Use of salmonella typhimurium-derived glutamate dehydrogenase and related biomaterials thereof in amino acid synthesis

Abstract

Disclosed in the present application is the use of Salmonella typhimurium-derived glutamate dehydrogenase and related biomaterials thereof in amino acid synthesis. Provided in the present application is the use of Salmonella typhimurium-derived glutamate dehydrogenase for improving the L-amino acid yield of a microorganism. In the present application, an optimized coding gene of the Salmonella typhimurium-derived glutamate dehydrogenase is further obtained by means of codon optimization, and the introduction of the optimized coding gene into a recipient microorganism can significantly improve the L-amino acid yield of the recipient microorganism.

Description

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

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the application of glutamate dehydrogenase derived from Salmonella Typhimurium and related biomaterials in amino acid synthesis. Background Technology

[0002] Amino acids, as the basic structural components of protein macromolecules, are essential for animal life activities and play a vital role in nutrition and health fields such as feed, food, medicine, culture media, and health foods. In recent years, with increasing public awareness of health, the demand for amino acids has been continuously increasing, driving the rapid development of the amino acid market. The continuous innovation and application of new technologies such as novel and efficient mutagenesis, precise gene editing and genome engineering, synthetic biology, and high-throughput screening have brought new opportunities for significantly improving the efficiency and level of microbial breeding. These technologies make the selection and modification of strains more precise and efficient, thereby improving the yield and quality of amino acids. Metabolic controlled fermentation technology, based on dynamic biochemistry and microbial genetics, obtains mutant strains suitable for producing certain products through artificial mutagenesis. Cultivating these strains under artificially controlled conditions allows for the selective and large-scale production of products needed by consumers. The application of biotechnology has significantly improved the yield and purity of amino acids. In China, the amino acid industry is also showing a growth trend, with the market size continuously expanding. In the future, with the increasing public awareness of the importance of healthy lifestyles, the continuous expansion of amino acid application fields, and the accelerated development of various amino acid application areas, global demand for amino acids will further increase. Meanwhile, the biosynthesis, fermentation, and extraction technologies of amino acids will be further optimized, leading to broader development prospects for amino acids and their derivatives. In summary, the current state of research on amino acid microbial fermentation is characterized by continuous technological advancements, expanding production scale, wide-ranging applications, and promising industry prospects. In the future, with further technological development and market expansion, the research and application of amino acid microbial fermentation will become more in-depth and widespread.

[0003] Invention Overview

[0004] The technical problem to be solved by this 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.

[0005] To solve this technical problem, this application provides the following technical solution: heterologous overexpression of the glutamate dehydrogenase A gene derived from Salmonella Typhimurium in Escherichia coli or Corynebacterium glutamicum to increase the L-amino acid production of Escherichia coli.

[0006] This application provides the use of glutamate dehydrogenase derived from Salmonella Typhimurium, wherein the use is the application of the glutamate dehydrogenase in increasing the L-amino acid production of microorganisms.

[0007] Furthermore, the glutamate dehydrogenase may be any of the following:

[0008] a1) A protein whose amino acid sequence contains SEQ ID NO.4;

[0009] a2) A protein 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) and is related to glutamate dehydrogenase.

[0010] a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

[0011] Furthermore, a1) can also be a protein whose amino acid sequence is SEQ ID NO.4.

[0012] In this application, SEQ ID NO.4 consists of 447 amino acid residues.

[0013] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0015] Further, the connection described in a3) may be a peptide bond formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in a1) or a2). Alternatively, the connection described in a3) may be a peptide bond formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in a1) or a2).

[0016] In this application, the L-amino acid may 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.

[0017] In this application, the L-amino acid may be L-arginine or L-glutamic acid.

[0018] This application also provides the application of biomaterials related to the aforementioned glutamate dehydrogenase, said application being the application of the biomaterials in any of the following A1)-A3).

[0019] A1) The application of the biomaterial in constructing engineered bacteria that produce L-amino acids;

[0020] A2) The application of the biomaterial in the preparation of L-amino acids;

[0021] A3) The application of the biomaterial in regulating (enhancing) the production of L-amino acids in microorganisms;

[0022] The biomaterial may be at least one of the following B1)-B3):

[0023] B1) The nucleic acid molecule encoding the glutamate dehydrogenase (which may also be described as the gene encoding glutamate dehydrogenase from Salmonella typhimurium);

[0024] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0025] B3), recombinant vectors containing the nucleic acid molecules described in B1), and / or recombinant vectors containing the expression cassette described in B2).

[0026] Furthermore, in the aforementioned applications, the nucleic acid molecule described in B1) can be G1) or G2).

[0027] The G1 nucleotide sequence contains the nucleic acid molecule of SEQ ID NO. 5;

[0028] Nucleic acid molecules that have more than 80% identity with the nucleic acid molecules described in G2) and G1).

[0029] Furthermore, in the aforementioned applications, the nucleic acid molecule described in G1) may be a DNA molecule whose nucleotide sequence is SEQ ID NO.5.

[0030] Furthermore, in the aforementioned applications, the nucleic acid molecule described in G2) may be a nucleic acid molecule whose nucleotide sequence is SEQ ID NO.3.

[0031] In some embodiments, the nucleic acid molecule B1) can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0032] In some specific embodiments, the nucleic acid molecule B1) is the cDNA gene (cDNA molecule) of the glutamate dehydrogenase. This cDNA gene includes the coding sequence (CDS) of the glutamate dehydrogenase. In one specific embodiment, the coding sequence is SEQ ID NO: 5.

[0033] The aforementioned biological materials are also protected under this application.

[0034] Furthermore, in the biological material, the nucleotide sequence of the nucleic acid molecule described in G1) may be SEQ ID NO.5.

[0035] Furthermore, in the biological material described above, the nucleic acid molecule described in G2 is not a nucleic acid molecule whose nucleotide sequence is SEQ ID NO.3.

[0036] This application also provides recombinant microorganisms, which may be any of the following:

[0037] C1) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0038] C2) Recombinant microorganisms containing the expression cassette described in B2);

[0039] C3) Recombinant microorganisms containing the recombinant vector described in B3);

[0040] C4) Recombinant microorganisms containing the glutamate dehydrogenase.

[0041] This 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:

[0042] D1) Escherichia coli bacteria;

[0043] D2), Escherichia coli;

[0044] D3), ​​Corynebacterium spp.;

[0045] D4), Corynebacterium glutamicum.

[0046] Furthermore, compared to the recipient microorganism, the recombinant microorganism contains a glutamate dehydrogenase encoding gene derived from Salmonella typhimurium.

[0047] Furthermore, the glutamate dehydrogenase encoding gene from Salmonella typhimurium is a nucleic acid molecule whose nucleotide sequence includes SEQ ID NO.5.

[0048] Furthermore, compared to the recipient microorganism, the recombinant microorganism exhibits enhanced glutamate dehydrogenase activity.

[0049] Furthermore, compared to the recipient microorganism, the recombinant microorganism has an increased content of glutamate dehydrogenase.

[0050] This application also provides a composition containing the above-described recombinant microorganisms.

[0051] The active ingredient in the above composition may be the recombinant microorganism and / or its metabolites and / or its culture.

[0052] The culture can 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).

[0053] In the above text, 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, collecting the remaining components of the fermentation broth, and obtaining the metabolite of the recombinant microorganism.

[0054] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.

[0055] The above composition can be the culture described above. The above composition can also be a microbial agent.

[0056] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0057] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0058] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0059] This application also provides the use of a substance in the preparation of L-amino acids, said substance being the above-described recombinant microorganism, the recombinant microorganism prepared by the above-described method, or the above-described combination.

[0060] Furthermore, the recombinant microorganism may be recombinant Escherichia coli or recombinant Corynebacterium glutamicum.

[0061] Furthermore, in the aforementioned application, the recombinant microorganism may be recombinant Escherichia coli, and the L-amino acid may be L-arginine.

[0062] Furthermore, in the aforementioned applications, the recombinant microorganism may also be recombinant Corynebacterium glutamicum, and the L-amino acid may be L-glutamic acid.

[0063] This application also provides a method for increasing the L-amino acid production of recipient microorganisms, the method comprising introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into the recipient microorganisms to increase the L-amino acid production of the recipient microorganisms.

[0064] In this application, the recipient microorganism may be a microorganism that produces the L-amino acid.

[0065] Furthermore, the recipient microorganism may be a prokaryotic microorganism.

[0066] Furthermore, the recipient microorganism may be bacteria.

[0067] Furthermore, the recipient microorganism may be a Gram-negative bacterium;

[0068] Furthermore, the recipient microorganism may be a bacterium of the genus Escherichia;

[0069] Furthermore, the recipient microorganism may be Escherichia coli.

[0070] Furthermore, the recipient bacterium, *Escherichia coli*, can be *Escherichia coli* that produces the L-amino acid.

[0071] Furthermore, the *E. coli* contains the ynjI site.

[0072] Among the recombinant microorganisms mentioned above, the recombinant microorganisms obtained by using Escherichia coli as the recipient bacteria are 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.

[0073] Furthermore, the recipient Escherichia coli may be Escherichia coli CGMCC NO.25402 and / or Escherichia coli W3110.

[0074] The Escherichia coli CGMCC NO.25402 can be the Escherichia coli strain with the accession number CGMCC No.25402 in the patent procedure.

[0075] Furthermore, the recipient microorganism may also be a Gram-positive bacterium.

[0076] Furthermore, the recipient microorganism may be a bacterium of the genus Corynebacterium.

[0077] Furthermore, the recipient microorganism may be Corynebacterium glutamicum.

[0078] Furthermore, the recipient bacterium, *Corynebacterium glutamicum*, can be a *Corynebacterium glutamicum* that produces the L-amino acid.

[0079] Furthermore, the *Corynebacterium glutamicum* contains the poxB site.

[0080] 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.

[0081] Furthermore, the recipient Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC NO.21220 and / or Corynebacterium glutamicum ATCC13032.

[0082] The *Corynebacterium glutamicum* strain CGMCC NO.21220 is a *Corynebacterium glutamicum* strain with accession number CGMCC No.21220 that has been deposited for patent procedures.

[0083] The Corynebacterium glutamicum ATCC13032 is the Corynebacterium glutamicum with ATCC number 1303290.

[0084] This application also provides a method for preparing L-amino acids, the method including the step of preparing L-amino acids using recombinant microorganisms as fermentation strains, wherein the recombinant microorganisms may be the recombinant microorganisms described above or the recombinant microorganisms prepared by the above method.

[0085] Furthermore, the method includes the steps of culturing the recombinant microorganism in a culture medium, collecting the culture product, and obtaining L-amino acids.

[0086] The term "culture product" refers to the collective name for liquid or solid products (all substances within the culture container) that have grown a microbial community after artificial inoculation and cultivation. In other words, it is the product obtained through the growth and / or amplification of microorganisms. It can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. It can also be a mixture containing a certain amount of culture medium, microbial cell metabolites, and with the microbial cells removed.

[0087] In this application, "Escherichia coli" and "Escherichia coli" are used interchangeably.

[0088] In this application, "Corynebacterium glutamicum" and "Corynebacterium glutamicum" are used interchangeably.

[0089] In some embodiments of this application, the recombinant microorganism may be recombinant Escherichia coli, which may be used as a fermentation strain to prepare L-arginine.

[0090] In some embodiments of this application, the recombinant microorganism may be recombinant Corynebacterium glutamicum, which may be used as a fermentation strain to prepare L-glutamic acid.

[0091] Preservation instructions for Escherichia coli strain with accession number CGMCC No. 25402

[0092] Classification and nomenclature: Escherichia coli

[0093] Strain number: YP004-8

[0094] Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0095] Abbreviation of depositary institution: CGMCC

[0096] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101

[0097] Date of preservation: July 25, 2022

[0098] CGMCC Registration Number: CGMCC No. 25402

[0099] Depositor: Ningxia Eppen Biotechnology Co., Ltd.

[0100] Address of the depositor: Yanghe Industrial Park, Yongning County, Yinchuan City, Ningxia Hui Autonomous Region

[0101] Ningxia Yipin Biotechnology Co., Ltd., the depositor of Escherichia coli YP004-8, has authorized Inner Mongolia Yipin Biotechnology Co., Ltd. to use this strain.

[0102] Preservation instructions for Corynebacterium glutamicum strain with accession number CGMCC No. 21220

[0103] Classification and nomenclature: Corynebacterium glutamicum

[0104] Strain number: YPGLU001

[0105] Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0106] Abbreviation of depositary institution: CGMCC

[0107] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101

[0108] Date of preservation: November 23, 2020

[0109] CGMCC Registration Number: CGMCC No. 21220

[0110] Depositor: Ningxia Eppen Biotechnology Co., Ltd.

[0111] Address of the depositor: Yanghe Industrial Park, Yongning County, Yinchuan City, Ningxia Hui Autonomous Region

[0112] Ningxia Yipin Biotechnology Co., Ltd., the depositor of Corynebacterium glutamicum YPGLU001, has authorized Inner Mongolia Yipin Biotechnology Co., Ltd. to use this strain.

[0113] Implementation methods of this application

[0114] I. Terminology in this application:

[0115] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0116] For ease of understanding this application, several terms and abbreviations used herein are defined as follows:

[0117] In this application, "identity" refers to the identity of an amino acid sequence or nucleotide sequence. The identity of an amino acid sequence (or nucleotide sequence) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0118] Specifically, the 70% or more similarity can be 75% or more similarity. Specifically, the 75% or more similarity can be 80% or more similarity. Specifically, the 80% or more similarity can be 85% or more similarity. Specifically, the 85% or more similarity can be 90% or more similarity. Specifically, the 90% or more similarity can be 91% or more similarity, 92% or more similarity, 93% or more similarity, 94% or more similarity, 95% or more similarity, 96% or more similarity, 97% or more similarity, 98% or more similarity, or 99% or more similarity. More specifically, the 70% or more identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0119] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. II. Implementation Examples

[0120] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0121] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0122] The recipient bacteria in the following examples are of four types, as follows:

[0123] Escherichia coli W3110, or simply E. coli W3110 or W3110, was purchased from the American Type Culture Collection (ATCC), strain number 27325;

[0124] Escherichia coli CGMCC NO.25402, also known as Escherichia coli CGMCC NO.25402 L-arginine high-yield strain CGMCC NO.25402 or CGMCC NO.25402, is a strain that has been deposited under a patent procedure, with the accession number CGMCC NO.25402.

[0125] Corynebacterium glutamicum ATCC13032, also known as ATCC13032, was purchased from the American Type Culture Collection (ATCC), with strain number 13032;

[0126] Corynebacterium glutamicum CGMCC No. 21220, also known as Corynebacterium glutamicum CGMCC No. 21220, high glutamate-producing bacteria CGMCC NO. 21220, or CGMCC NO. 21220, is a strain that has been deposited through a patent procedure with the accession number CGMCC NO. 21220.

[0127] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0128] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The t-test is used, and different letters indicate significant differences.

[0129] Example 1: Construction of recombinant Escherichia coli overexpressing glutamate dehydrogenase derived from Salmonella Typhimurium.

[0130] Based on the genome sequence of Escherichia coli W3110 (Gene ID: 998010) published by NCBI, the gdhA gene (abbreviated as gdhA(SA)) from Salmonella Typhimurium was overexpressed in the genomes of W3110 and the L-arginine high-yielding strain CGMCC NO.25402 using CRISPR / Cas9 gene editing technology, in order to further investigate the effect of the gdhA(SA) gene on L-arginine production.

[0131] The predicted inner membrane protein encoding gene (ynjI, db_xref="GI:85675112") of wild-type Escherichia coli W3110 was inserted with gdhA (abbreviated as gdhA(EC), SEQ ID NO.1), gdhA (SEQ ID NO.3) from Salmonella typhimurium, gdhA (SEQ ID NO.5) from Salmonella typhimurium after codon optimization, rocG (SEQ ID NO.25) from Bacillus subtilis, and gdh (SEQ ID NO.27) from Corynebacterium glutamicum ATCC13032.

[0132] The DNA molecule shown in SEQ ID NO.1 encodes a protein with the amino acid sequence of SEQ ID NO.2, and the protein is named *Escherichia coli* wild-type strain W3110 glutamate dehydrogenase. The DNA molecule shown in SEQ ID NO.3 encodes a protein with the amino acid sequence of SEQ ID NO.4, and the protein is named *Salmonella typhimurium* wild-type strain SL7207 glutamate dehydrogenase. SEQ ID NO.5 is a codon-optimized nucleotide sequence of SEQ ID NO.3, but the amino acid sequence of the protein it encodes remains SEQ ID NO.4. The DNA molecule shown in SEQ ID NO.25 encodes a protein with the amino acid sequence of SEQ ID NO.26, and the protein is named *Bacillus subtilis* strain subtilis str.168 (CP053102.1) glutamate dehydrogenase. The DNA molecule shown in SEQ ID NO.27 encodes a protein with the amino acid sequence of SEQ ID NO.28, and the protein is named *Corynebacterium glutamicum* ATCC13032 glutamate dehydrogenase.

[0133] I. Construction of sgRNA

[0134] Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting a suitable sgRNA target sequence, linearized pGRB cloning vector terminal sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0135] Amplifying the sgRNA fragment requires no template; only a PCR annealing process is needed. The system and procedure are as follows: PCR reaction system: sgRNA-1F 10 μL, sgRNA-1R 10 μL; PCR reaction procedure: denaturation at 95℃ for 5 min, annealing at 50℃ for 1 min. After annealing, the target fragment is recovered using a DNA purification kit, its DNA concentration is determined, and the concentration is diluted to 100 ng / μL.

[0136] pGRB plasmid (Addgene product, catalog number: 71539) was extracted and digested with Spe I and dephosphorylated to prevent self-ligation. The digestion system consisted of 5 μL 10×Buffer, 2.5 μL Spe I, 3000-5000 ng pGRB plasmid DNA, and ddH2O to a final volume of 50 μL. After digestion at 37°C for 3 h, the DNA was recovered by agarose gel electrophoresis and then dephosphorylated. The dephosphorylation system consisted of 5 μL 10×Buffer, 1000-2000 ng linearized pGRB plasmid DNA, 2.5 μL CIAP, and ddH2O to a final volume of 50 μL. After treatment at 37°C for 1 h, the linearized pGRB plasmid was recovered using a DNA purification kit. Recombination of sgRNA and pGRB plasmid was then performed using a Gibson Assembly kit (New England Biosciences). Recombinant system: 2.5 μL NEB assembly enzyme, 2 μL linearized cloning vector, and 0.5 μL sgRNA. After assembly at 50℃ for 30 min, the product was transformed into DH5α competent cells, plasmid was extracted, and identified by sequencing using sgRNA-PF and sgRNA-PR primers. The constructed plasmid was named pGRB-sgRNA-1 plasmid.

[0137] The primers used in this experiment were designed as follows (synthesized by Invitrogen Shanghai). The uppercase letters of sgRNA-1F and sgRNA-1R represent the homologous arm sequences of the pGRB cloning vector, and the lowercase letters represent the target sequences of the sgRNA.

[0138] sgRNA-1F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTctttaaaggtattggctccaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID NO. 54);

[0139] sgRNA-1R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACtggagccaatacctttaaagACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID NO. 55);

[0140] sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID NO.56);

[0141] sgRNA-PR: 5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID NO. 57).

[0142] II. Preparation of the integration fragment overexpressing the gdhA gene

[0143] The gdhA(EC) overexpression integration homologous arm DNA fragment Up-pBJ23119-gdhA(EC)-Down (SEQ ID NO.6, 2565bp) was prepared. In SEQ ID NO.6, positions 1-544 represent ΔynjI-UP, positions 545-621 represent the pBJ23119 promoter, positions 622-1965 represent gdhA(EC), and positions 1966-2565 represent ΔynjI-DOWN.

[0144] A 2565bp DNA fragment, Up-pBJ23119-gdhA(SA)-Down (SEQ ID NO.7), was prepared to integrate the gdhA(SA) overexpression homologous arm. In SEQ ID NO.7, positions 1-544 represent the UP fragment, positions 545-621 represent the PBJ23119 promoter, positions 622-1965 represent gdhA(SA), and positions 1966-2565 represent the Down fragment.

[0145] The gdhA(SA, optimized) overexpression integration homologous arm DNA fragment Up-pBJ23119-gdhA(SA, optimized)-Down (SEQ ID NO.8) 2565bp was prepared. In SEQ ID NO.8, positions 1-544 are the UP fragment, positions 545-621 are pBJ23119, positions 622-1965 are gdhA(SA, optimized), and positions 1966-2565 are the DOWN fragment.

[0146] The rocG (Bacillus subtilis, BS) overexpression integration homologous arm DNA fragment Up-pBJ23119-rocG-Down (SEQ ID NO. 29, 2496bp) was prepared. In SEQ ID NO. 29, positions 1-544 are the UP fragment, positions 545-621 are the pBJ23119 promoter, positions 622-1896 are rocG (Bacillus subtilis), and positions 1897-2496 are the DOWN fragment.

[0147] A 2565bp DNA fragment, Up-pBJ23119-gdhA(CG)-Down (SEQ ID NO.30), was prepared and integrated with the homologous arm of gdh (Corynebacterium glutamicum, CG) overexpression. In SEQ ID NO.30, positions 1-544 represent ΔynjI(gdhA)-UP, positions 545-621 represent pBJ23119, positions 622-1965 represent gdhA(CG), and positions 1966-2565 represent ΔynjI(gdhA)-DOWN.

[0148] III. Preparation and Transformation of Competent Behaviors

[0149] The pREDCas9 plasmid (Addgene product, catalog number #71541) was electroporated into E. coli electroporation competent cells. Note: The pREDCas9 plasmid is temperature-sensitive and will be lost at excessively high temperatures; therefore, strains containing pREDCas9 were cultured at 32°C. The revived cells after electroporation were plated onto LB agar plates containing zizomycin and incubated overnight at 32°C. Single colonies growing on antibiotic-resistant plates are considered positive recombinants.

[0150] W3110-Cas9 is a recombinant bacterium obtained by introducing the pREDCas9 plasmid into Escherichia coli W3110.

[0151] L-arginine producing strain CGMCC NO.25402-Cas9 is a recombinant strain obtained by introducing the pREDCas9 plasmid into the Escherichia coli strain with accession number CGMCC NO.25402.

[0152] W3110-Cas9, L-arginine CGMCC NO.25402-Cas9 competent cells were prepared, and when the cells grew to OD... 600 =0.1mM IPTG was added to a final concentration of 0.1mM to induce λ-Red-mediated homologous recombination. Culture continued until OD... 600 When the concentration of the bacterial culture medium was 0.6, bacterial cells were collected and each type of bacterial cell was divided into five groups and transformed into 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 transformed cells were then plated onto 2-YT plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C. Single colonies produced by culture were identified by rTaq PCR using primers P7 and P8, P7 and P9, P7 and P10, P7 and P42, and P7 and P46, respectively. Positive transformants were those that amplified fragments of 1641bp (SEQ ID NO.9), 1281bp (SEQ ID NO.10), 1281bp (SEQ ID NO.11), 1559bp (SEQ ID NO.31), and 1619bp (SEQ ID NO.32), respectively. The original bacteria were those that could not amplify any fragments.

[0153] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and arabinose to a final concentration of 0.2% to eliminate the pGRB-sgRNA-1 plasmid. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected. These colonies were then transferred to 2-YT medium and incubated at 42°C to eliminate the pREDCas9 plasmid. Colonies that grew on antibiotic-free 2-YT medium but not on spectinomycin (100 mg / L) were selected. rTaq PCR was used to identify the plasmids using primers P11 and P12, P13 and P14, P15 and P16, P43 and P44, and P47 and P48. PCR amplification yielded plasmids of 1884 bp (SEQ ID NO. 12), 1644 bp (SEQ ID NO. 13), 1824 bp (SEQ ID NO. 14), and 1190 bp (SEQ ID NO. 15). NO.33 and 1089bp (SEQ ID NO.34) are positive transformants that have eliminated the pREDCas9 plasmid, while those that cannot amplify the fragment are the original bacteria.

[0154] The primer sequences mentioned above are as follows (synthesized by Invitrogen Shanghai):

[0155] P7: 5'-GCACTGTAGTTGATGAAAGC-3' (SEQ ID NO. 15),

[0156] P8: 5'-AAATTCTTTGGCGTAATCTG-3' (SEQ ID NO. 16),

[0157] P9: 5'-CGCCATAAAGCCCACTTCGC-3' (SEQ ID NO. 17),

[0158] P10: 5'-AGCCATGAAACCCACTTCGC-3' (SEQ ID NO. 18),

[0159] P11: 5'-CGTGTGCAGTTCAGCTCTGC-3' (SEQ ID NO. 19),

[0160] P12: 5'-AAGCGCGTTTTCTTGCTGAA-3' (SEQ ID NO. 35),

[0161] P13: 5'-GGAATGATGCGCAAGCTTTC-3' (SEQ ID NO.36),

[0162] P14:5’-ATGCGGTGTTCCGCAAGTGT-3’(SEQ ID NO.45),

[0163] P15:5’-ACCCTGCCGATGGGCGGCGG-3’(SEQ ID NO.46),

[0164] P16:5’-ATGCGGTGTTCCGCAAGTGT-3’(SEQ ID NO.47),

[0165] P42:5’-TCCCGTTTATCGAGCAAATAAG-3’(SEQ ID NO.48),

[0166] P43:5’-CAGCTTTGGTATGGTCACC-3’(SEQ ID NO.49),

[0167] P44:5’-AGGCGATGAGTTGCTGTGTG-3’(SEQ ID NO.50),

[0168] P46:5’-ACGGATACGCGTGCGCGAC-3’(SEQ ID NO.51),

[0169] P47:5’-GTGCGATATCGCTCTTCCTT-3’(SEQ ID NO.52),

[0170] P48:5’-AGGCGATGAGTTGCTGTGTG-3’(SEQ ID NO.53)。

[0171] Wild-type Escherichia coli strains W3110, which integrated pBJ23119-gdhA(EC), pBJ23119-gdhA(SA), pBJ23119-gdhA(SA, optimized), pBJ23119-rcoG(BS), and pBJ23119-gdh(CG) 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 strains CGMCC NO.25402, which integrate pBJ23119-gdhA(EC), pBJ23119-gdhA(SA), pBJ23119-gdhA(SA, optimized), pBJ23119-rcoG(BS), and pBJ23119-gdh(CG) into their 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.

[0172] Recombinant strains W3110 / pBJ23119-gdhA(EC) and YPR / pBJ23119-gdhA(EC) contain two copies of the gdhA(EC) gene shown in SEQ ID NO.1; recombinant strains W3110 / pBJ23119-gdhA(SA) and YPR / pBJ23119-gdhA(SA) each contain one copy of the gdhA(EC) gene and one copy of the gdhA(SA) gene; recombinant strains W3110 / pBJ23119-gdhA(SA, optimized) and YPR / pBJ23119-gdhA(SA, optimized) each contain one copy of the gdhA(EC) gene and one copy of the gdhA(EC) gene. The recombinant strains W3110 / pBJ23119-rcoG(BS) and YPR / pBJ23119-rcoG(BS) each contain one copy of the gdhA(EC) gene and one copy of the rcoG(BS) gene; the recombinant strains W3110 / pBJ23119-gdh(CG) and YPR / pBJ23119-gdh(CG) each contain one copy of the gdhA(EC) gene and one copy of the gdh(CG) gene.

[0173] Compared to L-arginine-producing bacterium CGMCC NO.25402, YPR / pBJ23119-gdhA(SA, optimized) is a recombinant bacterium obtained by modifying L-arginine-producing bacterium CGMCC NO.25402 as follows: An expression cassette for the glutamate dehydrogenase encoding gene (gdhA(SA, optimized)) was introduced into the ynjI site of L-arginine-producing bacterium CGMCC NO.25402. Compared to wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA(SA, optimized) is a recombinant bacterium obtained by modifying wild-type Escherichia coli W3110 as follows: An expression cassette for the glutamate dehydrogenase encoding gene (gdhA(SA, optimized)) was introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the above-mentioned glutamate dehydrogenase encoding gene (gdhA(SA, optimized)) expression cassette is positions 545-1965 of SEQ ID NO.8. Wherein, SEQ ID... NO.8, bits 545-621 are pBJ23119, and bits 622-1965 are gdhA(SA, optimized).

[0174] Compared to L-arginine-producing bacterium CGMCC NO.25402, YPR / pBJ23119-gdhA(SA) is a recombinant bacterium obtained by modifying L-arginine-producing bacterium CGMCC NO.25402 as follows: An expression cassette for the glutamate dehydrogenase encoding gene gdhA(SA) was introduced into the ynjI site of L-arginine-producing bacterium CGMCC NO.25402. Compared to wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA(SA) is a recombinant bacterium obtained by modifying wild-type Escherichia coli W3110 as follows: An expression cassette for the glutamate dehydrogenase encoding gene gdhA(SA) was introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the above-mentioned expression cassette for the glutamate dehydrogenase encoding gene gdhA(SA) is positions 545-1965 of SEQ ID NO.7. Among them, the 545th to 621st positions of SEQ ID NO.7 are pBJ23119, and the 622nd to 1965th positions are gdhA(SA).

[0175] Compared to L-arginine-producing bacterium CGMCC NO.25402, YPR / pBJ23119-gdhA(EC) is a recombinant bacterium obtained by modifying L-arginine-producing bacterium CGMCC NO.25402 as follows: An expression cassette for the glutamate dehydrogenase encoding gene gdhA(EC) was introduced into the ynjI site of L-arginine-producing bacterium CGMCC NO.25402. Compared to wild-type Escherichia coli W3110, W3110 / pBJ23119-gdhA(EC) is a recombinant bacterium obtained by modifying wild-type Escherichia coli W3110 as follows: An expression cassette for the glutamate dehydrogenase encoding gene gdhA(EC) was introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the above-mentioned expression cassette for the glutamate dehydrogenase encoding gene gdhA(EC) is positions 545-1965 of SEQ ID NO.6. In SEQ ID NO.6, bits 545-621 are the pBJ23119 promoter, and bits 622-1965 are gdhA(EC).

[0176] Compared to L-arginine-producing bacterium CGMCC NO.25402, YPR / pBJ23119-rocG(BS) is a recombinant bacterium obtained by modifying L-arginine-producing bacterium CGMCC NO.25402 as follows: An expression cassette for the glutamate dehydrogenase encoding gene rocG(BS) was introduced into the ynjI site of L-arginine-producing bacterium CGMCC NO.25402. Compared to wild-type Escherichia coli W3110, W3110 / pBJ23119-rocG(BS) is a recombinant bacterium obtained by modifying wild-type Escherichia coli W3110 as follows: An expression cassette for the glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) was introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the above-mentioned glutamate dehydrogenase encoding gene rocG(BS) expression cassette is positions 545-1896 of SEQ ID NO.29. In SEQ ID NO.29, bits 545-621 are the pBJ23119 promoter, and bits 622-1896 are rocG(BS).

[0177] Compared to L-arginine-producing bacterium CGMCC NO.25402, YPR / pBJ23119-gdh (Corynebacterium glutamicum, CG) is a recombinant bacterium obtained by modifying L-arginine-producing bacterium CGMCC NO.25402 as follows: an expression cassette for the glutamate dehydrogenase encoding gene gdh(CG) was introduced into the ynjI site of L-arginine-producing bacterium CGMCC NO.25402. Compared to wild-type Escherichia coli W3110, W3110 / pBJ23119-gdh(CG) is a recombinant bacterium obtained by modifying wild-type Escherichia coli W3110 as follows: an expression cassette for the glutamate dehydrogenase encoding gene gdh(CG) was introduced into the ynjI site of wild-type Escherichia coli W3110. The nucleotide sequence of the above-mentioned expression cassette for the glutamate dehydrogenase encoding gene gdh(CG) is positions 545-1965 of SEQ ID NO.30. In SEQ ID NO.30, bits 545-621 are the pBJ23119 promoter, and bits 622-1965 are gdh(CG).

[0178] Example 2: Construction of recombinant Corynebacterium glutamicum containing Salmonella typhimurium glutamate dehydrogenase activity.

[0179] In this embodiment, the poxB gene was knocked out in the ATCC13032 and glutamate-producing strain CGMCC NO.21220, while simultaneously inserting a glutamate dehydrogenase gene via homologous recombination. The GenBank accession number for the pyruvate dehydrogenase gene is CAF21272.1 (BCT 27-FEB-2015), and this enzyme converts pyruvate to acetyl-CoA and CO2.

[0180] I. Preparation of an integrated fragment containing upstream and downstream homologous arms and the target gene

[0181] The integrated fragments containing upstream and downstream homologous arms and the target gene were prepared as follows:

[0182] The gdhA(SA) overexpression integration fragment Up-pBJ23119-gdhA(SA)-Down (SEQ ID NO.41, 2715bp) was prepared. In SEQ ID NO.41, positions 1-694bp represent the poxB-UP fragment, positions 695-771bp represent the PBJ23119 promoter, positions 772-2115 represent gdhA(SA), and positions 2116-2715 represent the poxB-Down fragment.

[0183] The gdhA(SA, optimized) overexpression integration fragment Up-pBJ23119-gdhA(SA, optimized)-Down (SEQ ID NO.42, 2715bp) was prepared. In SEQ ID NO.42, positions 1-694bp represent the poxB-UP fragment, positions 695-771bp represent pBJ23119, positions 772-2115 represent gdhA(SA, optimized), and positions 2116-2715 represent the poxB-Down fragment.

[0184] The rocG (Bacillus subtilis) overexpression integration fragment Up-pBJ23119-rocG(BS)-Down (SEQ ID NO.43, 2647bp) was prepared. In SEQ ID NO.43, positions 1-694bp are the poxB-UP fragment, positions 695-771 are the pBJ23119 promoter, positions 772-2046 are the rocG (Bacillus subtilis) fragment, and positions 2047-2647 are the poxB-Down fragment.

[0185] The gdh (Corynebacterium glutamicum, CG) overexpression integration fragment Up-pBJ23119-gdh(CG)-Down (SEQ ID NO.44, 2715bp) was prepared. In SEQ ID NO.44, positions 1-694 represent poxB-UP, positions 695-771bp represent pBJ23119, positions 772-2115 represent gdh(CG), and positions 2116-2715 represent poxB-Down.

[0186] II. Preparation of recombinant vectors and transformation of recipient strains

[0187] Integrate the fragments with those processed by Xbal I / BamH The pK18mobsacB plasmid (Addgene product, containing kanamycin resistance as a selection marker), purified after I enzyme digestion, was ligated with NEBuilder enzyme (NEB product) at 50℃ for 30 min. The ligation product was transformed into E. coli DH5α, and the resulting single clones were identified by PCR using M13 primers (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain positive integration plasmids, named pK18-pBJ23119-gdh(SA), pK18-pBJ23119-gdh(SA, optimized), pK18-pBJ23119-rocG(BS), and pK18-pBJ23119-gdh(CG). These positive integration plasmids contain a kanamycin resistance marker, and recombinants integrated into the genome can be obtained through kanamycin screening.

[0188] The correctly sequenced integration plasmids pK18-pBJ23119-gdh(SA), pK18-pBJ23119-gdh(SA, optimized), pK18-pBJ23119-rocG(BS), and pK18-pBJ23119-gdh(CG) were electroporated into Corynebacterium glutamicum ATCC13032 and CGMCC NO.21220, respectively. They were cultured in a 100 μg / mL kanamycin medium with the following composition: 150 g / L sucrose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L urea, 2.5 g / L sodium chloride, 20 g / L agar powder, and the remainder water, pH 7.0. Culture conditions: 32℃. Single colonies generated from the culture were identified by PCR using primers P25 and P26, P29 and P30, P57 and P58, and P61 and P62, respectively. Colonies amplified by P25 and P26 with a size of 1190 bp (SEQ ID NO. 20) were identified as pBJ23119-gdh(SA) integration positive strains; colonies amplified by P29 and P30 with a size of 1371 bp (SEQ ID NO. 21) were identified as pBJ23119-gdh(SA, optimized) integration positive strains; colonies amplified by P57 and P58 with a size of 771 bp (SEQ ID NO. 37) were identified as pBJ23119-rocG(BS) integration positive strains; and colonies amplified by P61 and P62 with a size of 1290 bp (SEQ ID NO. 38) were identified as pBJ23119-gdh(GC) integration positive strains. Colonies that could not be amplified were considered the original strains.

[0189] Positive strains were cultured on a medium containing 150 g / L sucrose (the medium consisted of 150 g / L sucrose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L urea, 2.5 g / L sodium chloride, 20 g / L agar powder, with the remainder being water, pH 7.0. Its purpose was to eliminate PK18 plasmids integrated into the genome). Single colonies 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).

[0190] Primer pairs P27 and P28 amplified strains with a size of 1524 bp (identifying pBJ23119-gdh(SA) integration, SEQ ID NO.22), which were positive strains of pBJ23119-gdh(SA) integration into the poxB site of the Corynebacterium glutamicum genome ATCC13032 or CGMCC 21220, and named them ATCC13032-gdh(SA) and YPGLU-gdhA(SA), respectively.

[0191] Primer pairs P31 and P32 amplified strains with a size of 1704 bp (identifying pBJ23119-gdh(SA, optimized) integration, SEQ ID NO.23), which were positive strains of pBJ23119-gdh(SA, optimized) integration into the poxB site of the genome of Corynebacterium glutamicum ATCC13032 or CGMCC NO.21220, and named them ATCC13032-gdh(SA, optimized) and YPGLU-gdhA(SA, optimized), respectively.

[0192] The primer pairs P59 and P60 amplified strains with a size of 1237 bp (identifying pBJ23119-rocG(BS) integration, SEQ ID NO.39), which were positive strains of pBJ23119-rocG(BS) integration into the poxB site of the Corynebacterium glutamicum genome ATCC13032 or CGMCC 21220, and named them ATCC13032-rocG(BS) and YPGLU-rocG(BS), respectively.

[0193] Primer pairs P63 and P64 amplified strains with a size of 939 bp (identifying pBJ23119-gdh(GC) integration, SEQ ID NO.40), which were positive strains of pBJ23119-gdh(GC) integration into the poxB site of the Corynebacterium glutamicum genome ATCC13032 or CGMCC 21220, and named them ATCC13032-gdh(GC) and YPGLU-gdh(GC), respectively.

[0194] The primer sequences mentioned above are as follows (synthesized by Invitrogen Shanghai):

[0195] P25: 5'-GAACAAAACCATTGCCATCA-3' (SEQ ID NO.58),

[0196] P26: 5'-GAAGGATGGAAGCGCATAC-3' (SEQ ID NO.59),

[0197] P27: 5'-GCGATGCTTAAACGTCATGG-3' (SEQ ID NO. 60),

[0198] P28: 5'-GTTGCAGGCATCATCGTTGC-3' (SEQ ID NO.61),

[0199] P29: 5'-AGGCATGGTTGGGCACGATG-3' (SEQ ID NO.62),

[0200] P30: 5'-GGTCAGCGCGTTTTTGAAGG-3' (SEQ ID NO.63),

[0201] P31: 5'-GGTGAAGTTATGCGTTTCTG-3' (SEQ ID NO. 64),

[0202] P32: 5'-CACGCAGTTGGAGCAAACGC-3' (SEQ ID NO. 65).

[0203] P57: 5'-CAAAATGATCCGACGATCTG-3' (SEQ ID NO. 66),

[0204] P58:5'-AGGACTGAGCTAGCTGTCA-3' (SEQ ID NO.67),

[0205] P59: 5'-TAGCTTCCTGGCCAAATTCA-3' (SEQ ID NO. 68),

[0206] P60: 5'-GCCATGAGCAGGGGATAT-3' (SEQ ID NO.69),

[0207] P61: 5'-CCAAAATGATCCCGACGATCT-3' (SEQ ID NO.70),

[0208] P62: 5'-TAGCTCGGTCATGAAGGACT-3' (SEQ ID NO.71),

[0209] P63: 5'-GTTGAGGTCTTCCGTGAGCG-3' (SEQ ID NO.72),

[0210] P64: 5'-ATGAGCAGGGGATATGCGTT-3' (SEQ ID NO. 24).

[0211] Both recombinant strains ATCC13032-gdhA(SA) and YPGLU-gdhA(SA) contain one copy of the gdhA gene from *Salmonella Typhimurium* as shown in SEQ ID NO. 3. Specifically, ATCC13032-gdhA(SA) and YPGLU-gdhA(SA) are recombinant strains obtained by knocking out the coding region of the poxB gene in the genomes of *Corynebacterium glutamicum* ATCCC13032 and CGMCC NO. 21220, respectively, and simultaneously introducing an expression cassette of the glutamate dehydrogenase-encoding gene gdhA(SA) at the poxB site, while keeping other nucleotides in the genomes of *Corynebacterium glutamicum* ATCC13032 and CGMCC NO. 21220 unchanged. The nucleotide sequence of the above-mentioned glutamate dehydrogenase-encoding gene gdhA(SA) expression cassette is positions 545-1965 of SEQ ID NO. 7. Among them, the 545th to 621st positions of SEQ ID NO.7 are pBJ23119, and the 622nd to 1965th positions are gdhA(SA).

[0212] Both recombinant strains ATCC13032-gdh(SA, optimized) and YPGLU-gdh(SA, optimized) contain one copy of the codon-optimized gdhA gene derived from Salmonella Typhimurium, as shown in SEQ ID NO.5. Specifically, the recombinant strains ATCC13032-gdh(SA, optimized) and YPGLU-gdh(SA, optimized) were obtained by knocking out the coding region of the poxB gene in the genomes of Corynebacterium glutamicum ATCCC13032 and CGMCC NO.21220, respectively, and simultaneously introducing the expression cassette of the glutamate dehydrogenase encoding gene (gdhA(SA, optimized)) at the poxB site, while keeping other nucleotides in the genomes of Corynebacterium glutamicum ATCCC13032 and CGMCC NO.21220 unchanged. The nucleotide sequence of the above-mentioned glutamate dehydrogenase encoding gene (gdhA(SA, optimized)) expression cassette is positions 545-1965 of SEQ ID NO.8. Among them, positions 545-621 of SEQ ID NO.8 are pBJ23119, and positions 622-1965 are gdhA(SA, optimized).

[0213] Both recombinant strains ATCC13032-rocG(BS) and YPGLU-rocG(BS) contain one copy of the rocG gene from Bacillus subtilis. Specifically, ATCC13032-rocG(BS) and YPGLU-rocG(BS) are recombinant strains obtained by knocking out the coding region of the poxB gene in the genomes of Corynebacterium glutamicum ATCCC13032 and CGMCC NO.21220, respectively, and simultaneously introducing the expression cassette of the glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) at the poxB site, while keeping other nucleotides in the genomes of Corynebacterium glutamicum ATCCC13032 and CGMCC NO.21220 unchanged. The nucleotide sequence of the above-mentioned glutamate dehydrogenase encoding gene rocG (Bacillus subtilis) expression cassette is positions 545-1896 of SEQ ID NO.29. In SEQ ID NO.29, positions 545-621 are the pBJ23119 promoter, and positions 622-1896 are rocG (Bacillus subtilis).

[0214] Both recombinant strains ATCC13032-gdh(GC) and YPGLU-gdh(GC) contain two copies of the gdh gene from Corynebacterium glutamicum ATCC13032. Specifically, ATCC13032-gdh(GC) and YPGLU-gdh(GC) are recombinant strains obtained by knocking out the coding region of the poxB gene in the genomes of Corynebacterium glutamicum ATCCC13032 and CGMCC NO.21220, respectively, and simultaneously introducing an expression cassette of the glutamate dehydrogenase encoding gene gdh(CG) at the poxB site, while keeping other nucleotides in the genomes of Corynebacterium glutamicum ATCC13032 and CGMCC NO.21220 unchanged. The nucleotide sequence of the above-mentioned glutamate dehydrogenase encoding gene gdh(CG) expression cassette is positions 545-1965 of SEQ ID NO.30. In SEQ ID NO.30, bits 545-621 are the pBJ23119 promoter, and bits 622-1965 are gdh(CG).

[0215] Example 3: Fermentation Experiment

[0216] I. L-Arginine Fermentation Experiment

[0217] The strains of *Escherichia coli* W3110, W3110 / pBJ23119-gdhA (EC), W3110 / pBJ23119-gdhA (SA), W3110 / pBJ23119-gdhA (SA, optimized), W3110 / pBJ23119-rocG (Bacillus subtilis), W3110 / pBJ23119-gdh (GC), and CGMCC were compared. Strain NO.25402, along with YPR / pBJ23119-gdhA(EC), YPR / pBJ23119-gdhA(SA), YPR / pBJ23119-gdhA(SA, optimized), YPR / pBJ23119-rocG(BS), and YPR / pBJ23119-gdh(GC), were inoculated into a 5L BLBIO-5GC-4-H fermenter (Shanghai Bailun Biotechnology Co., Ltd.) using L-arginine fermentation medium and the culture conditions provided in Table 1. Each strain was repeated three times. After fermentation, the L-arginine content was determined by high-performance liquid chromatography (HPLC), and the results were taken as the average of the three replicates, as shown in Tables 2 and 3.

[0218] L-Arginine fermentation medium: The solvent is water, and the solutes and their concentrations are as follows: glucose 8 g / L, FM902 yeast extract 3 g / L, K2HPO4·3H2O 6 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 0.05 g / L, betaine 0.5 g / L, VB12 0.005 g / L, defoamer 0.3 mL / L, ammonium sulfate 3 g / L, pH 7.2.

[0219] Table 1. L-Arginine Fermentation Control Process

[0220] Table 2. L-arginine production of engineered strains

[0221] Table 3. L-arginine production of engineered strains

[0222] Table 4. Significant difference analysis of fermentation results between recombinant strains of Salmonella Typhimurium before and after optimization of the gene encoding glutamate dehydrogenase.

[0223] As shown in the fermentation results above, regardless of whether the recipient strain was the high-L-arginine-producing strain CGMCC NO.25402 or the wild-type Escherichia coli W3110, the L-arginine yield after introducing the glutamate dehydrogenase encoding gene (gdh) from Salmonella Typhimurium was significantly higher than that of the recipient strain. However, overexpression of the rocG gene from Bacillus subtilis and the gdh gene from Corynebacterium glutamicum had no significant effect (Tables 2 and 3). Meanwhile, the L-arginine yield of the recombinant strain introduced with the optimized Salmonella Typhimurium glutamate dehydrogenase encoding gene (SEQ ID NO.5) was significantly higher than that of the recombinant strain introduced with the wild-type Salmonella Typhimurium glutamate dehydrogenase encoding gene (SEQ ID NO.4) (Table 4).

[0224] II. L-Glutamic Acid Fermentation Experiment

[0225] Fermentation experiments were conducted using 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 fermentation media as shown below and control processes as shown in Table 5 in a BLBIO-5GC-4-H fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.). Each strain was replicated three times. After fermentation, the L-glutamic acid content was determined by high-performance liquid chromatography (HPLC), and the results were taken as the average of the three replicates, as shown in Tables 6 and 7.

[0226] The fermentation medium formula is as follows: glucose 5.0 g / L, phosphate 0.38 g / L, magnesium sulfate 1.85 g / L, potassium chloride 1.6 g / L, biotin 550 μg / L, vitamin biotin B1 300 μg / L, ferrous sulfate 10 mg / L, manganese sulfate 10 g / dL, KH2PO4 2.8 g / L, vitamin C 0.75 mg / L, vitamin B12 2.5 μg / L, para-aminobenzoic acid 0.75 mg / L, defoamer 0.0015 mL / dL, betaine 1.5 g / L, sugarcane molasses 7 mL / L, corn steep liquor 77 mL / L, aspartic acid 1.7 g / L, and hair powder 2 g / L.

[0227] Table 5 L-Glutamic Acid Fermentation Control Process

[0228] Table 6 Results of L-glutamic acid fermentation experiments

[0229] Table 7 Results of L-glutamic acid fermentation experiments

[0230] Table 8. Significant differences in L-glutamate fermentation results between recombinant strains of *Salmonella typhimurium* before and after optimization of the gene encoding glutamate dehydrogenase.

[0231] The results showed that the introduction of the glutamate dehydrogenase encoding gene (gdh) from *Salmonella typhimurium* into *Corynebacterium glutamicum* ATCC13032 and CGMCC NO.21220 significantly increased the L-glutamate production of the recipient bacteria. However, overexpression of the rocG gene from *Bacillus subtilis* and the gdh gene from *Corynebacterium glutamicum* had no significant effect (Tables 6 and 7). Meanwhile, the recombinant strain with optimized *Salmonella typhimurium* (SEQ ID NO.5) showed significantly higher L-glutamate production than the recombinant strain without optimized glutamate dehydrogenase encoding gene (SEQ ID NO.4) (Table 8).

[0232] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Industrial applicability

[0233] 1. This application discloses for the first time the use of glutamate dehydrogenase and its encoding gene derived from Salmonella Typhimurium in increasing the L-amino acid production of microorganisms. Experimental verification in this application shows that glutamate dehydrogenase derived from Salmonella Typhimurium can significantly increase the L-amino acid production in recipient bacteria such as Escherichia coli or Corynebacterium glutamicum.

[0234] 2. This application obtained an optimized coding gene for glutamate dehydrogenase from Salmonella Typhimurium through codon optimization. Introducing the optimized coding gene or an expression cassette containing the optimized coding gene into recipient microorganisms significantly increases the L-amino acid yield of the recipient microorganisms. The glutamate dehydrogenase gene optimized in this application has produced unexpected technical effects and has great application potential in the field of microbial fermentation of L-amino acids.

[0235] Cross-reference to related applications

[0236] [Correction 04.12.2025 in accordance with Rule 91] This application claims priority to Chinese Patent Application No. 202411585569.9, filed on November 8, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. The application of glutamate dehydrogenase derived from Salmonella typhimurium, characterized by: The application is the use of the glutamate dehydrogenase in increasing the L-amino acid production of microorganisms.

2. The application according to claim 1, characterized in that: The glutamate dehydrogenase comprises at least one of the following: a1) A protein whose amino acid sequence contains SEQ ID NO.4; a2) A protein 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) and is related to the glutamate dehydrogenase. a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

3. The application according to claim 1 or 2, characterized in that: The L-amino acid is at least one or more selected from 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. Preferably, the L-amino acid comprises L-arginine or L-glutamic acid.

4. The application of biomaterials related to the glutamate dehydrogenase described in any one of claims 1 to 3, characterized in that: The application includes the use of the biomaterial in at least one of A1)-A3) below. A1) The application of the biomaterial in constructing engineered bacteria that produce L-amino acids; A2) The application of the biomaterial in the preparation of L-amino acids; A3) The application of the biomaterial in regulating the production of L-amino acids in microorganisms; The biomaterial comprises at least one of the following B1)-B3): B1) A nucleic acid molecule encoding the glutamate dehydrogenase described in claim 1. B2), an expression cassette containing the nucleic acid molecule described in B1), B3), recombinant vectors containing the nucleic acid molecules described in B1), and / or recombinant vectors containing the expression cassette described in B2).

5. The application according to claim 4, characterized in that: B1) The nucleic acid molecule comprises at least one of the following: The G1 nucleotide sequence contains the nucleic acid molecule of SEQ ID NO. 5; The nucleic acid molecules described in G2) and G1) have more than 80% identity and encode the nucleic acid molecule of the glutamate dehydrogenase described in claim 1.

6. A biomaterial, characterized in that: The biomaterial is the biomaterial described in claim 4 or 5.

7. Recombinant microorganisms, characterized by: The recombinant microorganism comprises at least one of the following: C1) Recombinant microorganisms containing the nucleic acid molecules described in claim 4 (B1); C2) Recombinant microorganisms containing the expression cassette described in claim 4 (B2); C3) Recombinant microorganisms containing the recombinant vector described in claim 4 (B3); C4) Recombinant microorganisms containing the glutamate dehydrogenase described in claim 1.

8. The method for preparing the recombinant microorganism according to claim 7, characterized in that: Preparation by a method comprising the following steps: introducing a nucleic acid molecule encoding a glutamate dehydrogenase derived from Salmonella Typhimurium as described in claim 1 or introducing the biological material as described in claim 6 into a recipient microorganism, wherein the recipient microorganism is a bacterium of the genus Escherichia and / or a bacterium of the genus Corynebacterium; Preferably, the Escherichia coli bacteria is Escherichia coli; or / and The Corynebacterium species mentioned is Corynebacterium glutamicum.

9. The method according to claim 8, characterized in that: The glutamate dehydrogenase derived from Salmonella typhimurium contains the amino acid sequence shown in SEQ ID NO.

4.

10. The application of a substance in the preparation of L-amino acids, characterized in that: The substance is the recombinant microorganism of claim 7, or the recombinant microorganism prepared by the method of claim 8 or 9.

11. A method for increasing the L-amino acid production of recipient microorganisms, characterized in that: The method comprises introducing a nucleic acid molecule encoding a glutamate dehydrogenase derived from Salmonella Typhimurium of claim 1 or introducing the biological material of claim 6 into the recipient microorganism to increase the L-amino acid production of the recipient microorganism.

12. A method for preparing L-amino acids, characterized in that: The method includes the step of preparing L-amino acids using recombinant microorganisms as fermentation strains, wherein the recombinant microorganisms are the recombinant microorganisms of claim 7 or the recombinant microorganisms prepared by the method of claim 8 or 9.

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