that can relieve feedback inhibition.

[0006] The present invention provides an isozyme aroG mutant of 3-deoxy-D-arabinopimelic acid-7-phosphate synthase, characterized in that aroG derived from Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Yersinia kristensenii, or Cronobacter sakazakii has any of the following mutations: Q151F, Q151F+D146N, Q151F+P150L, ​​Q151F+S180F, Q151F+F209A, Q151F+S211F, or Q151F+S180F+S211.

[0007] Preferably, the amino acid sequence thereof has the above mutations based on SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0008] The present invention provides a gene encoding the isozyme aroG mutant, and preferably the nucleotide sequence of the gene has the above mutation based on SEQ ID NO.1.

[0009] The present invention further provides a recombinant vector containing the encoding nucleic acid, preferably a pTrc99a plasmid.

[0010] The present invention also provides a recombinant genetically engineered bacterium containing the recombinant vector. Preferably, the bacterium is Escherichia coli. In particular, the bacterium is used to produce tryptophan.

[0011] The present invention provides the isozyme aroG mutant, or use of the encoding gene thereof in preparing tryptophan.

[0012] The present invention also provides the use of the isozyme aroG mutant in the preparation of food, medicine, fine chemicals, health products or cosmetics added with tryptophan.

[0013] The isozyme aroG mutant of 3-deoxy-D-arabinopimelic acid-7-phosphate synthase obtained in the present invention can increase the yield of tryptophan gene-edited engineered bacteria, can be used to prepare tryptophan, and further used in the synthesis of food, medicine, fine chemicals, health products or cosmetics, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG1 shows the docking conformation of aroG synthetase and inhibitor Phe in Example 1.

[0015] FIG2 is the agarose gel electrophoresis result of the aroG gene in the PCR amplification system in Example 2.

[0016] FIG3 is an agarose gel electrophoresis image of pTrc99a_aroG constructed in Example 2.

[0017] Figure 4 SDS-PAGE electrophoresis of wild-type and mutant aroG proteins.

[0018] Figure 5 Detection results of the anti-feedback inhibition effect of aroG mutant.

[0019] Figure 6 is a comparison of aroG sequences from five different bacterial genera.

[0020] FIG7 shows the results of the anti-feedback inhibition effect test of aroG multi-site mutants.

[0021] FIG8 is a graph comparing the anti-feedback inhibition effects of wild-type and mutant aroG at different inhibitor concentrations. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0023] 1. Related nucleotide sequence information in the sequence table of the embodiments of the present invention:

[0024] The sequence information of SEQ ID NO.1 is the nucleotide sequence of the DHAP synthase gene aroG of Escherichia coli MG1655.

[0025] The sequence information of SEQ ID NO.2 is the amino acid sequence of the DHAP synthase gene aroG of Escherichia coli MG1655.

[0026] The sequence information of SEQ ID NO.3 is the amino acid sequence of the DHAP synthase gene aroG of Klebsiella aerogenes.

[0027] The sequence information of SEQ ID NO.4 is the amino acid sequence of the DHAP synthase gene aroG of Proteus mirabilis.

[0028] The sequence information of SEQ ID NO.5 is the amino acid sequence of the DHAP synthase gene aroG of Yersinia kristensenii.

[0029] The sequence information of SEQ ID NO.6 is the amino acid sequence of the DHAP synthase gene aroG of Cronobacter sakazakii.

[0030] 2. Materials and methods involved in the embodiments of the present invention:

[0031] (1) Culture medium:

[0032] Solid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L.

[0033] Seed culture medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0034] Fermentation medium: glucose 20 g / L (distributed), yeast powder 3 g / L, (NH4)2SO4 4 g / L, KH2PO4 2.5 g / L, sodium chloride 1 g / L, MgSO4·7H2O 1 g / L, citric acid 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 1.2 mg / L, V H 0.1mg / L, V B1 0.5 mg / L, trace element mixture 1 mL / L, phenol red solution 2%, add water to make up the volume, adjust the pH to 7.0-7.2, and place the prepared culture medium in a 121℃, 0.1 MPa, wet heat sterilization for 20 minutes.

[0035] (2) Shake flask fermentation process

[0036] Shake tube activation culture: Pipette 100 μL of bacterial solution into a shake tube containing 10 mL of LB liquid medium and culture with shaking at 37°C and 220 rpm for about 12 hours.

[0037] Shake flask seed culture: Use a pipette to take 600 μL of bacterial solution and inoculate it into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Incubate at 37°C and 220 rpm for 3-4 h.

[0038] Shake flask fermentation: pipette 600 μL of seed solution into a 500 mL triangular flask containing 30 mL of fermentation medium, and wait for the bacteria to grow to OD 600 When the value was about 0.8, 0.2 mM IPTG inducer was added and the culture was shaken at 37°C and 220 rpm for 24 h.

[0039] Example 1: Mining of aroG anti-feedback inhibition sites

[0040] First, the three-dimensional crystal structure of aroG synthetase (Gene ID: 945605) was obtained from the RCSB Protein Data Bank database (PDB ID: 8e0x), and the three-dimensional structure of the feedback inhibitor phenylalanine (Phe) was obtained from the NCBIPubchem database (CAS: 63-91-2). The structures of the protein receptor aroG and the small molecule ligand Phe were then manipulated, including removing crystal water from the protein structure, removing excess metal ions and other cofactors and coenzymes from the crystal structure, repairing missing amino acids in the peptide chain and missing atoms in the amino acids, removing repetitive structures in the multimeric protein structure, and minimizing the energy of the protein structure and small molecule ligand to achieve their respective most stable states.

[0041] Then, Autodock Vina software was used to perform flexible molecular docking of aroG synthase and Phe. The flexible residues of aroG protein were set to sites near the inhibitor Phe binding pocket, such as Asp146, Pro150, Ser189, Phe209, etc. The size of the docking box was set to The coordinates of the center of the docking box are center_x = 34.706, center_y = 414.253, and center_z = 39.641. The energy range of the docking conformation screening is 0-4, and 20 docking conformations are finally output for analysis and comparison.

[0042] Among the 20 docking conformations output, the one with low binding energy and reasonable docking conformation was selected as the basic conformation for feedback inhibition site screening, focusing on the inhibitor Phe binding pocket. The key to relieving aroG's anti-feedback inhibition lies in relieving the binding of the inhibitor Phe. The docking conformations of aroG and Phe were analyzed. The C-terminal carboxyl group and N-terminal amino group of the inhibitor bind to residues such as Ser180, Asp6, and Asp7 in the active pocket of the aroG protein, transmitting the feedback inhibition signal to the catalytic active center of aroG, causing a change in the enzyme's conformation and a significant decrease in enzyme activity. The amino acid sites Q151, A154, and G178, which have small side chains around the inhibitor binding pocket and are close to the inhibitor's C-terminus, N-terminus, or benzene ring, were selected and mutated into residues with larger side chains or stronger hydrophobicity (such as Phe or Glu). This hinders the binding of the inhibitor and plays a role in anti-feedback inhibition. Finally, through rational design, the aroG anti-feedback mutants A154E, A154F, Q151F, and G178F were obtained.

[0043] Example 2: Construction of wild-type and mutant expression vectors pTrc99a-aroG

[0044] The DAHP synthase gene aroG (Gene ID: 945605) used in this study was derived from Escherichia coli MG1655. Genomic DNA from E. coli was extracted. Based on the published genome and pTrc99a plasmid sequences, primer pairs aroG-F and aroG-R were designed. Using the extracted E. coli genomic DNA as a template, a standard PCR amplification system and procedure was used to amplify the aroG gene. The amplified product was sequence-verified by agarose gel electrophoresis. Figure 2 shows the electrophoresis results.

[0045] After sequence size verification, the PCR system was digested with DPN I DNA methylase at 37°C for 30 minutes to remove the MG1655 template DNA. The aroG amplified product was then purified using a nucleic acid purification kit to remove proteins, salt ions, and small DNA fragments. Primer pairs pTrc99a-F and pTrc99a-R were designed to linearize the pTrc99a plasmid, resulting in homologous fragments of appropriate length between the aroG target gene and the linearized vector pTrc99a. The target gene and vector were ligated using the ClonExpress II One-Step Cloning Kit from Novizan at 37°C for 30 minutes. After successful ligation, the product was transformed into Escherichia coli DH5α and sequence verified using primer pairs pTrc99a_aroG-F and pTrc99a_aroG-R. The agarose gel electrophoresis pattern is shown in Figure 3. After band size verification, the product was sent to a sequencing company for sequencing, which confirmed the correct sequence information for the constructed pTrc99a_aroG.

[0046] The aroG mutant genes A154E, A154F, Q151F, and G178F used in the present invention were amplified by designing site-directed mutagenesis PCR primers (the sequence information of the mutant primers is shown in Table 1), using the pTrc99a_aroG wild-type plasmid as a template and a standard PCR amplification system and procedure to obtain the aroG mutant expression vector.

[0047] The sequences of all primers involved in the construction of wild-type and mutant aroG expression vectors are shown in Table 1 .

[0048] Table 1 Related primer sequences in Example 2

[0049] Example 3: Detection of feedback inhibition performance of aroG mutant enzyme

[0050] (1) Culture medium:

[0051] Solid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L.

[0052] Seed culture medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0053] Fermentation medium: glucose 20 g / L (distributed), yeast powder 3 g / L, (NH4)2SO4 4 g / L, KH2PO4 2.5 g / L, sodium chloride 1 g / L, MgSO4·7H2O 1 g / L, citric acid 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 1.2 mg / L, VH 0.1mg / L, V B1 0.5 mg / L, trace element mixture 1 mL / L, phenol red solution 2%, add water to make up the volume, adjust the pH to 7.0-7.2, and place the prepared culture medium in a 121℃, 0.1 MPa, wet heat sterilization for 20 minutes.

[0054] (2) Shake flask fermentation process

[0055] Shake tube activation culture: Pipette 100 μL of bacterial solution into a shake tube containing 10 mL of LB liquid medium and culture with shaking at 37°C and 220 rpm for about 12 hours.

[0056] Shake flask seed culture: Use a pipette to take 600 μL of bacterial solution and inoculate it into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Incubate at 37°C and 220 rpm for 3-4 h.

[0057] Shake flask fermentation: pipette 600 μL of seed solution into a 500 mL triangular flask containing 30 mL of fermentation medium, and wait for the bacteria to grow to OD 600 When the value was about 0.8, 0.2 mM IPTG inducer was added and the culture was shaken at 37°C and 220 rpm for 24 h.

[0058] (3) Verification of aroG protein induced expression

[0059] After activating the strains carrying the pTrc99a_aroG wild-type and mutant plasmids, transfer them to the primary seed medium at a ratio of 2% for cultivation. After 3-4 hours, transfer them to the fermentation medium at a ratio of 2% for cultivation. After 2-3 hours of cultivation, measure the OD value of the bacterial solution. 600 When the induction value was around 0.6-0.8, 0.2 mM IPTG was added. Samples were taken after 24 hours of induction shake flask fermentation and subjected to SDS-PAGE electrophoresis to verify the induced expression of aroG protein. The electrophoretogram is shown in Figure 4. The expression levels of the wild-type and mutant pTrc99a_aroG were higher than those of the plasmid-free chassis strain.

[0060] (4) Product tryptophan detection method

[0061] HPLC Detection of Tryptophan: Centrifuge 1 mL of fermentation broth at 12,000 rpm for 5 minutes, and collect the supernatant. Sample Preparation: Dilute a 5 g / L L-tryptophan standard to 0.12, 0.25, 0.5, and 0.75 g / L, respectively. Dilute the fermentation broth samples by appropriate multiples to ensure the measured values ​​fall between those of the standards. Chromatographic Separation Conditions: Use a C18 column, 10% v / v acetonitrile as the mobile phase, 30°C column temperature, 0.8 mL / min flow rate, 10 min retention time, and detection at 278 nm.

[0062] The shake flask fermentation results for wild-type and mutant aroG are shown in Figure 5. The Q151F mutant produced a higher yield of the product tryptophan than the wild-type, approximately four times that of the wild-type, demonstrating a certain degree of resistance to feedback inhibition. The amount of product tryptophan produced by the A154F mutant was comparable to that of the wild-type, indicating that the mutation at this position in aroG did not have a positive effect, enhanced resistance to feedback, or significantly altered enzyme activity. The A154E and G178F mutants showed no accumulation of the product tryptophan, indicating that directed evolution at these two sites resulted in the loss of aroG enzyme activity and the inability to produce the product tryptophan.

[0063] Example 4: Screening for feedback inhibition sites by protein sequence structure homology comparison

[0064] The amino acid sequence of aroG from Escherichia coli MG1655 (as shown in SEQ ID NO: 1) and the protein structure were compared with the aroG synthase sequences from Klebsiella aerogenes, Proteus mirabilis, Yersinia kristensenii, and Cronobacter sakazakii (as shown in SEQ ID NO: 2 to 6, respectively), and the sequence homologies were 92%, 85%, 87%, and 89%, respectively. Observing the sequence comparison diagram in Figure 6, it can be found that in the α6 region of aroG from five different bacterial genera, the Q151 site, as a conserved amino acid residue, appears at this site. The Swiss-pdbviewer software was used to perform a virtual mutation on the Q151 site of aroG from five different bacterial genera and change it to an F residue. After minimizing the energy of the mutant structure, the AutoDock Vina software was used to perform molecular docking of the inhibitor phenylalanine and the wild-type and mutant aroG synthases, and the size of the docking box was set to The coordinates of the docking box center are center_x = 34.706, center_y = 414.253, and center_z = 39.641. The docking conformation screening energy range was 0-4, and 20 docking conformations were ultimately output for analysis and comparison. The docking results showed that the affinity of aroG synthase after the Q151F mutation for the inhibitor Phe was lower than that before the mutation. This suggests that the Q151F mutation can confer feedback inhibition resistance in aroG synthases from different bacterial species. Mutations in the α6 region of aroG synthases from different bacterial species are important for enhancing feedback inhibition resistance and enhancing flux in the Trp metabolic pathway.

[0065] Example 5: Detection of anti-feedback inhibition performance of aroG multi-site mutant enzyme

[0066] First, using a laboratory-constructed and preserved strain T29 that can produce high tryptophan as the chassis, gene editing was performed through a two-step homologous recombination method to knock out the aroG gene on the chassis strain.

[0067] The pOH5899 plasmid used in the following examples was constructed and maintained in the laboratory.

[0068] The pKD46 plasmid used in the following examples is a product of miaolingbio (http: / / www.miaolingbio.com / ), with the product number being P0098.

[0069] 5.1 Construction of △aroG knockout cassettes aroG-1 and aroG-2

[0070] (1) Using plasmid pOH5899 as a template, primers aroG-5899-F / aroG-5899-R were used for PCR amplification and verified by agarose gel electrophoresis to obtain a single target band aroG-cat-SacB.

[0071] (2) After the single target band aroG-cat-SacB was purified and recovered, restriction endonuclease DpnⅠ was added to digest the methylated plasmid template and reacted at 37°C for 30 minutes.

[0072] (3) The fragment aroG-cat-SacB after template digestion was purified and sequenced to obtain the first recombinant fragment aroG-1 of the △aroG genotype.

[0073] (4) Using the laboratory-preserved strain T29 as a template, primers aroG-up-F and aroG-up-R were used for amplification, verified by agarose gel electrophoresis, and the template was digested with DpnⅠ and purified and recovered to obtain the aroG-up fragment.

[0074] (5) Using the laboratory-preserved strain T29 as a template, primers aroG-down-F and aroG-down-R were used for amplification, verified by agarose gel electrophoresis, and the template was digested with DpnⅠ and purified and recovered to obtain the aroG-down fragment.

[0075] (6) Using aroG-up and aroG-down as templates, overlapping PCR amplification was performed using primers aroG-up-F and aroG-down-R, and the fragment was verified by agarose gel electrophoresis. The template was digested with DpnⅠ and purified and recovered to obtain the aroG-2 fragment.

[0076] Amplification system: 25 μL of 2×Phanta Max Master Mix (Vazyme), 20 ng of DNA template, 1 μL each of upstream and downstream primers (10 μM), 20 μL of distilled water, and a total volume of 50 μL.

[0077] Amplification conditions were as follows: pre-denaturation at 95°C for 4 minutes (1 cycle); denaturation at 95°C for 15 seconds, annealing at 56°C for 15 seconds, and extension at 72°C for 1 minute (30 cycles); and extension at 72°C for 5 minutes (1 cycle).

[0078] 5.2 Construction of T29△aroG strain

[0079] (1) Prepare the chassis strain T29 into a chemical transformation competent state using conventional preparation methods, referring to the following book: J. Sambrook, DW Russell. Molecular Cloning Laboratory Guide [M]. Science Press, 2002.

[0080] (2) The plasmid pKD46 was transformed into the chassis strain and cultured at 30°C for 10-12 h. The transformants were picked and inoculated into LB medium, and ampicillin and arabinose were added (final concentrations of 50 mg / L and 2 g / L, respectively).

[0081] (3) The inoculated strain was prepared into a competent state for electroporation transformation and ampicillin and arabinose (final concentrations of 50 mg / L and 2 g / L, respectively) were added during the competent state culture.

[0082] (4) The first recombinant fragment aroG-1 was electroporated into competent cells and plated onto chloramphenicol and ampicillin dual-resistance plates. The cells were cultured at 30°C overnight. Transformants were selected and inoculated into LB medium supplemented with ampicillin, chloramphenicol, and arabinose (final concentrations of 50 mg / L, 25 mg / L, and 2 g / L, respectively) to prepare competent cells for electroporation.

[0083] (5) The transformants were prepared into competent cells for electroporation and prepared for use. Ampicillin, chloramphenicol, and arabinose were added during the competent culture (final concentrations were 50 mg / L, 25 mg / L, and 2 g / L, respectively).

[0084] (6) The second recombinant fragment aroG-2 was transformed into the competent cell by electroporation. 500 μL of the incubated bacterial solution was inoculated into a 30 mL Erlenmeyer flask containing sucrose culture medium and cultured at 37°C, 200 rpm, for 18-24 h.

[0085] (7) After incubation, there is a probability that flocculent precipitates will appear in the triangular flask. Take 20uL from the shake flask and streak it onto a plate containing sucrose. Incubate at 37°C overnight. The next day, pick a single colony on the plate and copy it onto an LB plate, a chloramphenicol resistance plate, and an ampicillin resistance plate. Select a single colony that grows on the LB plate but cannot grow on the chloramphenicol resistance plate and the ampicillin resistance plate. Use primers aroG-VF and aroG-VR for colony PCR. The positive strain is the one with an amplification length of 1000bp. The PCR product is sequenced and verified to obtain the T29△aroG strain.

[0086] The colony PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 minutes (1 cycle); denaturation at 95°C for 15 seconds, annealing at 56°C for 15 seconds, and extension at 72°C for 1 minute (30 cycles); and extension at 72°C for 5 minutes (1 cycle).

[0087] 5.3 Detection of the Feedback Resistance of Multi-site Mutation of aroG

[0088] (1) After the first round of screening, the anti-feedback performance of aroG-Q151F was significantly improved compared with the wild type. In order to further improve the anti-feedback performance and catalytic activity of aroG, we screened other mutation sites with potential positive stacking effects in the aroG anti-feedback inhibitor binding pocket, including D146N, P150L, ​​S180F, F209A, and S211F. As shown in Figure 6, these sites are also conserved sites in the aroG amino acid sequences of the five different bacterial species mentioned above.

[0089] (2) Next, the above mutation sites were superimposed on the basis of the constructed plasmid pTrc99a-aroG-Q151F. The method for constructing the multi-site mutation plasmid is as described in Example 2.

[0090] (3) The aroG multi-site mutation plasmid was transformed into the T29△aroG strain to obtain the T29△aroG strain carrying the Q151F+D146N, Q151F+P150L, ​​Q151F+S180F, Q151F+F209A, Q151F+S211F, and Q151F+S180F+S211F plasmids.

[0091] (4) Finally, the difference in anti-feedback inhibition ability and catalytic activity of different aroG multi-site mutant enzymes was verified by detecting the tryptophan content of the product through shake flask fermentation. The materials and operation procedures required for the shake flask fermentation experiment and the method for detecting the tryptophan product are as described in Example 3.

[0092] The shake flask fermentation results are shown in Figure 7. The tryptophan production accumulated by the T29△aroG strain carrying the wild-type and mutant aroG plasmids after 16 hours of shake flask fermentation was significantly higher than that of T29△aroG. This reflects that aroG synthase plays an important role in the tryptophan anabolic process, catalyzing the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form DAHP and phosphate, promoting the metabolic flow to the aromatic amino acid synthesis pathway, and facilitating the accumulation of the product tryptophan.

[0093] Secondly, by comparing the shake flask fermentation results of the wild-type and mutant aroG, it was found that the accumulated tryptophan production by fermentation with the single-site mutation Q151F and the multiple-site mutations Q151F+D146N, Q151F+P150L, ​​Q151F+S180F, Q151F+F209A, Q151F+S211F, and Q151F+S180F+S211F was higher than that of the wild-type aroG synthase, indicating that the changes in the above-mentioned sites have a promoting effect on the improvement of aroG's anti-feedback inhibition ability or the enhancement of its catalytic activity.

[0094] By observing the fermentation results of all aroG multi-site mutant enzymes, it can be found that Q151F+P150L has the highest tryptophan production compared with the Q151F single-site mutation and other multi-site mutations, which indicates that Q151F+P150L has a better effect on enhancing the anti-feedback inhibition ability or catalytic activity of aroG synthase than other mutants.

[0095] To further explore the enhancing effect of the Q151F+P150L site mutation on the anti-feedback inhibition ability of aroG synthase, we added different concentrations of phenylalanine (aroG synthase inhibitor) to the initial shake flask culture medium. The results of 16-h shake flask fermentation are shown in Figure 8.

[0096] First, compared to the wild-type, the Q151F and Q151F+P150L mutant enzymes accumulated higher levels of Trp during 16-hour shake flask fermentation in media containing different inhibitor concentrations. This suggests that the aroG mutant enzyme has stronger catalytic activity, can produce more target compounds in the same chassis strain, and has higher production efficiency. Second, when the concentration of the inhibitor phenylalanine gradually increased, the Q151F and Q151F+P150L enzymes showed a smaller decrease in tryptophan production than the wild-type enzyme, indicating that high concentrations of the inhibitor reduced the inhibitory effect of the aroG mutant enzyme and enhanced its ability to resist feedback inhibition.

[0097] The sequences of all primers included in the construction of T29△aroG strain and aroG multi-site mutant enzyme expression vector are shown in Table 2.

[0098] Table 2 Related primer sequences in Example 5

[0099] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An aroG mutant, an isoenzyme of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, characterized in that, aroG derived from Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Yersinia kristensenii, or Cronobacter sakazakii has any one of the following mutations: Q151F, Q151F+D146N, Q151F+P150L, Q151F+S180F, Q151F+F209A, Q151F+S211F, Q151F+S180F+S211.

2. The aroG mutant isoenzyme according to claim 1, characterized in that, Its amino acid sequence has the above mutations based on SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.

6.

3. A gene encoding the aroG mutant isozyme as described in claim 1 or 2, preferably the nucleotide sequence of said gene has a mutation based on SEQ ID NO.

1.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 3.

5. The recombinant vector according to claim 5, characterized in that, It is the pTrc99a plasmid.

6. A recombinant genetic engineering bacterium, characterized in that, Contains the recombinant vector as described in claim 4.

7. The recombinant genetically engineered bacterium according to claim 6, characterized in that, It is Escherichia coli.

8. The recombinant genetic engineering bacterium according to claim 6, wherein It is used for the production of tryptophan.

9. Use of the aroG mutant isozyme as described in claim 1, or its encoding gene in the preparation of tryptophan.

10. Use of the aroG mutant isozyme as described in claim 1 in the synthesis of foods, drugs, fine chemicals, health products, or cosmetics added with tryptophan.