Application of succinate dehydrogenase mutant in glutamic acid production

Abstract

The invention discloses application of a succinate dehydrogenase mutant in glutamic acid production, and belongs to the technical field of genetic engineering. In order to improve the glutamic acid yield of microorganisms, the invention provides a succinate dehydrogenase mutant, and the succinate dehydrogenase mutant at least comprises protein obtained by mutation of the 301st amino acid residue site of succinate dehydrogenase. Recombinant bacterium verification results show that compared with original target microorganisms, the glutamic acid yield of recombinant corynebacterium glutamicum expressing the succinate dehydrogenase mutant or overexpressing the succinate dehydrogenase is remarkably increased, and an unexpected technical effect is achieved.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the application of a succinate dehydrogenase mutant in glutamic acid production. Background Technology

[0002] Glutamic acid is an acidic amino acid that serves as an essential nutrient for human growth, possessing not only unique physiological functions but also distinctive roles in the food industry. Although glutamic acid exists naturally in food, large-scale production as a commercial product, "monosodium glutamate", requires an efficient and inexpensive industrial production method.

[0003] Currently, Corynebacterium glutamicum ( corynebacterium glutamicum It is the mainstay of large-scale industrial production of glutamic acid. As a GRAS (Granitized As Safe) biosafety bacterium, it plays an important role in the field of amino acid fermentation and has been safely used for nearly 60 years.

[0004] The most common method for producing glutamic acid is fermentation, which offers advantages such as a wide availability of raw materials, low production costs, controllable product quality, and a single product. However, the fermentation performance of current glutamic acid-producing strains remains poor, resulting in unsatisfactory glutamic acid conversion rates. Furthermore, the industrial demand for glutamic acid is extremely high, and existing strains are simply insufficient to meet the needs of large-scale industrial production. Therefore, further improvement of strains to enhance growth and increase glutamic acid conversion rates remains a crucial issue that needs to be addressed.

[0005] sdhA The gene-encoded succinate dehydrogenase does not directly catalyze the synthesis of glutamate, and its effect on glutamate production is unknown. Summary of the Invention

[0006] The technical problem to be solved by this application is: how to increase the glutamic acid production of microorganisms. The technical problem to be solved by this application is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To address the aforementioned technical problems, this application provides a succinate dehydrogenase mutant, wherein the succinate dehydrogenase mutant comprises at least one of the following: A1) The mutant comprises a protein obtained by mutating the 301st amino acid residue of succinate dehydrogenase; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) has an amino acid sequence that is more than 98% identical to that of the protein in A1) and has succinate dehydrogenase function. A3) A fusion protein with the same function is obtained by linking a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2); The succinate dehydrogenase described herein comprises a protein with the amino acid sequence SEQ ID NO: 3.

[0008] Furthermore, the succinate dehydrogenase is a protein with the amino acid sequence SEQ ID NO: 3.

[0009] In this application, A1) contains a protein obtained by mutating the proline residue at position 301 of the sequence shown in SEQ ID NO:3 to a serine residue.

[0010] In this application, A1) can be a protein obtained by mutating the proline residue at position 301 of the sequence shown in SEQ ID NO:3 to a serine residue.

[0011] Specifically, the protein described in A1) may also be a protein whose amino acid sequence is SEQ ID NO: 4.

[0012] Furthermore, A2) The protein described does not include the following type: a protein whose amino acid sequence is SEQ ID NO: 3.

[0013] The protein of this application is a succinate dehydrogenase mutant obtained by mutating amino acid position 301 of SEQ ID NO:3 of Corynebacterium glutamicum succinate dehydrogenase (wild type, amino acid sequence SEQ ID NO:3) from proline (P) to serine (S). The amino acid sequence of the obtained succinate dehydrogenase mutant is SEQ ID NO:4.

[0014] In this application, SEQ ID NO:4 consists of 673 amino acid residues.

[0015] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0016] 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.

[0017] 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 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 in A1) or A2). A linker peptide may also be present between the tag and the protein. The linker peptide forms peptide bonds by dehydration condensation with both the N-terminus of the tag and the C-terminus of the protein. Alternatively, the linker peptide forms peptide bonds by dehydration condensation with both the C-terminus of the tag and the N-terminus of the protein.

[0018] This application also provides biological materials, said biological materials comprising at least one of the following: B1) The nucleic acid molecule encoding the above-mentioned succinate dehydrogenase mutant; B2) Expression cassettes and / or constructs containing the nucleic acid molecules described in B1), B3) recombinant vectors containing the nucleic acid molecules described in B1) and / or recombinant vectors containing the expression cassettes and / or constructs described in B2). B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5), a whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3) or the recombinant microorganism described in B4).

[0019] Further, the nucleic acid molecule described in B1) includes at least one of the following: C1) A nucleic acid molecule whose coding sequence contains SEQ ID NO: 2; C2) is a nucleic acid molecule that has more than 70% similarity to the nucleic acid molecule described in C1) and encodes the protein described in claim 1.

[0020] C3) hybridizes with the DNA molecule defined by C1) under strict conditions and encodes a nucleic acid molecule of the succinate dehydrogenase mutant of claim 1.

[0021] This application also provides a method for increasing the glutamic acid production of microorganisms, the method comprising at least one of the following: D1) Induce the target microorganism to express the above-mentioned succinate dehydrogenase mutant; D2) Mutate the succinate dehydrogenase encoding gene in the target microorganism into the nucleic acid molecule described in B1) above, wherein the target microorganism contains the succinate dehydrogenase encoding gene. D3) Introducing the biomaterial described in claim 3 or 4 into the target microorganism; D4) Increase the expression level of the coding gene of the succinate dehydrogenase mutant as described in claim 1 in the target microorganism or increase the content of the succinate dehydrogenase mutant as described in claim 1 in the target microorganism; D5), overexpression of succinate dehydrogenase with the amino acid sequence SEQ ID NO:3.

[0022] Furthermore, the target microorganism described in D1) may contain the coding gene for succinate dehydrogenase (wild type), or it may not contain the coding gene for succinate dehydrogenase (wild type).

[0023] Further, the mutation described in D2) specifically involves mutating the deoxyribonucleotide at position 901 of the nucleotide sequence in the target microorganism from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T). The coding sequence of the mutated succinate dehydrogenase (i.e., the succinate dehydrogenase mutant) is shown in SEQ ID NO: 2. This mutation causes the amino acid at position 301 of the wild-type succinate dehydrogenase of Corynebacterium glutamicum (the amino acid sequence of the wild-type succinate dehydrogenase is shown in SEQ ID NO: 3) to change from proline (P) to serine (C). The amino acid sequence of the succinate dehydrogenase mutant is shown in SEQ ID NO: 4.

[0024] In this application, the coding sequence of the wild-type succinate dehydrogenase gene is SEQ ID NO: 1; the corresponding amino acid sequence is SEQ ID NO: 3. In this application, the coding sequence of the mutant succinate dehydrogenase gene is SEQ ID NO: 2; the corresponding amino acid sequence is SEQ ID NO: 4, also referred to as SdhA in this application. P301S express.

[0025] In this application, the target microorganism is at least one of the following: E1) Microorganisms capable of producing glutamic acid; E2), bacteria; E3), Gram-positive bacteria; E4), Corynebacterium spp.; E5), Corynebacterium glutamicum.

[0026] This application also provides recombinant microorganisms containing or expressing the above-mentioned succinate dehydrogenase mutant and / or overexpressing recombinant microorganisms containing succinate dehydrogenase with the amino acid sequence SEQ ID NO: 3.

[0027] Preferably, the recombinant microorganism comprises at least one of Corynebacterium glutamicum, Pantotheca, and Corynebacterium pingeri.

[0028] In this application, the overexpression may be achieved by introducing the encoding gene of succinate dehydrogenase with the amino acid sequence SEQ ID NO: 3 into the recipient bacteria, wherein the recipient bacteria may contain the encoding gene of succinate dehydrogenase with the amino acid sequence SEQ ID NO: 3.

[0029] The overexpression may be the encoding gene of succinate dehydrogenase containing more than two (greater than or equal to two) copies of the amino acid sequence SEQ ID NO: 3.

[0030] This application also provides compositions that may contain the recombinant microorganisms described above. These compositions can be used to prepare glutamic acid.

[0031] The active ingredient in the above composition may be the recombinant microorganism and / or its metabolites and / or its culture.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] The above composition may be the culture described above. The above composition may also be a microbial agent.

[0036] 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.

[0037] 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.

[0038] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0039] This application also provides a method for preparing glutamic acid, the method comprising at least the step of preparing glutamic acid using the recombinant microorganisms described above.

[0040] Further, the method includes the steps of culturing the recombinant microorganism or composition in a culture medium, collecting the culture product, and obtaining glutamic acid. The glutamic acid described in this application may be L-glutamic acid.

[0041] This application also provides applications that include at least one of the following: G1) Application of the above-mentioned succinate dehydrogenase mutant in the preparation of glutamic acid; G2), Application of the above-mentioned succinate dehydrogenase mutant in increasing microbial glutamate production; G3), the application of the above-mentioned biomaterials in the construction of engineered bacteria that produce glutamic acid; G4), Application of the above-mentioned biomaterials in the preparation of glutamic acid; G5), the application of the above-mentioned biomaterials in increasing the yield of glutamic acid in microorganisms; G6), the application of the above-mentioned biomaterials in constructing recombinant microorganisms with high glutamic acid production; G7), the application of the above-mentioned recombinant microorganisms in the preparation of glutamic acid.

[0042] Furthermore, the microorganism or the engineered bacteria is any one of the following: E1) Microorganisms capable of producing glutamic acid; E2), bacteria; E3), Gram-positive bacteria; E4), Corynebacterium spp.; E5), Corynebacterium glutamicum; E6) Pantothecin; E7) Corynebacterium pingeri.

[0043] In the recombinant microorganisms described in this application, the recombinant microorganism obtained by using Corynebacterium glutamicum as the target microorganism 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 and / or construct described in B2) into the BBD29_11950-BBD29_11955 spacer region of the target Corynebacterium glutamicum.

[0044] The recombinant Corynebacterium glutamicum can also be obtained by mutating the deoxyribonucleotide at position 901 of the coding gene (coding sequence is SEQ ID NO: 1) of the target Corynebacterium glutamicum succinate dehydrogenase from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T), thus obtaining a mutant containing succinate dehydrogenase (SdhA). P301SRecombinant bacteria encoding the gene (SEQ ID NO: 2) of .

[0045] The recombinant Corynebacterium glutamicum can also be obtained by transforming a recombinant plasmid containing an expression cassette and / or construct of the coding gene for the succinate dehydrogenase mutant into Corynebacterium glutamicum, thereby obtaining the coding gene for the succinate dehydrogenase mutant. sdhA C901T Recombinant bacteria of the gene (SEQ ID NO: 2) and / or recombinant bacteria expressing a succinate dehydrogenase mutant (SEQ ID NO: 4).

[0046] In some embodiments of this application, the Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC29950.

[0047] In this application, "mutated succinate dehydrogenase", "succinate dehydrogenase mutant", and "succinate dehydrogenase mutant (SdhA)" are used. P301S ) and "SdhA P301S The meaning of "" is the same.

[0048] The beneficial technical effects achieved by this application are as follows: This application provides a succinate dehydrogenase mutant (SdhA) that increases glutamate production in target microorganisms. P301S The results of the recombinant bacterial validation showed that, compared with the original target microorganism, the recombinant Corynebacterium glutamicum expressing or overexpressing the succinate dehydrogenase mutant significantly increased the glutamate production, achieving unexpected technical results.

[0049] Preservation Instructions Classification and nomenclature: Corynebacterium glutamicum ( Corynebacterium glutamicum ) Strain number: YP077-2 Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Abbreviation of depositary institution: CGMCC Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101 Deposit date: March 7, 2024 CGMCC Registration Number: 29950 Detailed Implementation I. Terminology in this application: Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0050] 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.

[0051] For ease of understanding this application, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) 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, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0052] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more 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% consistency.

[0053] 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.

[0054] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "including" and "comprise" are used interchangeably.

[0055] The term "microorganism capable of producing glutamate" refers to a microorganism that possesses the ability to produce and accumulate glutamate within itself using external substances (such as culture medium), and may further include the ability to secrete glutamate into the culture system. Thus, glutamate can be collected when the microorganism is cultured in a culture medium.

[0056] The term "culture product" refers to the collective term 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 by growing and / or amplifying 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.

[0057] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least 3 amino acids in length. Proteins, peptides, or polypeptides can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.

[0058] As used in this article, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thus forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively.

[0059] The term "biomaterial" refers to any material that carries genetic information and is capable of self-replication or replication within a biological system, such as genes, plasmids, microorganisms, animals, and plants.

[0060] As is generally understood in the art, the term "promoter" typically refers to a DNA molecule containing an RNA polymerase binding site and / or a transcription start site that assists or promotes the transcription of transcribed DNA. In prokaryotes, the promoter sequence is located at the 5' end of the transcription start site (TSS), covering a region approximately 40 bp in length, and structurally generally includes the transcription start site (denoted as +1). District 35 District 10 and 35 districts and The spacer region between the 10 regions. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application can include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0061] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. Promoters that drive expression in all or most tissues of the receptor are called “constitutive” promoters. Promoters that drive expression at certain times or stages of development are called “developmental” promoters. “Inducible” promoters are promoters that initiate transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, and synthetic.

[0062] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0063] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0064] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.

[0065] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0066] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0067] II. Implementation Examples The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.

[0068] 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.

[0069] Corynebacterium glutamicum ( Corynebacterium glutamicum CGMCC No. 29950, also known as Corynebacterium glutamicum CGMCC29950, is a strain that has been deposited using a patent procedure, with the accession number CGMCC NO. 29950.

[0070] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0071] Example 1: Constructing a system containing point mutations sdhA Recombinant vectors of gene coding regions Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI (GenBank: CP016335.1 (26-APR-2017), two pairs of amplified strains were designed and synthesized. sdhA Primers for the gene coding region were used in Corynebacterium glutamicum CGMCC29950 via allele substitution. sdhA A point mutation is introduced into the coding region of the gene (SEQ ID No. 1), wherein the point mutation is a mutation that... sdhA The cytosine (C) at position 901 of the gene's nucleotide sequence (SEQ ID No. 1) is mutated to thymine (T), resulting in the DNA molecule shown in SEQ ID No. 2 (the mutated one). sdhA Gene sequence, named sdhA C901T ).

[0072] The DNA molecule shown in SEQ ID No. 1 encodes a protein with the amino acid sequence of SEQ ID No. 3 (the protein name is protein SdhA). The DNA molecule shown in SEQ ID No. 2 encodes a mutant protein with the amino acid sequence of SEQ ID No. 4 (the mutant protein name is SdhA). P301S The mutant protein SdhA P301S The proline (P) at position 301 in the amino acid sequence (SEQ ID No. 4) is mutated to serine (S).

[0073] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Invitrogen Shanghai): P1:5'-cagtgccaagcttgcatgcctgcaggtcgactctagTGGTACGGTCCTTGACGCTG-3' (lowercase part is the homologous arm, located in the skeletal carrier); P2: 5'-CAGGTGGAGTTCACAGACAGGCCGGTTGGGTGGAAC-3' (The bases in bold are the mutation positions); P3: 5'-GTTCCACCCAACCGGCCTGTCTGTGAACTCCACCTG-3' (The bases in bold are the mutation positions); P4:5'-cagctatgaccatgattacgaattcgagctcggtacccGAGCGCACGGATCTTGTCG-3' (lowercase part is the homologous arm, located in the skeletal carrier).

[0074] Construction method: Using Corynebacterium glutamicum ATCC13869 as a template, PCR amplification was performed using primers P1 / P2 and P3 / P4, respectively, to obtain two sequences of 846 bp and 826 bp, respectively, each containing a mutant base. sdhA Gene fragments ( sdhA Up-1 and sdhA Down-1).

[0075] PCR amplification system: 5×HiFi with Mg 2+ Buffer 10μL, dNTP Mixture (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, add ddH2O to a total volume of 50μL.

[0076] PCR amplification program: 95℃ pre-denaturation for 5 min, (98℃ denaturation for 20 s; 60℃ annealing for 15 s; 72℃ extension for 30 s; 30 cycles), 72℃ extension for 5 min.

[0077] The two DNA fragments mentioned above ( sdhA Up-1 and sdhA Down-1 was separated and purified by agarose gel electrophoresis, and then compared with enzyme digestion ( Xbal I / BamH I) The purified pK18mobSacB vector (purchased from Biovector, catalog number: BiovectorpK18mobSacB, abbreviated as vector pK18) was ligated with NEBuilder enzyme (purchased from NEB) at 50℃ for 30 min. The ligation product was transformed by heat shock, and the resulting single clones were identified by PCR using primers P1 / P4. Those that amplified a 1636 bp fragment (SEQ ID No. 5, where position 830 C is mutated to T) were considered positive recombinant vector pK18. sdhA C901T The recombinant vector contained a kanamycin resistance marker. The vector was sent to a sequencing company for sequencing identification, and the recombinant vector pK18- containing the correct point mutation (C901T) was then analyzed. sdhA C901T Save for future use.

[0078] Recombinant vector pK18- sdhA C901T The presence of a mutation site (C901T) will lead to [a specific disease / problem] in Corynebacterium glutamicum CGMCC29950. sdhA The mutation of cytosine (C) at position 901 of the gene coding region to thymine (T) ultimately leads to the mutation of proline (P) at position 301 of the protein SdhA to serine (S).

[0079] The recombinant vector pK18- sdhA C901T It is the pK18mobsacB vector Xbal I and / BamH The fragment between the I restriction sites (AGGATCCCC) was replaced with the DNA fragment shown in SEQ ID No. 5 (where position 830 C is mutated to T), and the other sequences of the pK18mobsacB vector were kept unchanged to obtain the recombinant vector. sdhA C901T Contains the mutant gene shown in SEQ ID No. 2 sdhA C901T The mutation site (C901T).

[0080] Example 2: Constructing a system containing sdhA C901T engineered strains with mutated genes The allelic substitution plasmid (pK18-) constructed in Example 1 was used. sdhA C901T The bacteria were transformed into Corynebacterium glutamicum strain CGMCC29950 via electroporation (sequencing confirmed that this strain retains the wild-type chromosome). sdhA The gene coding sequence and wild-type Corynebacterium glutamicum strain ATCC13869 were cultured for 40 h on solid culture plates containing kanamycin (50 mg / L) (the culture medium composition is shown in Table 1, with 2% agar powder added). Single colonies were identified using primers P1 / P4 from Example 1. Strains that amplified a 1636 bp band were considered positive strains. Positive strains were streaked onto medium containing 15% sucrose. After growth, single colonies were cultured on media containing and without kanamycin. Strains that grew on the non-kanamycin medium but not on the kanamycin medium were further amplified by PCR using primers P1 / P4. Multiple identified DNA fragments (1636 bp) were sequenced. Through sequence alignment, strains with a mutated base sequence (C901T) were identified as positive strains with successful allelic substitution. The positive strains obtained from *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* strain ATCC13869 were named YPG- sdhA -1、 sdhA -1.

[0081] Recombinant strain YPG- sdhA -1、 sdhA -1 all contain the mutant gene shown in SEQ ID No. 2. sdhA C901T The sequence indicates that the protein shown in SEQ ID No. 4 is expressed. Recombinant bacteria YPG- sdhA The only difference between -1 and Corynebacterium glutamicum CGMCC29950 is: YPG- sdhA -1 represents Corynebacterium glutamicum CGMCC29950. sdhA Sequence replacement sdhA C901T Strains obtained by mutating sequences while keeping other sequences unchanged; recombinant bacteria sdhA The only difference between -1 and wild-type Corynebacterium glutamicum ATCC13869 is: sdhA -1 represents wild-type Corynebacterium glutamicum ATCC13869. sdhA Sequence replacement sdhA C901T The strain was obtained by mutating the sequence while keeping other sequences unchanged.

[0082] Table 1. Composition of Corynebacterium glutamicum culture medium (solvent is water)

[0083] Example 3: Constructing genome overexpression sdhA C901T engineered strains with mutated genes Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, four pairs of amplified upstream and downstream homologous arm fragments were designed and synthesized. sdhA C901T Primers for the gene promoter and coding regions were inserted into the genomes of *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* ATCC13869 via homologous recombination. sdhA C901T Gene copy.

[0084] The primers were designed as follows (synthesized by Invitrogen Shanghai): P5:5'-cagtgccaagcttgcatgcctgcaggtcgactctagACGAGTTCATCAGCACGAGTC-3' (lowercase part is the homologous arm, located in the skeletal carrier). P6:5'-ATCACGCTATAGTTGCGCCGACGTTTTTCCTCATGATTTGTGGC-3', P7:5'-AAATCATGAGGAAAACGTCGGCGCAACTATAGCGTGATC-3', P8:5'-GTTTCAGAGTGAGTGCTCATGGCACCTCCAGTGTCGCTG-3', P9:5'-ACAGCGACACTGGAGGTGCCATGAGCACTCACTCTGAAAC-3', P10:5'-GCCACGCCCGCAAAAGATTTACTTGTAGTTCCTTGTCTGCAGTG-3', P11:5'-AGACAAGGAACTACAAGTAAATCTTTTGCGGGCGTGGCAT-3', P12:5'-cagctatgaccatgattacgaattcgagctcggtacccCTTATGTGCGGACTGCTTTAGTG-3' (lowercase part is the homologous arm, located in the skeletal carrier).

[0085] Construction method: Using the genome of *Corynebacterium glutamicum* ATCC13869 as a template, PCR amplification was performed using primers P5 and P6 to obtain a 607bp upstream homologous arm fragment (corresponding to the coding region of the BBD29_11950 gene and the upstream spacer region on the *Corynebacterium glutamicum* ATCC13869 genome (sequence shown as SEQ ID No. 6, 1-607); PCR amplification was performed using primers P11 and P12 to obtain a 570bp downstream homologous arm fragment (corresponding to the coding region of the BBD29_11955 gene and part of the coding region of BBD29_11960 on the *Corynebacterium glutamicum* ATCC13869 genome (sequence shown as SEQ ID No. 6, 3126-3695); PCR amplification was performed using primers P7 and P8 to obtain a 496bp promoter fragment (sequence shown as SEQ ID No. 6, 608-1103). The plasmid pK18- sdhA C901T Using primers P9 and P10 as templates, PCR amplification was performed to obtain... sdhA C901T The gene coding region fragment is 2022 bp (the sequence is shown as SEQ ID No. 6, 1104-3125, where cytosine C at position 2004 is mutated to thymine T).

[0086] After the PCR reaction, the upstream and downstream homologous arm fragments obtained from the amplification were analyzed. sdhA C901T The gene promoter and coding region fragments were recovered by electrophoresis using a column-based DNA gel extraction kit. The four recovered fragments were ligated with the pK18mobsacB plasmid, purified after digestion with Xbal I and BamHI, using NEBuilder enzyme (NEB) at 50 °C for 30 min. The ligation product was transformed into DH5α cells, and the resulting single clones were identified by PCR using primers P5 and P12. A fragment of 3695 bp was identified as a positive integration plasmid (recombinant vector), and the resulting recombinant vector was named pK18- sdhA C901T OE, this positive integration plasmid contains a kanamycin resistance marker, and recombinants integrated into the genome can be obtained through kanamycin screening. pK18- sdhA C901T The structure of OE is as follows: the fragment between the XbalI and BamH I restriction sites of pK18mobsacB is replaced with a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:6, while keeping the other nucleotide sequences of pK18mobsacB unchanged.

[0087] The correctly sequenced integrative plasmid pK18- sdhA C901TOE electroporation was used to transform *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* ATCC13869. The cultures were then incubated on solid culture plates for 40 hours. Single colonies were identified by PCR using primers P13 and P14. Strains containing a 2507 bp fragment (sequence shown in SEQ ID No. 7) were considered positive strains, while those without the fragment were considered the original strains.

[0088] The positive strain was streaked onto a solid culture plate containing 15% sucrose and cultured for 40 h. Single colonies produced were further identified by PCR using primers P15 and P16. The PCR amplification of a strain containing a 2191 bp fragment (sequence shown in SEQ ID No. 8) was confirmed. sdhA C901T Positive strains of *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* ATCC13869 whose genes and promoters have been integrated into the BBD29_11950-BBD29_11955 spacer region of the genome (documented in GenBank:CP016335.1(26-APR-2017)).

[0089] The PCR identification primers are shown below (synthesized by Invitrogen Shanghai): P13: 5'-TTGCCTCAATGACCTTAACAACG-3' (corresponding to the outer side of the upstream homologous arm). P14: 5'-CGTTGTTCAGAGGTCCAACAC-3' (corresponding to) sdhA (inside the gene) P15: 5'-ACGTAATGGTGAAAAGCGCTG-3' (corresponding to) sdhA (inside the gene) P16: 5'-TCGCGCCGATTCATGAGTTC-3' (corresponding to the outer side of the downstream homologous arm).

[0090] The starting strain was Corynebacterium glutamicum CGMCC29950, which contained a point mutation gene. sdhA C901T The bacterium was named YPG- sdhA -2. Using wild-type Corynebacterium glutamicum ATCC13869 as the starting strain, containing a point mutation gene... sdhA C901T The bacteria were named sdhA -2.

[0091] Recombinant strain YPG- sdhA -2 and sdhA -2 both contain the mutation shown in SEQ ID No. 2. sdhA C901T Genes. Specifically, recombinant bacteria YPG- sdhA -2 is an insertion at the BBD29_11950-BBD29_11955 spacer site in the genome of Corynebacterium glutamicum CGMCC29950. sdhA C901T The recombinant bacteria were obtained by modifying the gene and its promoter sequence (as shown in SEQ ID No. 6, 608-3125, where 608-1103 is the promoter region and 1104-3125 is the coding region, with cytosine C at position 2004 mutated to thymine T), while keeping other nucleotides of the Corynebacterium glutamicum CGMCC29950 genome unchanged; sdhA -2 is an insertion at the BBD29_11950-BBD29_11955 interstitial site in the genome of wild-type Corynebacterium glutamicum ATCC13869. sdhA C901T The recombinant bacteria were obtained by modifying the gene and its promoter sequence (as shown in SEQ ID No. 6, 608-3125, where 608-1103 is the promoter region and 1104-3125 is the coding region, where cytosine C at position 2004 is mutated to thymine T), while keeping other nucleotides of the wild-type Corynebacterium glutamicum ATCC13869 genome unchanged.

[0092] Example 4: Constructing plasmid overexpression sdhA engineered strains with mutated genes Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, an amplification method was designed and synthesized. sdhA C901T Primers for the gene coding region and promoter region were used to overexpress *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* ATCC13869 using the expression vector pXMJ19. sdhA C901T Gene.

[0093] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Invitrogen Shanghai): P17:5'-cagaataattaagcttgcatgcctgcaggtcgacGGCGCAACTATAGCGTGATC-3' (The lowercase nucleotide sequence is a homologous sequence of pXMJ19). P18:5'-ccaaaacagccaagctgaattcgagctcggtaccTTACTTGTAGTTCCTTGTCTGCAGTG-3' (The lowercase nucleotide sequence is a homologous sequence of pXMJ19).

[0094] With plasmid pK18- sdhA C901T Using OE as a template, PCR amplification was performed using primers P17 and P18 to obtain... sdhA C901T The gene promoter and coding region fragments were purified and ligated with the expression vector pXMJ19 (purchased from TaKaRa, containing chloramphenicol resistance) recovered by double digestion with Xbal I and BamHI at 50°C for 30 min using NEBuilder enzyme (purchased from NEB). The ligation product was transformed into DH5α competent cells and plated onto 2-YT agar plates containing chloramphenicol (34 mg / L) and incubated at 37°C for 12 h. Single clones were identified by PCR using primers P17 and P18. Cells that could amplify a 2518 bp fragment (sequence shown in SEQ ID No. 9) were considered to contain chloramphenicol. sdhA C901T Positive transformants pXMJ19- of gene promoter and coding region sequences sdhA C901T .

[0095] The correctly sequenced pXMJ19- sdhA C901T Plasmids were electroporated into *Corynebacterium glutamicum* CGMCC29950 and wild-type *Corynebacterium glutamicum* ATCC13869, respectively. After 40 hours of incubation on solid culture plates, single colonies were identified by PCR using primers P17 and P18. Strains amplified by PCR containing a 2518 bp fragment were considered positive. Using *Corynebacterium glutamicum* CGMCC29950 as the starting strain, plasmid pXMJ19-... sdhA C901T The bacterium was named YPG- sdhA -3. Using wild-type Corynebacterium glutamicum ATCC13869 as the starting strain, containing plasmid pXMJ19- sdhA C901T The bacteria were named sdhA -3.

[0096] Example 5: L-glutamic acid fermentation experiment The strains constructed in Examples 2-4, Corynebacterium glutamicum CGMCC29950, and wild-type Corynebacterium glutamicum ATCC13869 were fermented in 500 mL baffle shake flasks under the culture medium shown in Table 2 and the control conditions shown in Table 3. After fermentation, the L-glutamic acid production was detected using an SBA-Biosensor Analyzer (Shandong Academy of Sciences Institute of Biology). Each strain was repeated three times, and the results are shown in Table 4.

[0097] Table 2. Shake flask fermentation medium formulation (solvent is water)

[0098] Table 3. Fermentation Control Conditions

[0099] Table 4. L-glutamic acid production and significance analysis

[0100] Note: P<0.05 in the table indicates a significant difference compared to the starting strain; P<0.01 indicates a highly significant difference compared to the starting strain.

[0101] The fermentation results are shown in Table 4. The results indicate that the fermentation of Corynebacterium glutamicum effectively inhibits the growth of bacteria in Corynebacterium glutamicum. sdhA Point mutations in the coding region of a gene sdhA C901T and overexpression sdhA C901T Genes that contribute to increasing L-glutamic acid production.

[0102] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A succinate dehydrogenase mutant, characterized by: The succinate dehydrogenase mutant comprises at least one of the following: A1) The mutant comprises a protein obtained by mutating the 301st amino acid residue of succinate dehydrogenase: A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) has an amino acid sequence that is more than 98% identical to that of the protein in A1) and has succinate dehydrogenase function. A3) A fusion protein with the same function is obtained by linking a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2); The succinate dehydrogenase described herein comprises a protein with the amino acid sequence SEQ ID NO:

3.

2. The mutant according to claim 1, characterized in that, The A1) contains a protein obtained by mutating the proline residue at position 301 of the sequence shown in SEQ ID NO:3 to a serine residue.

3. A biomaterial, characterized in that: The biomaterial comprises at least one of the following: B1) A nucleic acid molecule encoding the succinate dehydrogenase mutant of claim 1 or 2; B2) Expression cassettes and / or constructs containing the nucleic acid molecules described in B1), B3) recombinant vectors containing the nucleic acid molecules described in B1) and / or recombinant vectors containing the expression cassettes and / or constructs described in B2). B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5), a whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3) or the recombinant microorganism described in B4).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule described herein contains at least one of the following: C1) A nucleic acid molecule whose coding sequence contains SEQ ID NO: 2; C2) A nucleic acid molecule that has more than 70% similarity to the nucleic acid molecule described in C1) and encodes the protein described in claim 1; C3) hybridizes with the DNA molecule defined by C1) under strict conditions and encodes a nucleic acid molecule of the succinate dehydrogenase mutant of claim 1.

5. A method for increasing the yield of glutamic acid from microorganisms, characterized in that: The method includes at least one of the following: D1) To induce the target microorganism to express the succinate dehydrogenase mutant as described in claim 1 or 2; D2) Mutate the coding gene for succinate dehydrogenase in the target microorganism into the nucleic acid molecule described in claim 3 or 4, wherein the target microorganism contains the coding gene for succinate dehydrogenase. D3) Introducing the biomaterial described in claim 3 or 4 into the target microorganism; D4) Increase the expression level of the coding gene of the succinate dehydrogenase mutant as described in claim 1 or 2 in the target microorganism or increase the content of the succinate dehydrogenase mutant as described in claim 1 or 2 in the target microorganism; D5), overexpression of succinate dehydrogenase with the amino acid sequence SEQ ID NO:

3.

6. The method according to claim 5, characterized in that: The target microorganism is at least one of the following: E1) Microorganisms capable of producing glutamic acid; E2), bacteria; E3), Gram-positive bacteria; E4), Corynebacterium spp.; E5), Corynebacterium glutamicum.

7. Recombinant microorganisms, characterized by: The recombinant microorganism contains or expresses the succinate dehydrogenase mutant of claim 1 or 2 and / or overexpresses the recombinant microorganism containing the succinate dehydrogenase whose amino acid sequence is SEQ ID NO:

3. Preferably, the recombinant microorganism contains at least one of Corynebacterium glutamicum, Pantotheca acuminata, and Corynebacterium pekinensis.

8. A method for preparing glutamic acid, characterized in that: The method includes at least the step of preparing glutamic acid using the recombinant microorganisms of claim 7.

9. Application, characterized in that: The application includes at least one of the following: G1), the application of the succinate dehydrogenase mutant according to claim 1 or 2 in the preparation of glutamic acid; G2), the application of the succinate dehydrogenase mutant according to claim 1 or 2 in increasing the glutamate production of microorganisms; G3), the application of the biomaterials described in claim 3 or 4 in the construction of engineered bacteria that produce glutamic acid; G4), the application of the biomaterial described in claim 3 or 4 in the preparation of glutamic acid; G5), the application of the biomaterials described in claim 3 or 4 in increasing the yield of glutamic acid in microorganisms; G6), the application of the biomaterials described in claim 3 or 4 in the construction of recombinant microorganisms with high glutamate production; G7) The application of the recombinant microorganisms described in claim 7 in the preparation of glutamic acid.

10. The application according to claim 9, characterized in that: The microorganism is, or the engineered bacteria is, any of the following: E1) Microorganisms capable of producing glutamic acid; E2), bacteria; E3), Gram-positive bacteria; E4), Corynebacterium spp.; E5), Corynebacterium glutamicum; E6) Pantothecin; E7) Corynebacterium pingeri.

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