L-histidine production strain and construction method and application thereof

Abstract

The application provides an L-histidine production strain and a construction method and application thereof. The strain takes a wild-type Corynebacterium glutamicum as a starting strain, overexpresses NCgl1505 genes 、 NCgl2093 genes , NCgl2096 genes , NCgl2094 genes 、 NCgl2097 genes , NCgl2100 genes , NCgl0218 genes 、 NCgl2101 genes , NCgl0799 genes, NCgl2102 genes and NCgl1504 Y56M，T235P genes and efflux proteins NCgl1262 genes; heterologously introduced pntAB genes; knocked out NCgl0909 genes, NCgl2632 genes and NCgl0580 genes; overexpressed NCgl1610-1609-1608 operons; the synthesis efficiency and production intensity of the strain are significantly improved through an ATP optimization strategy.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to an L-histidine producing strain, its construction method, and its application. Background Technology

[0002] L-histidine ( L-Histidine L-histidine is a basic amino acid containing an imidazole ring, with wide applications in medicine, food, feed, and cosmetics. Statistics show that the global market demand for L-histidine is steadily increasing, creating an urgent need for efficient, low-cost, and sustainable production technologies.

[0003] Currently, the main problems faced in metabolic engineering include: L-histidine affects key enzymes. NCgl1504 Feedback inhibition prevents L-histidine from being produced efficiently; insufficient supply of ATP and NADPH also limits L-histidine production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an L-histidine producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-histidine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-histidine producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An L-histidine-producing strain, named HIS-13, is derived from wild-type Corynebacterium glutamicum. Corynebacterium glutamicum The overexpression strain was obtained through gene editing using metabolic engineering techniques. NCgl1505 Gene 、NCgl2093 Gene 、NCgl2096 Gene 、NCgl2094 Gene 、NCgl2097 Gene 、NCgl2100 Gene 、NCgl0218 Gene 、NCgl2101 Gene 、NCgl0799 Genes and NCgl2102 Genes, after overexpression mutation NCgl1504 Y56M，T235P Genes; Heterologous introduction of E. coli Escherichia coli Source pntAB Genes; overexpression of efflux proteins NCgl1262 Gene; knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes; overexpression NCgl1610-1609-1608 Manipulator.

[0008] Preferably, the above-mentioned L-histidine producing strain is found in wild-type Corynebacterium glutamicum. Corynebacterium glutamicum Using P on the genome tuf Startup driver NCgl1505 Gene 、NCgl2093 Gene 、NCgl2096 Gene 、 NCgl2094 Gene 、NCgl2097 Gene 、NCgl2100 Gene 、NCgl0218 Gene 、NCgl2101 Gene 、NCgl0799 Genes and NCgl2102 Gene overexpression, using P tuf After promoter-driven mutation NCgl1504 Y56M，T235P Gene overexpression; heterologous introduction of E. coli-derived genes pntAB Genes and P tuf Promoter-driven overexpression; using P sod Promoter-driven efflux proteins NCgl1262 Gene overexpression; knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes; using P tuf Startup driver NCgl1610-1609-1608 Operator overexpression.

[0009] Preferably, the above-mentioned L-histidine producing strain, wherein NCgl2102 Gene, NCgl2101 Genes and NCgl2100 Genes NCgl2102-2101-2100 Formal overexpression of natural operators; NCgl2097 Genes and NCgl2096 Genes NCgl2097-2096 Formal overexpression of natural operators; NCgl2094 Genes and NCgl2093 Genes NCgl2094-2093 The form of natural operator overexpression.

[0010] Preferably, the above-mentioned L-histidine producing strain, the wild-type Corynebacterium glutamicum... Corynebacterium glutamicum Wild-type Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032.

[0011] Preferably, the above-mentioned L-histidine producing strain, wherein NCgl0909 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing; NCgl2632 The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; NCgl0580 The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing.

[0012] Preferably, the above-mentioned L-histidine producing strain, wherein NCgl1610-1609-1608 The nucleotide sequence of the operon is shown in SEQ ID NO.4 of the sequence listing; NCgl1610 The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing; NCgl1609 The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing; NCgl1608 The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing.

[0013] Preferably, the above-mentioned L-histidine producing strain, wherein NCgl2102-2101-2100 The nucleotide sequence of the natural operon is shown in SEQ ID NO. 8 of the sequence listing; NCgl2102 The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing; NCgl2101 The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing; NCgl2100 The nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing; NCgl2097-2096 The nucleotide sequence of the natural operon is shown in SEQ ID NO.12 of the sequence listing; NCgl2097 The nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing; NCgl2096 The nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing; NCgl2094-2093 The nucleotide sequence of the natural operon is shown in SEQ ID NO. 15 of the sequence listing; NCgl2094 The nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing; NCgl2093 The nucleotide sequence of the gene is shown in SEQ ID NO.17 of the sequence listing; NCgl0218 The nucleotide sequence of the gene is shown in SEQ ID NO. 18 of the sequence listing; NCgl0799 The nucleotide sequence of the gene is shown in SEQ ID NO. 19 of the sequence listing; NCgl1505 The nucleotide sequence of the gene is shown in SEQ ID NO.20 of the sequence listing; NCgl1504 Y56M，T235P The nucleotide sequence of the gene is shown in SEQ ID NO. 21 of the sequence listing; *Escherichia coli* Escherichia coli Source pntAB The nucleotide sequence of the gene is shown in SEQ ID NO. 22 of the sequence listing; *Escherichia coli* Escherichia coli Source pntA The nucleotide sequence of the gene is shown in SEQ ID NO. 23 of the sequence listing; *Escherichia coli* Escherichia coli Source pntB The nucleotide sequence of the gene is shown in SEQ ID NO. 24 of the sequence listing; efflux protein NCgl1262 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.25.

[0014] Preferably, the above-mentioned L-histidine producing strain, wherein P tuf The nucleotide sequence of the promoter is shown in SEQ ID NO. 37 of the sequence listing, P sod The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.26.

[0015] The above method for constructing L-histidine-producing strains involves using the wild-type Corynebacterium glutamicum as the starting strain. Corynebacterium glutamicum Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) Overexpression on the genome NCgl1505 Gene 、NCgl2093 Gene 、NCgl2096 Gene 、NCgl2094 Gene 、 NCgl2097 Gene 、NCgl2100 Gene 、NCgl0218 Gene 、NCgl2101 Gene 、NCgl0799 Gene 、NCgl2102 Genes, and the result of overexpression of endogenous mutations NCgl1504 Y56M，T235P Gene; (2) Heterogeneous introduction of Escherichia coli pntAB Gene; (3) Overexpression of efflux proteins NCgl1262 Gene; (4) Knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes, and overexpression NCgl1610-1609- 1608 Manipulator.

[0016] Preferably, the method for constructing the above-mentioned L-histidine-producing strain includes the following specific steps: 1) In NCgl3068 Sites using P tuf Starter driver N Cgl2102-2101-2100 Operator overexpression; 2) In NCgl1781 Sites using P tuf Startup driver NCgl2097-2096 Operator overexpression; 3) In NCgl1775 Sites using P tuf Startup driver NCgl2094-2093 Operator overexpression; 4) In NCgl1790 Sites using Ptuf Startup driver NCgl0218 Gene overexpression; 5) In NCgl1774 Sites using P tuf Startup driver NCgl0799 Gene overexpression; 6) In NCgl1773 Sites using P tuf Startup driver NCgl1505 Gene overexpression; 7) In NCgl1817 Sites using P tuf Startup driver NCgl1504 Y56M，T235P Gene overexpression; 8) In NCgl2998 Sites using P tuf Promoter-driven E. coli-derived pntAB Overexpression; 9) In NCgl2949 Sites using P sod Startup driver NCgl1262 Gene overexpression; 10) Knockout NCgl0909 Gene; 11) Knockout NCgl2632 Gene; 12) Knockout NCgl0580 Gene; 13) In NCgl2923 Sites using P tuf Startup driver NCgl1610-1609-1608 Operator overexpression.

[0017] The above NCgl3068 The nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing; NCgl1781 The nucleotide sequence is shown in SEQ ID NO.28 of the sequence listing; NCgl1775 The nucleotide sequence is shown in SEQ ID NO.29 of the sequence listing; NCgl1790 The nucleotide sequence is shown in SEQ ID NO.30 of the sequence listing; NCgl1773 The nucleotide sequence is shown in SEQ ID NO.31 of the sequence listing; NCgl1774 The nucleotide sequence is shown in SEQ ID NO.32 of the sequence listing; NCgl1817 The nucleotide sequence is shown in SEQ ID NO.33 of the sequence listing; NCgl2998 The nucleotide sequence is shown in SEQ ID NO.34 of the sequence listing; NCgl2949 The nucleotide sequence is shown in SEQ ID NO.35 of the sequence listing; NCgl2923 The nucleotide sequence is shown in the sequence listing SEQ ID NO.36.

[0018] Application of the above-mentioned L-histidine producing strains in the production of L-histidine.

[0019] Preferably, the above-mentioned application uses a fermentation method to produce L-histidine.

[0020] Preferably, in the above application, fermentation is carried out using a fermenter: the seed liquid is inoculated into the fermentation medium at a 20% inoculation rate, and the pH is maintained at 7.0 by adding ammonia water during fermentation.

[0021] Preferably, the above application follows these steps: (1) Slant seed culture: The strain was inoculated from a 20% glycerol preservation tube at -80℃ into a solid slant for activation culture. The culture conditions were 32℃ for 12h, and two generations were activated. The bacteria on the second generation solid slant were used as the starting bacteria for the seed tank. (2) Seed culture in fermenter: Second-generation solid slant cells were washed off with sterile physiological saline and all of them were inoculated into a 5 L fermenter for culture. The culture medium was brought to a final volume of 2 L. The culture conditions were 32℃, pH 7.0, dissolved oxygen 30-50%, and cultured until OD reached 2000. 600nm Reaching 15; (3) Fermentation culture in fermenter: Inoculate the seed liquid into the fermentation medium at a rate of 20%, and make the medium volume up to 2L. The culture conditions are 32℃, dissolved oxygen 30-50%, and the pH is maintained at around 7.0 by adding ammonia water during the fermentation process.

[0022] Preferably, the slant culture medium used in the above application is BHI medium.

[0023] Preferably, in the above application, the seed culture medium used is: MgSO4·7H2O 1g / L, yeast powder 4g / L, peptone 2g / L, K2HPO4·3H2O 4g / L, glutamic acid 1.3g / L, FeSO4·7H2O 8mg / L, MnSO4 6mg / L, VB1, VB3, and VB5 2mg / L each, and ammonium sulfate 8g / L.

[0024] Preferably, in the above application, the fermentation medium used is: MgSO4·7H2O 2g / L, yeast powder 4g / L, peptone 3g / L, K2HPO4·3H2O 6g / L, glutamic acid 1.3g / L, FeSO4·7H2O 30mg / L, MnSO4 10mg / L, VB1, VB3, and VB5 2mg / L each, and ammonium sulfate 8g / L.

[0025] All of the above-mentioned culture media can be prepared using standard methods.

[0026] Beneficial effects: The above-mentioned L-histidine-producing strains were constructed using an ATP optimization strategy, including knockout.NCgl0909 Gene, NCgl2632 Gene , NCgl0580 From the perspective of energy metabolism, knocking out genes that consume non-essential ATP reduces ATP loss, allowing more ATP to be used effectively for product synthesis; especially NCgl0580 After knockout, the transcriptional levels of several key genes in the glycolysis pathway were significantly upregulated, which is beneficial for ATP production; after overexpression of the mutant... NCgl1504 Y56M，T235P Genes, by altering the regulatory domains of enzymes, prevent the binding of L-histidine, thus changing the protein conformation and increasing histidine production; overexpression NCgl1610-1609-1608 The operon increases the level of oxidative phosphorylation, promoting ATP production; by using a specific promoter to overexpress a specific gene, combined with an ATP optimization strategy, the resulting strains show a significant improvement in synthesis efficiency and production intensity. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the L-histidine metabolic pathway and its modification. In the diagram, GUO: 5-(5-phosphate-D-ribocarbamoyl)-1-(5-phosphateribosyl)-imidazol-4-carboxamide; 2ER: 5-[(5-phosphate-1-deoxyribone-1-amino)methylacetamide]-1-(5-phosphateribosyl)imidazol-4-carboxamide. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0029] Unless otherwise specified, the percentage sign "%" in the examples refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of solution.

[0030] All technical means used in this invention are methods known to those skilled in the art. "Knockout" in this invention refers to the inactivation of a target gene through gene editing, while "introduction" refers to the insertion of a foreign gene into the genome of an engineered bacterium after linking it to a promoter through gene editing.

[0031] The starting strain used in the examples was Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032 (commercially available strain). The corresponding promoter and genes are listed in the sequence listing. Primers used in the construction of the strains are listed in Table 1, plasmids are listed in Table 2, and the high-fidelity enzyme PCR system in the examples is listed in Table 3.

[0032] The gene editing method used in the gene manipulation in this embodiment refers to the literature (An update of the suicideplasmid-mediated genome editing system in Corynebacterium glutamicum. microbial biotechnology). pK18mobsacB and pXT01 involved in this invention are both disclosed in that article, where pXT01 is pXTuf in the article.

[0033] Table 1 Primers used in strain construction

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] Table 2. Plasmids involved in the strain construction process.

[0042] Table 3 Overlap PCR Amplification System

[0043] Example 1 like Figure 1 As shown, the construction steps of an L-histidine-producing strain are as follows: Step 1: In NCgl3068 Sites using P tuf Starter driver N Cgl2102-2101-2100 Operator overexpression; Step 2: In NCgl1781 Sites using P tuf Startup driver NCgl2097-2096 Operator overexpression; Step 3: In NCgl1775 Sites using P tuf Startup driver NCgl2094-2093 Operator overexpression; Step 4: In NCgl1790 Sites using Ptuf Startup driver NCgl0218 Gene overexpression; Step 5: In NCgl1774 Sites using P tuf Startup driver NCgl0799 Gene overexpression; Step 6: In NCgl1773 Sites using P tuf Startup driver NCgl1505 Gene overexpression; Step 7: In NCgl1817 Sites using P tuf Startup driver NCgl1504 Y56M，T235P Gene overexpression; Step 8: In NCgl2998 Sites using P tuf Promoter-driven E. coli-derived pntAB Overexpression; Step 9: In NCgl2949 Sites using P sod Startup driver NCgl1262 Gene overexpression; Step 10: Knockout NCgl0909 Gene; Step 11: Knockout NCgl2632 Gene; Step 12: Knockout NCgl0580 Gene; Step 13: In NCgl2923 Sites using P tuf Startup driver NCgl1610-1609-1608 Operator overexpression; where, exogenous genes pntAB Derived from Escherichia coli Escherichia coli W3110.

[0044] The preparation method of the linear carrier is as follows: First, the carrier is placed in the linear carrier... Figure 1 pK18 shown mobsacB The DH5α strain containing the plasmid was cultured in shake tubes for 12 hours at 37°C. The plasmid was then extracted and recovered using a plasmid extraction kit; detailed operating procedures were described in the instructions for the Plasmid Mini KitⅠ(200) manufactured by OMEGA (Guangzhou) Co., Ltd. A double digestion method was then used, with the digestion system incubated at 32°C for 30 minutes. The digestion system consisted of 2000 ng of recombinant plasmid, 1 μL each of the two restriction endonucleases, 5 μL of 10×Q.cut Buffer, and ddH2O to a final volume of 50 μL. Finally, using the linearized product from the double digestion as a template, PCR was used to amplify the linearized vector required for homologous recombination.

[0045] Preparation method of recombinant DNA fragments: Recombinant DNA fragments generally need to contain upstream and downstream homologous arms and the target gene fragment to be integrated (gene knockout does not require the target gene fragment). The upstream and downstream homologous arms of the cleavage site are obtained by PCR amplification using the genome of *Corynebacterium glutamicum* as a template. The target gene is obtained by PCR amplification using the genome of the strain to which the gene belongs. Finally, the recombinant DNA fragment containing the upstream and downstream homologous arms of the cleavage site and the target gene fragment to be integrated is obtained through overlap PCR.

[0046] Method for recombinantly ligating DNA fragments with linearized vectors: Recombinases from the ClonExpress® II One Step Cloning Kit series are used to recombinantly ligate DNA fragments with linearized pK18. mobsacB Vector. The reaction system consisted of 4 μL of 5×CE II Buffer, 50 ng of linearized cloning vector, 20 ng of DNA fragment, 2 μL of Exnase® II, and ddH2O to a final volume of 20 μL. It was incubated in a 32°C water bath for 30 minutes. The recombinant pK18 vector carrying the DNA fragment was then... mobsacB plasmidization to E. coli DH5α Competent cells. Positive transformants were screened and cultured, and then plasmids were extracted and recovered using a plasmid extraction kit.

[0047] Ⅰ Knockout NCgl0909 Using genes as an example, this aims to illustrate the steps involved in gene knockout.

[0048] The specific steps are as follows: I-1. Using the genome of Corynebacterium glutamicum 13032 as a template, and with primers NCgl0909-S1 and NCgl0909-A2 as one group and primers NCgl0909-S3 and NCgl0909-A4 as another group, knockout cells were obtained by PCR amplification. base gene NCgl0909 The upstream and downstream homologous arms were purified and extracted using a DNA gel purification and recovery kit.

[0049] Ⅰ-2. Using the upstream and downstream homologous arms obtained in step 1, and with NCgl0909-S1 and NCgl0909-A4 as primers, overlap extension PCR amplification was performed, followed by purification and extraction using a DNA gel purification and recovery kit to obtain the knockout DNA. NCgl0909 The recombined fragment.

[0050] Ⅰ-3. Extract pK18 mobsacB The plasmid was prepared by double digestion with XbaⅠ and EcoRI to form a linear vector, which was then recovered.

[0051] Ⅰ-4. Compare the obtained overlapping segments with the linearized pK18mobsacB Transfected cells were transferred into DH5α competent cells, plated on 0.05 g / L kanamycin-resistant plates, and positive transformants were selected and pK18 was extracted. mobsacB -△ NCgl0909 Plasmid.

[0052] Ⅰ-5. The constructed pK18 mobsacB -△ NCgl0909 The plasmid was transformed into Corynebacterium glutamicum by electroporation. Corynebacterium glutamicum HIS-9 electrocompetent cells were plated on plates containing 0.01 g / L kanamycin and incubated at 32°C for 24 hours. Colony PCR was performed, and PCR fragments were examined by agarose gel electrophoresis. Colonies growing on kanamycin-resistant plates were single colonies that had undergone single crossover.

[0053] Ⅰ-6. Transfer the successfully integrated single colonies to BHI medium and incubate at 32℃. At 2h, 4h, and 6h, transfer 50μL of fermentation broth onto BHI plates containing 20% ​​sucrose and incubate at 32℃ for 24h. Spot the single colonies onto BHI plates containing 20% ​​sucrose and 0.01g / L kanamycin-resistant plates. Select single colonies that grow on the 20% sucrose BHI plate but not on the kanamycin-resistant plate for colony PCR verification. Agarose gel electrophoresis confirms the correct PCR fragment, indicating a double crossover. The successfully integrated single colony is identified as strain HIS-10. The gene knockout process is now complete.

[0054] Knockout NCgl2632 Genes and Knockout NCgl058 The 0 gene follows the same operating method as described above, except that the primers used are NCgl2632-S1, NCgl2632-A2, NCgl2632-S3, NCgl2632-A4 and NCgl0580-S1, NCgl0580-A2, NCgl0580-S3, NCgl0580-A4.

[0055] II. NCgl2923 ::P tufNCgl1610-1609-1608 This example aims to illustrate the steps involved in gene integration.

[0056] The specific steps are as follows: II-1. Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, primers NCgl1610-08-S1 and NCgl1610-08-A2 were used as one group, primers NCgl1610-08-S5 and NCgl1610-08-A6 as another group, and primers NCgl1610-08-S7 and NCgl1610-08-A8 as yet another group. P was obtained through PCR amplification. tufNCgl1610-1609-1608The upstream homologous arm, target gene, and downstream homologous arm were identified; using pXT01 plasmid as a template and primers NCgl1610-08-S3 and NCgl1610-08-A4 as a group, P was obtained. tuf The promoter fragment was purified and extracted using a DNA gel purification and recovery kit.

[0057] II-2, P obtained in step 1 tufNCgl1610-1609-1608 The upstream homologous arm, promoter, target gene, and downstream homologous arm were identified. Using NCgl1610-08-S1 and NCgl1610-08-A8 as primers, overlap extension PCR amplification was performed, followed by purification and extraction using a DNA gel purification and recovery kit to obtain the integrated P. tufNCgl1610-1609-1608 The recombined fragment.

[0058] II-3, Extract pK18 mobsacB The plasmid was prepared by double digestion with XbaⅠ and EcoRI to form a linear vector, which was then recovered.

[0059] II-4. Compare the obtained overlapping segments with the linearized pK18. mobsacB Transfected cells were transferred into DH5α competent cells, plated on 0.05 g / L kanamycin-resistant plates, and positive transformants were selected and pK18 was extracted. mobsacB- NCgl2923 ::P tufNCgl1610-1609-1608 Plasmid.

[0060] II-5. The constructed pK18 mobsacB - NCgl2923 ::P tufNCgl1610-1609-1608 The plasmid was transformed into HIS-12 electrocompetent cells by electroporation and plated on plates containing 0.01 g / L kanamycin, then incubated at 32°C for 24 hours. Colony PCR was performed, and the PCR fragments were examined by agarose gel electrophoresis. Colonies growing on kanamycin-resistant plates were single colonies that had undergone single crossover.

[0061] II-6. Successfully integrated single colonies were transferred to BHI medium and incubated at 32°C. 50 μL of fermentation broth was transferred at 2 h, 4 h, and 6 h and plated onto BHI plates containing 20% ​​sucrose, and incubated at 32°C for 24 h. Single colonies were then spotted onto BHI plates containing 20% ​​sucrose and 0.01 g / L kanamycin-resistant plates. Colonies that grew on the 20% sucrose BHI plates but not on the kanamycin-resistant plates were selected for colony PCR verification. Agarose gel electrophoresis confirmed the correct PCR fragments, indicating double crossover. The successfully integrated single colony was identified as strain HIS-13. The gene integration process was thus completed.

[0062] exist NCgl3068 At the site, integration of P tufNCgl2102-2101-2100 The primers used were: NCgl2100-02-S1, NCgl2100-02-A2, NCgl2100-02-S3, NCgl2100-02-A4, NCgl2100-02-S5, NCgl2100-02-A6, NCgl2100-02-S7, and NCgl2100-02-A8; similarly, in NCgl1781 At the site, integration of P tufNCgl2097- 2096 The primers used were: NCgl2096-97-S1, NCgl2096-97-A2, NCgl2096-97-S3, NCgl2096-97-A4, NCgl2096-97-S5, NCgl2096-97-A6, NCgl2096-97-S7, and NCgl2096-97-A8; NCgl1775 At the site, integration of P tufNCgl2094-2093 The primers used were: NCgl2093-94-S1, NCgl2093-94-A2, NCgl2093-94-S3, NCgl2093-94-A4, NCgl2093-94-S5, NCgl2093-94-A6, NCgl2093-94-S7, and NCgl2093-94-A8; NCgl1790 At the site, integration of P tufNCgl0218 The primers used were: NCgl0218-S1, NCgl0218-A2, NCgl0218-S3, NCgl0218-A4, NCgl0218-S5, NCgl0218-A6, NCgl0218-S7, and NCgl0218-A8; NCgl1773 At the site, integration of P tufNCgl1505 The primers used were: NCgl1505-S1, NCgl1505-A2, NCgl1505-S3, NCgl1505-A4, NCgl1505-S5, NCgl1505-A6, NCgl1505-S7, and NCgl1505-A8; NCgl1774 At the site, integration of P tufNCgl0799 The primers used were: NCgl0799-S1, NCgl0799-A2, NCgl0799-S3, NCgl0799-A4, NCgl0799-S5, NCgl0799-A6, NCgl0799-S7, and NCgl0799-A8; NCgl1817 At the site, integration of P tufNCgl1504 Y56M，T235P The primer used was NCgl1504. -S1 NCgl1504 -A2NCgl1504 -S3 NCgl1504 A4 NCgl1504 -S5 NCgl1504 -A6 NCgl1504 -S7 NCgl1504 -A8 NCgl1504 -S9 NCgl1504 -A10 NCgl1504 -S11 NCgl1504 -A12 ;exist NCgl2949 At the site, integration of P sodNCgl1262 The primers used were: NCgl1262-S1, NCgl1262-A2, NCgl1262-S3, NCgl1262-A4, NCgl1262-S5, NCgl1262-A6, NCgl1262-S7, and NCgl1262-A8. 。

[0063] III. Based on integration of Escherichia coli sources pntAB Using genes as an example, this study aims to illustrate gene editing strategies that introduce exogenous genes.

[0064] The specific steps are as follows: III-1. Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, primers EcpntAB-S1 and EcpntAB-A2 were used as one group, and primers EcpntAB-S7 and EcpntAB-A8 were used as another group. P was obtained by over-PCR amplification. tufpntAB The upstream and downstream homologous arms were obtained; using pXT01 plasmid as a template, primers EcpntAB-S3 and EcpntAB-A4 were used as a group, and P was obtained by PCR amplification. tufEcpntAB The promoter; using the E. coli W3110 genome as a template, and with primers EcpntAB-S5 and EcpntAB-A6 as a group, respectively, P was obtained by PCR amplification. tufEcpntAB The target gene fragment.

[0065] Ⅲ-2, P obtained in step 1 tufEcpntAB The upstream homologous arm, promoter, target gene, and downstream homologous arm were identified. Using EcpntAB-S1 and EcpntAB-A8 as primers, overlap extension PCR amplification was performed, followed by purification and extraction using a DNA gel purification and recovery kit to obtain the integrated P. tufEcpntAB The recombined fragment.

[0066] Ⅲ-3, Extract pK18 mobsacB The plasmid was prepared by double digestion with XbaⅠ and EcoRI to form a linear vector, which was then recovered.

[0067] III-4. Compare the obtained overlapping segments with the linearized pK18. mobsacB Transfected cells were transferred into DH5α competent cells, plated on 0.05 g / L kanamycin-resistant plates, and positive transformants were selected and pK18 was extracted. mobsacB - NCgl2998:: P tufEcpntAB Plasmid.

[0068] Ⅲ-5. The constructed pK18 mobsacB - NCgl2998:: P tufEcpntAB The plasmid was transformed into HIS-7 electrocompetent cells by electroporation and plated on plates containing 0.01 g / L kanamycin, then incubated at 32°C for 24 hours. Colony PCR was performed, and the PCR fragments were examined by agarose gel electrophoresis. Colonies growing on kanamycin-resistant plates were single colonies that had undergone single crossover.

[0069] III-6. Successfully integrated single colonies were transferred to BHI medium and incubated at 32°C. 50 μL of fermentation broth was transferred at 2 h, 4 h, and 6 h and plated onto BHI plates containing 20% ​​sucrose, incubated at 32°C for 24 h. Single colonies were then spotted onto BHI plates containing 20% ​​sucrose and 0.01 g / L kanamycin-resistant plates. Colonies that grew on the 20% sucrose BHI plate but not on the kanamycin-resistant plate were selected for colony PCR verification. Agarose gel electrophoresis confirmed the correct PCR fragments, indicating a double crossover. The successfully integrated single colony was identified as strain HIS-8. This completes the gene integration process for introducing the exogenous gene.

[0070] IV. Construct HIS-13. The construction process is as follows: From wild-type Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032 after expression PtufNCgl2102-2101-2100 The operon yields strain HIS-1, and the integration follows the procedure described in step II above; HIS-1 is expressed... PtufNCgl2097-2096 The operon yielded strain HIS-2, and the integration was performed using the method described in step II above; HIS-2 was expressed... PtufNCgl2094-2093 The operon yielded strain HIS-3, and the integration was performed using the method described in step II above; HIS-3 was expressed... PtufNCgl0218 The gene was obtained from strain HIS-4, and the integration was performed using the method described in step II above; HIS-4 was expressed... PtufNCgl0799 The gene was obtained from strain HIS-5, and the integration was performed using the method described in step II above; HIS-5 was expressed... PtufNCgl1505 The strain HIS-6 was obtained, and the integration was performed using the method described in step II above; HIS-6 was expressed... PtufNCgl1504 Y56M，T235P The strain HIS-7 was obtained, and the integration was performed using the method described in step II above; HIS-7 was expressed... PtufEcpntAB The strain HIS-8 was obtained, and the introduction of the exogenous gene followed the operation method described in step III above; HIS-8 was expressed... PsodNCgl1262 The strain HIS-9 was obtained, and the integration was performed using the method described in step II above; HIS-9 was then knocked out. NCgl0909 The strain HIS-10 was obtained, and the knockout process was performed using the method described in step I above; HIS-10 was knocked out. NCgl2632 The strain HIS-11 was obtained, and the knockout process was performed using the method described in step I above; HIS-11 was knocked out. NCgl0580 The strain HIS-12 was obtained, and the knockout procedure was performed using the method described in step I above; HIS-12 was expressed... PtufNCgl1610-1609-1608 The operon yields strain HIS-13, and the integration is performed using the method described in step II above.

[0071] Example 2 Using strains HIS-6 to HIS-13 from Example 1 as production strains, a fermenter validation experiment was conducted, as detailed below: (1) Slant seed culture: The strain was inoculated from a 20% glycerol preservation tube at -80℃ into a solid slant for activation culture. The culture conditions were 32℃ for 12h, and two generations were activated. The bacteria on the second generation solid slant were used as the starting bacteria for the seed tank. (2) Seed culture in fermenter: Second-generation solid slant cells were washed off with sterile physiological saline and all of them were inoculated into a 5 L fermenter for culture. The culture medium was brought to a final volume of 2 L. The culture conditions were 32℃, pH 7.0, dissolved oxygen 30-50%, and cultured until OD reached 2000. 600 nm Reaching 15; (3) Fermentation culture in a fermenter: The inoculum amount is 20% of the seed tank, the culture medium is kept to a volume of 2 L, the culture conditions are 32℃, dissolved oxygen 30-50%, and the pH is maintained at around 7.0 by adding ammonia water during the fermentation process.

[0072] The slant culture medium used was BHI medium.

[0073] The seed culture medium used was: MgSO4·7H2O 1g / L, yeast extract 4g / L, peptone 2g / L, K2HPO4·3H2O 4g / L, glutamic acid 1.3g / L, FeSO4·7H2O 8mg / L, MnSO4 6mg / L, VB1, VB3, and VB5 2mg / L each, and ammonium sulfate 8g / L.

[0074] The fermentation medium used was: MgSO4·7H2O 2g / L, yeast extract 4g / L, peptone 3g / L, K2HPO4·3H2O 6g / L, glutamic acid 1.3g / L, FeSO4·7H2O 30mg / L, MnSO4 10mg / L, VB1, VB3, and VB5 2mg / L each, and ammonium sulfate 8g / L.

[0075] The fermentation results of the strain are shown in Table 4.

[0076] Table 4

[0077] As shown in Table 4, the strains described in this application are used... NCgl1504 Y56M，T235P Effectively relieved the feedback inhibition of L-histidine; knockout NCgl0909 Gene, NCgl2632 Gene 、NCgl0580 Genes, by reducing ATP loss, allow more ATP to be used for product synthesis, and overexpression... NCgl1610-1609-1608 The operon increases the level of oxidative phosphorylation, promoting ATP production; the modification process works synergistically to significantly improve the synthetic efficiency and production intensity of the resulting strain.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification made by those skilled in the art based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An L-histidine-producing strain, characterized in that: Based on wild-type Corynebacterium glutamicum Corynebacterium glutamicum The overexpression strain was obtained through gene editing using metabolic engineering techniques. NCgl1505 Gene 、 NCgl2093 Gene , NCgl2096 Gene , NCgl2094 Gene , NCgl2097 Gene , NCgl2100 Gene , NCgl0218 Gene , NCgl2101 Gene , NCgl0799 Genes and NCgl2102 Genes, after overexpression mutation NCgl1504 Y56M，T235P Genes; Heterologous introduction of E. coli Escherichia coli Source pntAB Genes; overexpression of efflux proteins NCgl1262 Gene; knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes; overexpression NCgl1610-1609-1608 Manipulator.

2. The L-histidine-producing strain according to claim 1, characterized in that: wild-type Corynebacterium glutamicum Corynebacterium glutamicum Using P on the genome tuf Startup driver NCgl1505 Gene , NCgl2093 Gene 、 NCgl2096 Gene , NCgl2094 Gene , NCgl2097 Gene , NCgl2100 Gene , NCgl0218 Gene , NCgl2101 Gene , NCgl0799 Genes and NCgl2102 Gene overexpression, using P tuf After promoter-driven mutation NCgl1504 Y56M，T235P Gene overexpression; heterologous introduction of E. coli-derived genes pntAB Genes and P tuf Promoter-driven overexpression; using P sod Promoter-driven efflux proteins NCgl1262 Gene overexpression; knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes; using P tuf Startup driver NCgl1610-1609-1608 Operator overexpression.

3. The L-histidine-producing strain according to claim 1 or 2, characterized in that: The NCgl2102 Gene, NCgl2101 Genes and NCgl2100 Genes NCgl2102-2101-2100 Formal overexpression of natural operators; NCgl2097 Genes and NCgl2096 Genes NCgl2097-2096 Formal overexpression of natural operators; NCgl2094 Genes and NCgl2093 Genes NCgl2094-2093 The form of natural operator overexpression.

4. The L-histidine-producing strain according to claim 1 or 2, characterized in that: The NCgl0909 The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing; NCgl2632 The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; NCgl0580 The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing; NCgl0218 The nucleotide sequence of the gene is shown in SEQ ID NO. 18 of the sequence listing; NCgl0799 The nucleotide sequence of the gene is shown in SEQ ID NO. 19 of the sequence listing; NCgl1505 The nucleotide sequence of the gene is shown in SEQ ID NO.20 of the sequence listing; NCgl1504 Y56M，T235P The nucleotide sequence of the gene is shown in SEQ ID NO. 21 of the sequence listing; *Escherichia coli* Escherichia coli Source pntAB The nucleotide sequence of the gene is shown in SEQ ID NO.22 of the sequence listing; efflux protein NCgl1262 The nucleotide sequence of the gene is shown in SEQ ID NO. 25 of the sequence listing; NCgl2102 The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing; NCgl2101 The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing; NCgl2100 The nucleotide sequence of the gene is shown in SEQ ID NO. 11 of the sequence listing; NCgl2097 The nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing; NCgl2096 The nucleotide sequence of the gene is shown in SEQ ID NO. 14 of the sequence listing; NCgl2094 The nucleotide sequence of the gene is shown in SEQ ID NO. 16 of the sequence listing; NCgl2093 The nucleotide sequence of the gene is shown in SEQ ID NO. 17 of the sequence listing; NCgl1610-1609- 1608 The nucleotide sequence of the operon is shown in the sequence listing SEQ ID NO.

4.

5. The L-histidine-producing strain according to claim 2, characterized in that: The P tuf The nucleotide sequence of the promoter is shown in SEQ ID NO.37 of the sequence listing, P sod The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.

26.

6. The L-histidine-producing strain according to claim 3, characterized in that: The NCgl2102-2101-2100 The nucleotide sequence of the natural operon is shown in SEQ ID NO. 8 of the sequence listing; NCgl2097-2096 The nucleotide sequence of the natural operon is shown in SEQ ID NO.12 of the sequence listing; NCgl2094-2093 The nucleotide sequence of the natural operon is shown in SEQ ID NO. 15 of the sequence listing.

7. The method for constructing the L-histidine-producing strain according to any one of claims 1-6, characterized in that: The originating strain is wild-type Corynebacterium glutamicum. Corynebacterium glutamicum Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) Overexpression on the genome NCgl1505 Gene , NCgl2093 Gene , NCgl2096 Gene , NCgl2094 Gene 、 NCgl2097 Gene , NCgl2100 Gene , NCgl0218 Gene , NCgl2101 Gene , NCgl0799 Gene , NCgl2102 Genes, and the result of overexpression of endogenous mutations NCgl1504 Y56M，T235P Gene; (2) Heterogeneous introduction of Escherichia coli pntAB Gene; (3) Overexpression of efflux proteins NCgl1262 Gene; (4) Knockout NCgl0909 Gene, NCgl2632 Genes and NCgl0580 Genes, and overexpression NCgl1610-1609-1608 Manipulator.

8. The method for constructing the L-histidine-producing strain according to claim 7, characterized in that: The specific steps are as follows: 1) In NCgl3068 Sites using P tuf Starter driver N Cgl2102-2101-2100 Operator overexpression; 2) In NCgl1781 Sites using P tuf Startup driver NCgl2097-2096 Operator overexpression; 3) In NCgl1775 Sites using P tuf Startup driver NCgl2094-2093 Operator overexpression; 4) In NCgl1790 Sites using P tuf Startup driver NCgl0218 Gene overexpression; 5) In NCgl1774 Sites using P tuf Startup driver NCgl0799 Gene overexpression; 6) In NCgl1773 Sites using P tuf Startup driver NCgl1505 Gene overexpression; 7) In NCgl1817 Sites using P tuf Startup driver NCgl1504 Y56M，T235P Gene overexpression; 8) In NCgl2998 Sites using P tuf Promoter-driven E. coli-derived pntAB Overexpression; 9) In NCgl2949 Sites using P sod Startup driver NCgl1262 Gene overexpression; 10) Knockout NCgl0909 Gene; 11) Knockout NCgl2632 Gene; 12) Knockout NCgl0580 Gene; 13) In NCgl2923 Sites using P tuf Startup driver NCgl1610-1609-1608 Operator overexpression.

9. The use of the L-histidine-producing strain according to any one of claims 1-6 in the production of L-histidine.

10. The application according to claim 9, characterized in that: L-histidine was produced using a fermentation method.

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