Application of glutamate dehydrogenase from Salmonella typhimurium and related biomaterials in amino acid synthesis

Abstract

The present application discloses the application of glutamate dehydrogenase derived from Salmonella typhimurium and related biomaterials in amino acid synthesis, and belongs to the field of genetic engineering technology. The technical problem to be solved by the present application is: how to increase the amino acid production of microorganisms. To this end, the present application provides the application of glutamate dehydrogenase derived from Salmonella typhimurium in increasing the L-amino acid production of microorganisms. The present application discloses for the first time the use of glutamate dehydrogenase derived from Salmonella typhimurium and its encoding gene in increasing the L-amino acid production of microorganisms. The present application also obtains an optimized encoding gene of glutamate dehydrogenase derived from Salmonella typhimurium through codon optimization, and the introduction of the optimized encoding gene into a recipient microorganism can significantly increase the L-amino acid production of the recipient microorganism. The optimized encoding gene produces unexpected technical effects and has great application potential in the field of microbial fermentation of L-amino acids.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to the application of glutamate dehydrogenase derived from Salmonella typhimurium and related biomaterials in amino acid synthesis. Background Art

[0002] Amino acids, as the basic structure of protein macromolecules, are related to animal life activities and play a vital role in nutrition and health fields such as feed, food, medicine, culture medium, and health food. In recent years, with the improvement of people's health awareness, the demand for amino acids has also been increasing, which has promoted the rapid development of the amino acid market. The continuous innovation and application expansion of new technologies such as new and efficient mutagenesis, precise gene editing and genome engineering, synthetic biology and high-throughput screening have brought new opportunities for the substantial improvement of the efficiency and level of microbial breeding. These technologies make the selection and transformation of strains more accurate and efficient, thereby improving the yield and quality of amino acids. Metabolic controlled fermentation technology is based on dynamic biochemistry and microbial genetics. It obtains mutant strains suitable for the production of a certain product through artificial mutagenesis, and cultivates them under artificially controlled conditions, which can selectively produce a large number of products needed by people. The application of biotechnology has significantly improved the yield and purity of amino acids. In China, the amino acid industry has also shown a growth trend, and the market size has continued to expand. In the future, with the continuous improvement of the public's awareness of the importance of a healthy lifestyle, the continuous expansion of the application fields of amino acids, and the accelerated development of various amino acid application fields, the global demand for amino acids will further rise. At the same time, the amino acid biosynthesis process, fermentation process, and extraction technology will be further optimized, and amino acids and derivative products will have a broader development space. In summary, the current status of research on amino acid microbial fermentation is characterized by continuous technological progress, continuous expansion of production scale, wide application fields, and broad industry prospects. In the future, with the further development of science and technology and the continuous expansion of the market, the research and application of amino acid microbial fermentation will be more in-depth and extensive. Summary of the invention

[0003] The technical problem to be solved by the present application is: how to increase the fermentation yield of L-amino acids, for example, how to increase the yield of L-amino acids catalyzed by whole-cell microorganisms.

[0004] To solve this technical problem, the present application provides the following technical solution: heterologously overexpressing the glutamate dehydrogenase A gene from Salmonella typhimurium in Escherichia coli or Corynebacterium glutamicum to increase the L-amino acid production of Escherichia coli.

[0005] The present application provides the application of glutamate dehydrogenase derived from Salmonella typhimurium, and the application is the application of the glutamate dehydrogenase in increasing the L-amino acid production of microorganisms.

[0006] Furthermore, the glutamate dehydrogenase may be any one of the following:

[0007] a1), a protein whose amino acid sequence contains SEQ ID NO.4;

[0008] a2), a protein related to glutamate dehydrogenase, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1), which has more than 80% identity with the amino acid sequence shown in a1);

[0009] a3) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of a1) or a2).

[0010] Furthermore, a1) may also be a protein whose amino acid sequence is SEQ ID NO.4.

[0011] In the present application, SEQ ID NO. 4 consists of 447 amino acid residues.

[0012] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0014] Further, the connection described in a3) can be a peptide bond connection formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in a1) or a2). Or, the connection described in a3) can be a peptide bond connection formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in a1) or a2).

[0015] In the present application, the L-amino acid can be any one of L-arginine, L-glutamic acid, L-lysine, L-tryptophan, L-phenylalanine, L-glycine, L-alanine, L-cysteine, L-aspartic acid, L-histidine, L-isoleucine, L-leucine, L-methionine, L-aspartic acid, L-proline, L-glutamine, L-asparagine, L-proline, L-glutamine, L-serine, L-threonine, L-valine and L-tyrosine.

[0016] In the present application, the L-amino acid may be L-arginine or L-glutamic acid.

[0017] The present application also provides an application of the biomaterial related to the glutamate dehydrogenase, and the application can be the application of the biomaterial in any one of the following A1)-A3),

[0018] A1), application of the biomaterial in constructing an engineered bacterium producing L-amino acids;

[0019] A2), use of the biomaterial in the preparation of L-amino acids;

[0020] A3) Application of the biomaterial in regulating (increasing) the production of L-amino acids of microorganisms;

[0021] The biological material may be at least one of the following B1) to B3):

[0022] B1), a nucleic acid molecule encoding the glutamate dehydrogenase (also can be expressed as a gene encoding glutamate dehydrogenase derived from Salmonella typhimurium);

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

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

[0025] Furthermore, in the application, the nucleic acid molecule described in B1) may be G1) or G2)

[0026] G1) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.5;

[0027] G2) A nucleic acid molecule that is more than 80% identical to the nucleic acid molecule described in G1).

[0028] Furthermore, in the application, G1) the nucleic acid molecule may be a DNA molecule whose nucleotide sequence is SEQ ID NO.5.

[0029] Furthermore, in the application, G2) the nucleic acid molecule may be a nucleic acid molecule having a nucleotide sequence of SEQ ID NO.3.

[0030] The above-mentioned biological materials are also protected by this application.

[0031] Furthermore, in the biological material, the nucleotide sequence of the nucleic acid molecule in G1) may be SEQ ID NO.5.

[0032] Furthermore, in the biological material, G2) the nucleic acid molecule is not a nucleic acid molecule whose nucleotide sequence is SEQ ID NO.3.

[0033] The present application also provides a recombinant microorganism, which may be any of the following:

[0034] C1), a recombinant microorganism containing the nucleic acid molecule described in B1);

[0035] C2), a recombinant microorganism containing the expression cassette described in B2);

[0036] C3), a recombinant microorganism containing the recombinant vector described in B3);

[0037] C4), a recombinant microorganism containing the glutamate dehydrogenase.

[0038] The present application also provides a method for preparing the recombinant microorganism, wherein the recombinant microorganism is prepared according to a method comprising the following steps: introducing the above-mentioned biological material into a recipient microorganism, wherein the recipient microorganism may be any of the following:

[0039] D1), Escherichia bacteria;

[0040] D2), Escherichia coli ( Escherichia coli );

[0041] D3), ​​Corynebacterium;

[0042] D4), Corynebacterium glutamicum ( Corynebacterium glutamicum ).

[0043] Furthermore, compared with the recipient microorganism, the recombinant microorganism contains a glutamate dehydrogenase encoding gene derived from Salmonella typhimurium.

[0044] Furthermore, the glutamate dehydrogenase encoding gene derived from Salmonella typhimurium is a nucleic acid molecule having a nucleotide sequence comprising SEQ ID NO.5.

[0045] Furthermore, the activity of glutamate dehydrogenase of the recombinant microorganism is enhanced compared to that of the recipient microorganism.

[0046] Furthermore, compared with the recipient microorganism, the content of glutamate dehydrogenase in the recombinant microorganism is increased.

[0047] The present application also provides a composition, which contains the above-mentioned recombinant microorganism.

[0048] The active ingredient of the above composition may be the recombinant microorganism or / and the metabolite of the recombinant microorganism or / and the culture of the recombinant microorganism.

[0049] The above-mentioned culture may be a substance obtained by culturing the recombinant microorganism in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the recombinant microorganism and a substance secreted into a liquid culture medium, or a solid fermentation product containing the recombinant microorganism and a substance secreted into a solid culture medium).

[0050] In the above, the metabolite may be a product obtained by removing the recombinant microorganism from the culture, such as culturing the recombinant microorganism in a liquid fermentation medium, collecting the fermentation broth (containing the recombinant microorganism and substances secreted into the liquid culture medium), removing the recombinant microorganism from the fermentation broth, and collecting the remaining components of the fermentation broth to obtain the metabolite of the recombinant microorganism.

[0051] The active ingredients of the above composition may also contain other biological ingredients or non-biological ingredients. Those skilled in the art may determine the other active ingredients of the above composition according to the effects of the composition.

[0052] The above composition may be the culture. The above composition may also be a bacterial agent.

[0053] The above-mentioned bacterial agent refers to a live bacterial preparation made by expanding the target microorganisms and using a carrier as an adsorbent to adsorb the fermentation liquid or solid fermentation product of the bacteria.

[0054] The above-mentioned bacterial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0055] The bacterial agent may further include a carrier as required, and the carrier may be a solid carrier or a liquid carrier.

[0056] The present application also provides the use of a substance in the preparation of L-amino acids, wherein the substance is the above-mentioned recombinant microorganism, the recombinant microorganism prepared by the above-mentioned method, or the above-mentioned composition.

[0057] Furthermore, the recombinant microorganism may be a recombinant Escherichia coli or a recombinant Corynebacterium glutamicum.

[0058] Furthermore, in the application, the recombinant microorganism may be a recombinant Escherichia coli, and the L-amino acid may be L-arginine.

[0059] Furthermore, in the application, the recombinant microorganism may also be a recombinant Corynebacterium glutamicum, and the L-amino acid may be L-glutamic acid.

[0060] The present application also provides a method for increasing the L-amino acid production of a recipient microorganism, which may include introducing the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3) into the recipient microorganism to increase the L-amino acid production of the recipient microorganism.

[0061] In the present application, the recipient microorganism may be a microorganism that produces the L-amino acid.

[0062] Furthermore, the recipient microorganism may be a prokaryotic microorganism.

[0063] Furthermore, the recipient microorganism may be a bacterium.

[0064] Further, the recipient microorganism may be a Gram-negative bacterium;

[0065] Furthermore, the recipient microorganism may be a bacterium of the genus Escherichia;

[0066] Furthermore, the recipient microorganism may be Escherichia coli ( Escherichia coli ).

[0067] Furthermore, the Escherichia coli used as the recipient bacteria may be an Escherichia coli that produces the L-amino acid.

[0068] Furthermore, the Escherichia coli contains a ynjI site.

[0069] Among the recombinant microorganisms, the recombinant microorganism obtained by using Escherichia coli as the recipient bacterium is named recombinant Escherichia coli. The recombinant Escherichia coli can be a recombinant bacterium obtained by introducing the nucleic acid molecule described in B1) or the expression cassette described in B2) into the ynjI site of the recipient Escherichia coli.

[0070] Furthermore, the recipient Escherichia coli may be Escherichia coli CGMCC NO. 25402 and / or Escherichia coli W3110.

[0071] The Escherichia coli CGMCC NO. 25402 may be an Escherichia coli strain with the deposit number of CGMCC No. 25402 in the patent procedure.

[0072] Furthermore, the recipient microorganism may also be a Gram-positive bacterium.

[0073] Furthermore, the recipient microorganism may be a bacterium of the genus Corynebacterium.

[0074] Furthermore, the recipient microorganism may be Corynebacterium glutamicum ( Corynebacterium glutamicum ).

[0075] Furthermore, the Corynebacterium glutamicum used as the recipient bacterium may be Corynebacterium glutamicum that produces the L-amino acid.

[0076] Furthermore, the Corynebacterium glutamicum contains a poxB site.

[0077] Among the recombinant microorganisms, the recombinant microorganism obtained by using Corynebacterium glutamicum as the recipient bacterium is named recombinant Corynebacterium glutamicum. The recombinant Corynebacterium glutamicum can be a recombinant bacterium obtained by introducing the nucleic acid molecule described in B1) or the expression cassette described in B2) into the poxB site of the recipient Corynebacterium glutamicum.

[0078] Furthermore, the recipient Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC NO. 21220 and / or Corynebacterium glutamicum ATCC13032.

[0079] The Corynebacterium glutamicum CGMCC NO. 21220 is a Corynebacterium glutamicum strain with the deposit number of CGMCC No. 21220, which is deposited under patent procedure.

[0080] The Corynebacterium glutamicum ATCC13032 is the Corynebacterium glutamicum numbered 1303290 in ATCC.

[0081] The present application also provides a method for preparing L-amino acids, which may include the step of using a recombinant microorganism as a fermentation strain to prepare L-amino acids. The recombinant microorganism may be the above-mentioned recombinant microorganism or a recombinant microorganism prepared by the above-mentioned method.

[0082] Furthermore, the method comprises the steps of culturing the recombinant microorganism with a culture medium, collecting the culture product, and obtaining L-amino acids.

[0083] The term "culture product" refers to a general term for liquid or solid products (all materials in the culture container) having a microbial colony after artificial inoculation and cultivation. That is, the product obtained by growing and / or amplifying the microorganism can be a biological pure culture of the microorganism, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. It can also be a mixture containing a certain amount of culture medium, microbial cell metabolites and removing the microbial cell. In this application, "Escherichia coli" and "Escherichia coli" are common. In this application, "Corynebacterium glutamicum" and "Corynebacterium glutamicum" are common.

[0084] In some embodiments of the present application, the recombinant microorganism may be a recombinant Escherichia coli, and the recombinant Escherichia coli may be used as a fermentation strain to prepare L-arginine.

[0085] In some embodiments of the present application, the recombinant microorganism may be a recombinant Corynebacterium glutamicum, and the recombinant Corynebacterium glutamicum may be used as a fermentation strain to prepare L-glutamic acid.

[0086] The beneficial technical effects achieved by this application are as follows:

[0087] 1. This application discloses for the first time the use of glutamate dehydrogenase from Salmonella typhimurium and its encoding gene in increasing the L-amino acid production of microorganisms. This application demonstrates through experimental verification that glutamate dehydrogenase from Salmonella typhimurium can significantly increase the L-amino acid production in recipient Escherichia coli or Corynebacterium glutamicum.

[0088] 2. The present application obtains an optimized coding gene of glutamate dehydrogenase from Salmonella typhimurium through codon optimization. The optimized coding gene or the expression cassette containing the optimized coding gene is introduced into a recipient microorganism to significantly increase the L-amino acid production of the recipient microorganism. The glutamate dehydrogenase gene obtained by the optimization of the present application produces an unexpected technical effect and has great application potential in the field of microbial fermentation of L-amino acids.

[0089] Deposit description of Escherichia coli strain with deposit number CGMCC No. 25402

[0090] Classification name: Escherichia coli Escherichia coli

[0091] Strain ID: YP004-8

[0092] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0093] Abbreviation of depository unit: CGMCC

[0094] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0095] Date of deposit: July 25, 2022

[0096] CGMCC Registration Number: CGMCC No. 25402

[0097] Deposit description of Corynebacterium glutamicum strain with deposit number CGMCC No. 21220

[0098] Classification name: Corynebacterium glutamicum Corynebacterium glutamicum

[0099] Strain ID: YPGLU001

[0100] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0101] Abbreviation of depository unit: CGMCC

[0102] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0103] Date of deposit: November 23, 2020

[0104] CGMCC Registration Number: CGMCC No. 21220 DETAILED DESCRIPTION

[0105] 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.

[0106] 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.

[0107] There are four types of recipient bacteria in the following embodiments, which are as follows:

[0108] Escherichia coli W3110, referred to as E. coli W3110 or W3110, was purchased from the American Type Culture Collection (ATCC) with the strain number: 27325;

[0109] Escherichia coli ( Escherichia coli ) CGMCC NO. 25402, also known as Escherichia coli CGMCC NO.25402, L-arginine high-producing strain CGMCC NO. 25402 or CGMCC NO. 25402, which has been deposited under patent procedures with the deposit number CGMCC NO. 25402;

[0110] Corynebacterium glutamicum ATCC13032, also known as ATCC13032, was purchased from the American Type Culture Collection (ATCC) with the strain number 13032;

[0111] Corynebacterium glutamicum ( Corynebacterium glutamicum ) CGMCC No. 21220, also known as Corynebacterium glutamicum CGMCC No. 21220, glutamate high-producing bacteria CGMCC NO. 21220 or CGMCC NO. 21220. This strain has been deposited under the patent procedure and the deposit number is CGMCC NO. 21220.

[0112] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0113] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation and tested using t-test. Different letters indicate significant differences.

[0114] Example 1: Construction of recombinant Escherichia coli overexpressing glutamate dehydrogenase from Salmonella typhimurium

[0115] According to the Escherichia coli ( Escherichia coli ) W3110 genome sequence (Gene ID: 998010), and used CRISPR / Cas9 gene editing technology to overexpress the gdhA gene from Salmonella typhimurium (abbreviated as gdhA (SA), the same below) in the genomes of W3110 and the L-arginine high-producing strain CGMCC NO.25402, respectively, to further study the effect of the gdhA (SA) gene on L-arginine production.

[0116] gdhA from Escherichia coli W3110 (abbreviated as gdhA (EC), SEQ ID NO.1), gdhA from Salmonella typhimurium (SEQ ID NO.3), gdhA from Salmonella typhimurium after codon optimization (SEQ ID NO.5), rocG from Bacillus subtilis (SEQ ID NO.25) and gdh from Corynebacterium glutamicum ATCC13032 (SEQ ID NO.27) were inserted into the predicted inner membrane protein encoding gene (ynjI, db_xref="GI:85675112") site of wild-type Escherichia coli W3110.

[0117] Among them, the DNA molecule shown in SEQ ID NO.1 encodes a protein with an amino acid sequence of SEQ ID NO.2, and the name of the protein is glutamate dehydrogenase of wild-type Escherichia coli W3110 strain. The DNA molecule shown in SEQ ID NO.3 encodes a protein with an amino acid sequence of SEQ ID NO.4, and the name of the protein is glutamate dehydrogenase of wild-type Salmonella typhimurium SL7207 strain. SEQ ID NO.5 is the nucleotide sequence of SEQ ID NO.3 after codon optimization, and the amino acid sequence of the protein encoded by it is still SEQ ID NO.4. The DNA molecule shown in SEQ ID NO.25 encodes a protein with an amino acid sequence of SEQ ID NO.26, and the name of the protein is glutamate dehydrogenase of Bacillus subtilis str. 168 (CP053102.1) strain. The DNA molecule shown in SEQ ID NO.27 encodes a protein with an amino acid sequence of SEQ ID NO.28, and the name of the protein is glutamate dehydrogenase of Corynebacterium glutamicum ATCC13032.

[0118] 1. Construction of sgRNA

[0119] According to the Escherichia coli ( Escherichia coli )W3110 genome sequence, use CRISPRRGEN Tools (http: / / www.rgenome.net / cas-designer / ) to design sgRNA target sequences, and after selecting the appropriate sgRNA target sequence, add linearized pGRB cloning vector end sequences to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0120] To amplify the sgRNA fragment, no template is required, only the PCR annealing process is required. The system and procedure are as follows. PCR reaction system: sgRNA-1F 10μL, sgRNA-1R 10μL; PCR reaction procedure: 95℃ denaturation for 5min, 50℃ annealing for 1min. After annealing, the target fragment was recovered using a DNA purification kit, its DNA concentration was determined, and the concentration was diluted to 100ng / μL.

[0121] pGRB plasmid (Addgene product, catalog number: 71539) was extracted and used Spe I digestion and dephosphorylation treatment to prevent pGRB plasmid from self-ligation. Enzyme digestion system: 10×Buffer 5μL, SpeI 2.5μL, pGRB plasmid DNA 3000-5000 ng, supplemented with ddH2O to 50μL. After enzyme digestion at 37℃ for 3h, agarose gel electrophoresis was performed to remove the gel and then dephosphorylated. The dephosphorylation system was: 10×Buffer 5μL, linearized pGRB plasmid DNA 1000-2000 ng, CIAP 2.5μL, supplemented with ddH2O to 50μL. After treatment at 37℃ for 1h, the linearized pGRB plasmid was recovered using a DNA purification kit. The Gibson Assembly Kit (NewEngland) was then used to reconstitute sgRNA and pGRB plasmid. Recombination system: NEB assembly enzyme 2.5 μL, linearized cloning vector 2μL, sgRNA 0.5μL. After assembly at 50℃ for 30min, the product was transformed into DH5α competent cells, the plasmid was extracted, and sequencing was performed using sequencing primers sgRNA-PF and sgRNA-PR for identification. The constructed plasmid was named pGRB-sgRNA-1 plasmid.

[0122] The primers used in this experiment were designed as follows (synthesized by Shanghai Invitrogen). The uppercase letters of sgRNA-1F and sgRNA-1R are the homology arm sequences of the pGRB cloning vector, and the lowercase letters are the target sequences of sgRNA:

[0123] sgRNA-1F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTctttaaaggtattggctccaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID NO. 54);

[0124] sgRNA-1R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACtggagccaatacctttaaagACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID NO. 55);

[0125] sgRNA-PF: 5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID NO. 56);

[0126] sgRNA-PR: 5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID NO. 57).

[0127] 2. Preparation of integrated fragments for overexpressing the gdhA gene

[0128] The gdhA (EC) overexpression integrated homology arm DNA fragment Up-pBJ23119-gdhA (EC)-Down (SEQ ID NO.6, 2565 bp) was prepared, wherein the 1st to 544th positions of SEQ ID NO.6 were ΔynjI-UP, the 545th to 621st positions were pBJ23119 promoter, the 622th to 1965th positions were gdhA (EC), and the 1966th to 2565th positions were ΔynjI-DOWN.

[0129] The gdhA (SA) overexpression integration homology arm DNA fragment Up-pBJ23119-gdhA (SA)-Down (SEQ ID NO.7) 2565 bp was prepared, wherein the 1st to 544th positions of SEQ ID NO.7 were the UP fragment, the 545th to 621st positions were the PBJ23119 promoter, the 622th to 1965th positions were gdhA (SA), and the 1966th to 2565th positions were the Down fragment.

[0130] The gdhA (SA, optimized) overexpression integrated homology arm DNA fragment Up-pBJ23119-gdhA (SA, optimized)-Down (SEQ ID NO.8) 2565bp was prepared, wherein the 1st to 544th positions of SEQ ID NO.8 were the UP fragment, the 545th to 621st positions were pBJ23119, the 622th to 1965th positions were gdhA (SA, optimized), and the 1966th to 2565th positions were the DOWN fragment.

[0131] The rocG (Bacillus subtilis, BS) overexpression integration homology arm DNA fragment Up-pBJ23119-rocG-Down (SEQ ID NO.29, 2496 bp) was prepared. Among them, the 1st to 544th positions of SEQ ID NO.29 are the UP fragment, the 545th to 621st positions are the pBJ23119 promoter, the 622th to 1896th positions are rocG (Bacillus subtilis), and the 1897th to 2496th positions are the DOWN fragment.

[0132] A gdh (Corynebacterium glutamicum, CG) overexpression integrated homology arm DNA fragment Up-pBJ23119-gdhA (CG)-Down (SEQ ID NO.30) 2565 bp was prepared, wherein the 1st to 544th positions of SEQ ID NO.30 were ΔynjI (gdhA)-UP, the 545th to 621st positions were pBJ23119, the 622th to 1965th positions were gdhA (CG), and the 1966th to 2565th positions were ΔynjI (gdhA)-DOWN.

[0133] 3. Preparation and transformation of competent cells

[0134] The plasmid pREDCas9 (Addgene product, catalog number #71541) was electroporated into the electroporation competent E. coli. Note: pREDCas9 plasmid is a temperature-sensitive plasmid and will be lost if the temperature is too high, so the culture temperature of the strain containing pREDCas9 is 32 ℃. The revived bacteria after electroporation are spread on LB plates containing spectinomycin and cultured overnight at 32 ℃. Single colonies grown on the resistant plate are positive recombinants.

[0135] W3110-Cas9 is a recombinant bacterium obtained by introducing the pREDCas9 plasmid into Escherichia coli W3110.

[0136] The L-arginine producing bacteria CGMCC NO. 25402-Cas9 is a recombinant bacterium obtained by introducing the pREDCas9 plasmid into the Escherichia coli strain with the deposit number CGMCC NO.25402.

[0137] Prepare W3110-Cas9 and L-arginine CGMCC NO. 25402-Cas9 competent cells. When the cells grow to OD 600 =0.1Add IPTG at a final concentration of 0.1 mM to induce λ-Red-mediated homologous recombination. Continue culturing until OD 600 =0.6, the bacteria were collected and each group was divided into five groups and transformed with pGRB-sgRNA-1 plasmid and Up-pBJ23119-gdhA (EC)-Down, pGRB-sgRNA-1 plasmid and Up-pBJ23119-gdhA (SA)-Down, pGRB-sgRNA-1 plasmid and Up-pBJ23119-gdhA (SA, optimized)-Down, pGRB-sgRNA-1 plasmid and Up-pBJ23119-rocG-Down, and pGRB-sgRNA-1 plasmid and Up-pBJ23119-gdh (CG)-Down, respectively. The plates were spread on 2-YT plate medium containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C. The single colonies produced by the culture were identified by r Taq PCR using primers P7 and P8, P7 and P9, P7 and P10, P7 and P42, and P7 and P46, respectively. The fragments of 1641 bp (SEQ ID NO.9), 1281 bp (SEQ ID NO.10), 1281 bp (SEQ ID NO.11), 1559 bp (SEQ ID NO.31), and 1619 bp (SEQ ID NO.32) amplified by the corresponding PCR were positive transformants, and those without amplified fragments were original bacteria.

[0138] The positive transformants were inoculated in 2-YT medium containing spectinomycin (100 mg / L) and arabinose with a final concentration of 0.2% to eliminate the plasmid pGRB-sgRNA-1. The colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and then these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. The colonies that did not grow on spectinomycin (100 mg / L) but grew on 2-YT without resistance were selected and identified by r Taq PCR using primers P11 and P12, P13 and P14, P15 and P16, P43 and P44, P47 and P48, respectively. PCR amplified the fragments containing 1884 bp (SEQ ID NO.12), 1644 bp (SEQ ID NO.13), 1824 bp (SEQ ID NO.14), 1190 bp (SEQ ID NO.33) and 1089 bp (SEQ ID NO.34) are positive transformants that eliminated the pREDCas9 plasmid, and those that could not be amplified are original bacteria.

[0139] The primer sequences are as follows (synthesized by Shanghai Invitrogen):

[0140] P7: 5'-GCACTGTAGT TGATGAAAGC-3' (SEQ ID NO. 15),

[0141] P8: 5'-AAATTCTTG GCGTAATCTG-3' (SEQ ID NO. 16),

[0142] P9: 5'-CGCCATAAAG CCCACTTCGC-3' (SEQ ID NO. 17),

[0143] P10: 5'-AGCCATGAAACCCACTTCGC-3' (SEQ ID NO. 18),

[0144] P11: 5'-CGTGTGCAGT TCAGCTCTGC-3' (SEQ ID NO. 19),

[0145] P12: 5'-AAGCGCGTTTTCTTGCTGAA-3' (SEQ ID NO. 35),

[0146] P13: 5'-GGAATGATGC GCAAGCTTTC-3' (SEQ ID NO. 36),

[0147] P14:5’-ATGCGGTGTT CCGCAAGTGT-3’(SEQ ID NO.45),

[0148] P15:5’-ACCCTGCCGA TGGGCGGCGG-3’(SEQ ID NO.46),

[0149] P16:5’-ATGCGGTGTT CCGCAAGTGT-3’(SEQ ID NO.47),

[0150] P42: 5’-TCCCGTTTAT CGAGCAAATA AG-3’ (SEQ ID NO.48),

[0151] P43: 5’-CAGCTTTGGT ATGGTCACC-3’ (SEQ ID NO.49),

[0152] P44: 5’- AGGCGATGAG TTGCTGTGTG-3’ (SEQ ID NO.50),

[0153] P46: 5’- ACGGATACGC GTGCGCGAC-3’ (SEQ ID NO.51),

[0154] P47: 5’- GTGCGATATC GCTCTTCCTT-3’ (SEQ ID NO.52),

[0155] P48: 5’-AGGCGATGAG TTGCTGTGTG -3’ (SEQ ID NO.53)。

[0156] The wild-type Escherichia coli W3110 strains with pBJ23119-gdhA (EC), pBJ23119-gdhA (SA), pBJ23119-gdhA (SA, optimized), pBJ23119-rcoG (BS), and pBJ23119-gdh (CG) integrated into their genome were named W3110 / pBJ23119-gdhA (EC), W3110 / pBJ23119-gdhA (SA), W3110 / pBJ23119-gdhA (SA, optimized), W3110 / pBJ23119-rcoG (BS), and W3110 / pBJ23119-gdh (CG), respectively. The L-arginine-producing strain CGMCC NO. 25402 with pBJ23119-gdhA (EC), pBJ23119 -gdhA (SA), pBJ23119-gdhA (SA, optimized), pBJ23119-rcoG (BS), and pBJ23119-gdh (CG) integrated into the genome were named YPR / pBJ23119-gdhA (EC), YPR / pBJ23119-gdhA (SA), YPR / pBJ23119-gdhA (SA, optimized), YPR / pBJ23119-rcoG (BS), and YPR / pBJ23119-gdh (CG), respectively.

[0157] The recombinant bacteria W3110 / pBJ23119-gdhA (EC) and YPR / pBJ23119-gdhA (EC) contained two copies of the gdhA (EC) gene shown in SEQ ID NO.1; the recombinant bacteria W3110 / pBJ23119-gdhA (SA) and YPR / pBJ23119-gdhA (SA) contained one copy of the gdhA (EC) gene and one copy of the gdhA (SA) gene; the recombinant bacteria W3110 / pBJ23119-gdhA (SA, optimized) and YPR / pBJ23119-gdhA (SA, optimized) contained one copy of the gdhA (EC) gene and one copy of the gdhA (SA) gene. The gdhA (SA, optimized) gene of shellfish; the recombinant bacteria W3110 / pBJ23119-rcoG (BS) and YPR / pBJ23119-rcoG (BS) both contain one copy of the gdhA (EC) gene and one copy of the rocG (BS) gene; the recombinant bacteria W3110 / pBJ23119-gdh (CG) and YPR / pBJ23119-gdh (CG) both contain one copy of the gdhA (EC) gene and one copy of the gdh (CG) gene.

[0158] Compared with L-arginine producing bacteria CGMCC NO. 25402, YPR / pBJ23119-gdhA (SA, optimized) is a recombinant bacterium obtained by carrying out the following transformation on L-arginine producing bacteria CGMCC NO. 25402: an expression cassette of a glutamate dehydrogenase encoding gene (gdhA (SA, optimized) is introduced into the ynjI site of L-arginine producing bacteria CGMCC NO. 25402. Compared with wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA (SA, optimized) is a recombinant bacterium obtained by carrying out the following transformation on wild-type Escherichia coli W3110: an expression cassette of a glutamate dehydrogenase encoding gene (gdhA (SA, optimized) is introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the expression cassette of the glutamate dehydrogenase encoding gene (gdhA (SA, optimized) is SEQ ID NO. 8, positions 545-1965. Among them, SEQ ID NO.8 positions 545-621 are pBJ23119, and positions 622-1965 are gdhA (SA, optimized).

[0159] Compared with L-arginine producing bacteria CGMCC NO. 25402, YPR / pBJ23119-gdhA (SA) is a recombinant bacterium obtained by performing the following transformation on L-arginine producing bacteria CGMCC NO. 25402: an expression cassette of glutamate dehydrogenase encoding gene gdhA (SA) is introduced at the ynjI site of L-arginine producing bacteria CGMCC NO. 25402. Compared with wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA (SA) is a recombinant bacterium obtained by performing the following transformation on wild-type Escherichia coli W3110: an expression cassette of glutamate dehydrogenase encoding gene gdhA (SA) is introduced at the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the expression cassette of the above-mentioned glutamate dehydrogenase encoding gene gdhA (SA) is SEQ ID NO.7, positions 545-1965. Among them, positions 545-621 of SEQID NO.7 are pBJ23119, and positions 622-1965 are gdhA (SA).

[0160] Compared with L-arginine producing bacteria CGMCC NO. 25402, YPR / pBJ23119-gdhA (EC) is a recombinant bacterium obtained by performing the following transformation on L-arginine producing bacteria CGMCC NO. 25402: an expression cassette of glutamate dehydrogenase encoding gene gdhA (EC) is introduced at the ynjI site of L-arginine producing bacteria CGMCC NO. 25402. Compared with wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA (EC) is a recombinant bacterium obtained by performing the following transformation on wild-type Escherichia coli W3110: an expression cassette of glutamate dehydrogenase encoding gene gdhA (EC) is introduced at the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the expression cassette of the above-mentioned glutamate dehydrogenase encoding gene gdhA (EC) is SEQ ID NO. 6, positions 545-1965. Among them, positions 545-621 of SEQID NO.6 are pBJ23119 promoter, and positions 622-1965 are gdhA (EC).

[0161] Compared with L-arginine producing bacteria CGMCC NO. 25402, YPR / pBJ23119-rocG (BS) is a recombinant bacterium obtained by the following transformation in L-arginine producing bacteria CGMCC NO. 25402: an expression cassette of glutamate dehydrogenase encoding gene rocG (BS) is introduced at the ynjI site of L-arginine producing bacteria CGMCC NO. 25402. Compared with Escherichia coli wild type W3110, W3110 / pBJ23119-rocG (BS) is a recombinant bacterium obtained by the following transformation in Escherichia coli wild type W3110: an expression cassette of glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) is introduced at the ynjI site of Escherichia coli wild type W3110. The nucleotide sequence of the expression cassette of the above-mentioned glutamate dehydrogenase encoding gene rocG (BS) is SEQ ID NO. 29, positions 545-1896. Among them, positions 545-621 of SEQ ID NO.29 are pBJ23119 promoter, and positions 622-1896 are rocG (BS).

[0162] Compared with L-arginine producing bacteria CGMCC NO. 25402, YPR / pBJ23119-gdh (Corynebacterium glutamicum, CG) is a recombinant bacterium obtained by performing the following transformation on L-arginine producing bacteria CGMCC NO. 25402: an expression cassette of glutamate dehydrogenase encoding gene gdh (CG) is introduced at the ynjI site of L-arginine producing bacteria CGMCC NO. 25402. Compared with wild-type Escherichia coli W3110, W3110 / pBJ23119-gdh (CG) is a recombinant bacterium obtained by performing the following transformation on wild-type Escherichia coli W3110: an expression cassette of glutamate dehydrogenase encoding gene gdh (CG) is introduced at the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the expression cassette of the above-mentioned glutamate dehydrogenase encoding gene gdh (CG) is SEQ ID NO.30, positions 545-1965. Among them, positions 545-621 of SEQ ID NO.30 are pBJ23119 promoter, and positions 622-1965 are gdh (CG).

[0163] Example 2: Construction of a recombinant Corynebacterium glutamicum containing Salmonella typhimurium glutamate dehydrogenase activity

[0164] In this embodiment, the poxB gene of the genetically engineered strains ATCC13032 and glutamate high-producing bacteria CGMCC NO. 21220 was knocked out and the glutamate dehydrogenase gene was inserted by homologous recombination. The GenBank accession number of the pyruvate dehydrogenase encoding gene is CAF21272.1 (BCT 27-FEB-2015), which converts pyruvate into acetyl CoA and CO2.

[0165] 1. Prepare the integrated fragment containing upstream and downstream homology arms and target gene

[0166] The integrated fragments containing upstream and downstream homology arms and target genes were prepared as follows:

[0167] Prepare the gdhA (SA) overexpression integration fragment Up-pBJ23119-gdhA (SA)-Down (SEQ ID NO.41, 2715 bp), wherein the 1st to 694 bp of SEQ ID NO.41 is the poxB-UP fragment, the 695th to 771 bp is the PBJ23119 promoter, the 772th to 2115th is gdhA (SA), and the 2116th to 2715th is the poxB-Down fragment.

[0168] Prepare gdhA (SA, optimized) overexpression integration fragment Up-pBJ23119-gdhA (SA, optimized)-Down (SEQ ID NO.42, 2715 bp), wherein SEQ ID NO.42 bp 1-694 is poxB-UP fragment, bp 695-771 is pBJ23119, bp 772-2115 is gdhA (SA, optimized), and bp 2116-2715 is poxB-Down fragment.

[0169] Prepare the rocG (Bacillus subtilis) overexpression integration fragment Up-pBJ23119-rocG (BS)-Down (SEQ ID NO.43, 2647 bp). The 1st to 694 bp of SEQ ID NO.43 is the poxB-UP fragment, the 695th to 771st bp is the pBJ23119 promoter, the 772th to 2046th bp is the rocG (Bacillus subtilis), and the 2047th to 2647th bp is the poxB-Down fragment.

[0170] Prepare gdh (Corynebacterium glutamicum, CG) overexpression integration fragment Up-pBJ23119-gdh (CG) -Down (SEQ ID NO. 44, 2715 bp), wherein SEQ ID NO. 44 1-694 is poxB-UP, 695-771 bp is pBJ23119, 772-2115 is gdh (CG), and 2116-2715 is poxB-Down.

[0171] 2. Preparation of recombinant vector and transformation of recipient strain

[0172] The integration fragment was connected with the pK18mobsacB plasmid (Addgene product, which contains kanamycin resistance as a selection marker) that was purified after digestion with Xbal I / BamH I using NEBuilder enzyme (NEB product) at 50°C for 30 min. The single clone grown after the ligation product was transformed into Escherichia coli DH5α was cloned using M13 primers (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') were identified by PCR to obtain positive integration plasmids, which were named pK18-pBJ23119-gdh (SA), pK18-pBJ23119-gdh (SA, optimized), pK18-pBJ23119-rocG (BS), and pK18-pBJ23119-gdh (CG). The positive integration plasmids contain a kanamycin resistance marker, and recombinants with the plasmid integrated into the genome can be obtained by kanamycin screening.

[0173] The correctly sequenced integration plasmids pK18-pBJ23119-gdh (SA), pK18-pBJ23119-gdh (SA, optimized), pk18-pBJ23119-rocG (BS), and pK18-pBJ23119-gdh (CG) were electro-transformed into Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220, respectively, and cultured in a medium containing 100 μg / mL kanamycin. The composition of the medium was: sucrose 150 g / L, polypeptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, urea 2 g / L, sodium chloride 2.5 g / L, agar powder 20 g / L, and the rest was water, pH 7.0. Culture conditions: 32 ℃. The single colonies produced by the culture were identified by PCR using primers P25 and P26, P29 and P30, P57 and P58, and P61 and P62. The colonies with a size of 1190 bp (SEQ ID NO.20) amplified by PCR from P25 and P26 were pBJ23119-gdh (SA) integration-positive strains; the strains with a size of 1371 bp (SEQ ID NO.21) amplified by PCR from P29 and P30 were pBJ23119-gdh (SA, optimized) integration-positive strains; the strains with a size of 771 bp (SEQ ID NO.37) amplified by PCR from P57 and P58 were pBJ23119-rocG (BS) integration-positive strains; the strains with a size of 1290 bp (SEQ ID NO.38) amplified by PCR from P61 and P62 were pBJ23119-gdh (GC) integration-positive strains; the strains without amplified fragments were original strains.

[0174] The positive strains were cultured in a culture medium containing 150 g / L sucrose (the composition of the culture medium was: sucrose 150 g / L, polypeptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, urea 2 g / L, sodium chloride 2.5 g / L, agar powder 20 g / L, and the rest was water, pH 7.0. Function: to eliminate the PK18 plasmid integrated into the genome). The single colonies produced by the culture were further identified by PCR using primer pairs P27 and P28 (for identifying pBJ23119-gdh (SA) integration), P31 and P32 (for identifying pBJ23119-gdh (SA, optimized) integration), P59 and P60 (for identifying PBJ23119-rocG (BS) integration), and P63 and P64 (for identifying PBJ23119-gdh (GC) integration).

[0175] The strains amplified by primer pair P27 and P28 with a size of 1524 bp (identifying the integration of pBJ23119-gdh (SA), SEQ ID NO. 22) were positive strains in which pBJ23119-gdh (SA) was integrated into the poxB site on the genome of Corynebacterium glutamicum ATCC13032 or CGMCC 21220, and were named ATCC13032-gdh (SA) and YPGLU-gdhA (SA), respectively.

[0176] The strain with a size of 1704 bp amplified by primer pair P31 and P32 (identifying the integration of pBJ23119-gdh (SA, optimized), SEQ ID NO. 23) was a positive strain in which pBJ23119-gdh (SA, optimized) was integrated into the poxB site on the genome of Corynebacterium glutamicum ATCC13032 or CGMCC NO. 21220, and was named ATCC13032-gdh (SA, optimized) and YPGLU-gdhA (SA, optimized), respectively.

[0177] The strain with a size of 1237 bp (identifying pBJ23119-rocG (BS) integration, SEQ ID NO. 39) amplified by primer pair P59 and P60 was a positive strain in which pBJ23119-rocG (BS) was integrated into the poxB site on the genome of Corynebacterium glutamicum ATCC13032 or CGMCC 21220, and was named ATCC13032-rocG (BS) and YPGLU-rocG (BS), respectively.

[0178] The strains amplified by primer pair P63 and P64 with a size of 939 bp (identifying the integration of pBJ23119-gdh (GC), SEQ ID NO. 40) were positive strains in which pBJ23119-gdh (GC) was integrated into the poxB site on the genome of Corynebacterium glutamicum ATCC13032 or CGMCC 21220, and were named ATCC13032-gdh (GC) and YPGLU-gdh (GC), respectively.

[0179] The primer sequences mentioned above are as follows (synthesized by Shanghai Invitrogen):

[0180] P25: 5'-GAACAAAACC ATTGCCATCA-3' (SEQ ID NO. 58),

[0181] P26: 5'-GAAGGATGG AAGCGCATAC-3' (SEQ ID NO. 59),

[0182] P27: 5'-GCGATGCTTAAACGTCATGG-3' (SEQ ID NO. 60),

[0183] P28: 5'-GTTGCAGGCA TCATCGTTGC-3' (SEQ ID NO. 61),

[0184] P29: 5'-AGGCATGGTT GGGCACGATG-3' (SEQ ID NO. 62),

[0185] P30: 5'-GGTCAGCGCG TTTTTGAAGG -3' (SEQ ID NO. 63),

[0186] P31: 5'-GGTGAAGTTA TGCGTTTCTG -3' (SEQ ID NO. 64),

[0187] P32: 5'-CACGCAGTTG GAGCAAACGC-3' (SEQ ID NO. 65).

[0188] P57: 5'-CAAAATGATCCGACGATCTG-3' (SEQ ID NO.66),

[0189] P58: 5'-AGGACTGAGC TAGCTGTCA-3' (SEQ ID NO.67),

[0190] P59: 5'-TAGCTTCCTG GCCAAATTCA-3' (SEQ ID NO. 68),

[0191] P60: 5'-GCCATGAGCA GGGGATAT-3' (SEQ ID NO. 69),

[0192] P61: 5'-CCAAATGAT CCGACGATCT-3' (SEQ ID NO.70),

[0193] P62: 5'-TAGCTCGGTC ATGAAGGACT-3' (SEQ ID NO.71),

[0194] P63: 5'-GTTGAGGTCTTCCGTGAGCG-3' (SEQ ID NO.72),

[0195] P64: 5'-ATGAGCAGGG GATATGCGTT-3' (SEQ ID NO. 24).

[0196] The recombinant bacteria ATCC13032-gdhA (SA) and YPGLU-gdhA (SA) both contain a copy of the gdhA gene from Salmonella typhimurium shown in SEQ ID NO.3. Specifically, the recombinant bacteria ATCC13032-gdhA (SA) and YPGLU-gdhA (SA) are respectively obtained by knocking out the poxB gene coding region in the genome of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220, while introducing the expression cassette of the glutamate dehydrogenase encoding gene gdhA (SA) at the poxB site, while keeping the other nucleotides in the genome of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220 unchanged. The nucleotide sequence of the expression cassette of the glutamate dehydrogenase encoding gene gdhA (SA) is SEQ ID NO.7, positions 545-1965. Among them, positions 545-621 of SEQ ID NO.7 are pBJ23119, and positions 622-1965 are gdhA (SA).

[0197] The recombinant bacteria ATCC13032-gdh (SA, optimized) and YPGLU-gdh (SA, optimized) both contain one copy of the codon-optimized gdhA gene from Salmonella typhimurium as shown in SEQ ID NO.5. Specifically, the recombinant bacteria ATCC13032-gdh (SA, optimized) and YPGLU-gdh (SA, optimized) are respectively obtained by knocking out the poxB gene coding region in the genome of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220, and introducing the expression cassette of the glutamate dehydrogenase encoding gene (gdhA (SA, optimized) at the poxB site, while keeping the other nucleotides in the genome of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220 unchanged. The nucleotide sequence of the expression cassette of the glutamate dehydrogenase encoding gene (gdhA (SA, optimized) is SEQ ID NO.8, positions 545-1965. Among them, positions 545-621 of SEQ ID NO.8 are pBJ23119, and positions 622-1965 are gdhA (SA, optimized).

[0198] The recombinant bacteria ATCC13032-rocG (BS) and YPGLU-rocG (BS) both contain a copy of the rocG gene from Bacillus subtilis; specifically, the recombinant bacteria ATCC13032-rocG (BS) and YPGLU-rocG (BS) are respectively obtained by knocking out the poxB gene coding region in the genome of Corynebacterium glutamicum ATCCC13032 and CGMCC NO. 21220, and introducing the expression cassette of the glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) at the poxB site, while keeping the other nucleotides in the genome of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220 unchanged. The nucleotide sequence of the expression cassette of the glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) is SEQ ID NO. 29, positions 545-1896. Among them, positions 545-621 of SEQ ID NO.29 are pBJ23119 promoter, and positions 622-1896 are rocG (Bacillus subtilis).

[0199] The recombinant bacteria ATCC13032-gdh (GC) and YPGLU-gdh (GC) both contain two copies of the gdh gene from Corynebacterium glutamicum ATCC13032; specifically, the recombinant bacteria ATCC13032-gdh (GC) and YPGLU-gdh (GC) are obtained by knocking out the poxB gene coding region in the genomes of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220, and introducing the expression cassette of the glutamate dehydrogenase encoding gene gdh (CG) at the poxB site, while keeping the other nucleotides in the genomes of Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220 unchanged. The nucleotide sequence of the expression cassette of the glutamate dehydrogenase encoding gene gdh (CG) is SEQ ID NO. 30, positions 545-1965. Among them, positions 545-621 of SEQ ID NO.30 are pBJ23119 promoter, and positions 622-1965 are gdh (CG).

[0200] Example 3, fermentation experiment

[0201] 1. L-arginine fermentation experiment

[0202] The Escherichia coli W3110 strain, W3110 / pBJ23119-gdhA (EC), W3110 / pBJ23119-gdhA (SA), W3110 / pBJ23119-gdhA (SA, optimized), W3110 / pBJ23119-rocG (Bacillus subtilis), W3110 / pBJ23119-gdh (GC), CGMCC NO. The 25402 strain and YPR / pBJ23119-gdhA (EC), YPR / pBJ23119-gdhA (SA), YPR / pBJ23119-gdhA (SA, optimized), YPR / pBJ23119-rocG (BS), and YPR / pBJ23119-gdh (GC) were inoculated in a 5L fermenter of the BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) and fermented with L-arginine fermentation medium and the culture conditions provided in Table 1. Each strain was repeated three times. After the fermentation, the L-arginine content was detected by high performance liquid chromatography (HPLC), and the results were taken as the average of three replicates, as shown in Tables 2 and 3.

[0203] L-arginine fermentation medium: the solvent is water, the solutes and their concentrations are glucose 8g / L, FM902 yeast powder 3g / L, K2HPO4•3H2O 6g / L, MgSO4•7H2O 1g / L, FeSO4•7H2O 0.05g / L, betaine 0.5g / L, VB12 0.005g / L, defoamer 0.3mL / L, ammonium sulfate 3g / L, pH 7.2.

[0204]

[0205]

[0206]

[0207]

[0208] As shown in the above fermentation results, whether the recipient strain is the high-yield L-arginine strain CGMCC NO. 25402 or the wild-type Escherichia coli W3110, the L-arginine production after the introduction of the glutamate dehydrogenase encoding gene (gdh) from Salmonella typhimurium is significantly higher than that of the recipient strain, but overexpression of the rocG gene from Bacillus subtilis and the gdh gene of Corynebacterium glutamicum has no obvious effect (Table 2 and Table 3). At the same time, the L-arginine production of the recombinant bacteria introduced with the optimized glutamate dehydrogenase encoding gene (SEQ ID NO.5) of Salmonella typhimurium is significantly higher than that of the recombinant bacteria introduced with the glutamate dehydrogenase encoding gene (SEQ ID NO.4) of wild-type Salmonella typhimurium (Table 4).

[0209] L-Glutamic Acid Fermentation Experiment

[0210] ATCC13032, CGMCC NO. 21220, ATCC13032-gdh (SA), ATCC13032-gdh (SA, optimized), YPGLU-gdh (SA), YPGLU-gdh (SA, optimized), ATCC13032-rocG, ATCC13032-gdh (GC), YPGLU-rocG, YPGLU-gdh (GC) and the fermentation medium shown below in the fermentation tank of BLBIO-5GC-4-H model (purchased from Shanghai Bailun Biotechnology Co., Ltd.) and the control process shown in Table 5 were subjected to fermentation experiments. Each strain was repeated three times. After the fermentation was completed, the L-glutamic acid content was detected by high performance liquid chromatography (HPLC), and the results were averaged over three replicates, as shown in Tables 6 and 7.

[0211] Formula of fermentation medium: glucose 5.0g / L, phosphoric acid 0.38g / L, magnesium sulfate 1.85g / L, potassium chloride 1.6g / L, biotin 550μg / L, vitamin B1 300μg / L, ferrous sulfate 10mg / L, manganese sulfate 10g / dl, KH2PO42.8g / L, vitamin C 0.75mg / L, vitamin B12 2.5μg / L, p-aminobenzoic acid 0.75mg / L, defoaming agent 0.0015mL / dL, betaine 1.5g / L, cane molasses 7mL / L, corn steep liquor 77mL / L, aspartic acid 1.7g / L, hair powder 2g / L.

[0212]

[0213]

[0214]

[0215]

[0216] The results showed that the introduction of the glutamate dehydrogenase encoding gene (gdh) from Salmonella typhimurium into Corynebacterium glutamicum ATCC13032 and CGMCC NO. 21220 could significantly increase the L-glutamate production of the recipient bacteria, but overexpression of the rocG gene from Bacillus subtilis and the gdh gene of Corynebacterium glutamicum had no significant effect (Table 6 and Table 7). At the same time, the recombinant bacteria introduced with the optimized Salmonella typhimurium (SEQ ID NO.5) had a significantly higher L-glutamate production than the recombinant bacteria introduced with the glutamate dehydrogenase encoding gene (SEQ ID NO.4) before optimization (Table 8).

[0217] 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. Application of glutamate dehydrogenase derived from Salmonella typhimurium, characterized in that: The application is the application of the glutamate dehydrogenase in increasing the L-arginine production of Escherichia coli; Or, the application is the application of the glutamate dehydrogenase in increasing the L-glutamate production of Corynebacterium glutamicum; The glutamate dehydrogenase is a protein whose amino acid sequence is SEQ ID NO.

4.

2. The use of the biomaterial related to glutamate dehydrogenase according to claim 1, characterized in that: The application is the application of the biomaterial in the following A1) or A2), A1), application of the biomaterial in increasing the yield of L-arginine in Escherichia coli; A2), use of the biological material in increasing the yield of L-glutamic acid in Corynebacterium glutamicum; The biological material is any one of the following B1) to B4): B1), a nucleic acid molecule encoding the glutamate dehydrogenase according to claim 1, B2), an expression cassette containing the nucleic acid molecule described in B1), B3), a recombinant vector containing the nucleic acid molecule described in B1); B4), a recombinant vector containing the expression cassette described in B2).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule described is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO.

5.

4. Use of a recombinant Escherichia coli containing the nucleic acid molecule described in claim 3 B1) in the preparation of L-arginine.

5. Use of a recombinant Corynebacterium glutamicum containing the nucleic acid molecule described in B1) of claim 3 in the preparation of L-glutamic acid.

6. A method for increasing the L-arginine production of a recipient Escherichia coli, characterized in that: The method comprises introducing the biological material of claim 2 into the recipient Escherichia coli.

7. A method for increasing the yield of L-glutamic acid in a recipient Corynebacterium glutamicum, characterized in that: The method comprises introducing the biological material described in claim 2 into the recipient Corynebacterium glutamicum.

8. A method for preparing L-arginine, characterized in that: The method comprises the step of preparing L-arginine using a recombinant microorganism as a fermentation strain, wherein the recombinant microorganism is a recombinant Escherichia coli containing the nucleic acid molecule described in claim 3 B1).

9. A method for preparing L-glutamic acid, characterized in that: The method comprises the step of preparing L-glutamic acid using a recombinant microorganism as a fermentation strain, wherein the recombinant microorganism is a recombinant Corynebacterium glutamicum containing the nucleic acid molecule described in claim 3 B1).

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