A succinate dehydrogenase mutant and its application in increasing glutamate production

Abstract

The present application discloses a succinate dehydrogenase mutant and its application in increasing glutamate production, belonging to the field of microbial technology. The technical problem to be solved by the present application is: how to increase the glutamate production of microorganisms. To solve the above technical problem, the present application provides the following protein, which comprises a protein obtained by mutating the 133rd amino acid of succinate dehydrogenase from arginine (R) to cysteine (C); the protein may be a protein comprising the amino acid sequence of SEQ ID NO: 4. The present application provides a succinate dehydrogenase mutant (SdhA) with the ability to increase the glutamate production in a target microorganism. R133C ), the verification results of the recombinant bacteria showed that compared with the original target microorganism, the expression of the succinate dehydrogenase mutant (SdhA R133C )'s recombinant Corynebacterium glutamicum significantly increased its glutamate production, achieving unexpected technical results.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a succinate dehydrogenase mutant and its application in increasing glutamate production. Background Art

[0002] Glutamic acid is an acidic amino acid. It contains two carboxyl groups within its molecule and is chemically known as α-aminoglutaric acid. As a vital nutrient for human growth, glutamate not only has special physiological effects but also possesses unique functions in the food industry.

[0003] At present, the most commonly used production method of glutamic acid is a fermentation method, which mainly produces glutamic acid by fermentation with Corynebacterium glutamicum.Corynebacterium glutamicum is a heterotrophic aerobic type, a Gram-positive bacterium, with a fast growth rate, non-pathogenicity, and characteristics of weak degradation ability of its own metabolites.The fermentation method has advantages such as a wide range of raw material sources, low production cost, controllable product quality, and a single product.But the fermentation performance of the bacterial classification of producing glutamic acid is still relatively poor, and the glutamic acid conversion rate is undesirable.Glutamic acid is the main raw material for producing monosodium glutamate, and the industrial demand for glutamic acid is extremely high.Existing bacterial classification can not meet the demand of large-scale industrial production at all.Further bacterial strain is improved, and the production bacterial classification growth is improved, and improving the glutamic acid conversion rate is still a problem that needs to be solved at present. Summary of the Invention

[0004] 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 technical subject described herein, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, the present application provides the following protein, comprising a protein obtained by mutating the 133rd amino acid of succinate dehydrogenase from arginine (R) to cysteine (C) (i.e., a succinate dehydrogenase mutant);

[0006] The protein comprises any one of the following:

[0007] A1), a protein comprising the amino acid sequence of SEQ ID NO: 4;

[0008] A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of A1) and having an amino acid sequence identity of more than 70% with the protein of A1) and having the function of succinate dehydrogenase;

[0009] A3) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1) or A2).

[0010] Furthermore, the protein in A1) may also be a protein whose amino acid sequence is SEQ ID NO: 4. Furthermore, the protein in A2) does not include the following type: a protein whose amino acid sequence is SEQ ID NO: 3.

[0011] The protein disclosed herein is a succinate dehydrogenase mutant of Corynebacterium glutamicum (wild-type, amino acid sequence: SEQ ID NO: 3) obtained by mutating amino acid position 133 in SEQ ID NO: 3 from arginine (R) to cysteine (C). The amino acid sequence of the resulting succinate dehydrogenase mutant is SEQ ID NO: 4.

[0012] In the present application, SEQ ID NO: 4 consists of 673 amino acid residues.

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

[0014] A protein tag is a polypeptide or protein that is fused with a target protein using in vitro DNA recombination techniques to facilitate expression, detection, tracing, and / or purification of the target protein. Examples of protein tags include Flag, His, MBP, HA, myc, GST, and / or SUMO tags.

[0015] Furthermore, the linkage described in A3) can be achieved by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in A1) or A2) to form a peptide bond. Alternatively, the linkage described in A3) can be achieved by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in A1) or A2) to form a peptide bond. A linker peptide can also be included between the tag and the protein. The linker peptide can form peptide bonds with the N-terminus of the tag and the C-terminus of the protein, respectively, by dehydration condensation. Alternatively, the linker peptide can form peptide bonds with the C-terminus of the tag and the N-terminus of the protein, respectively.

[0016] The present application also provides a biological material related to the protein, wherein the biological material comprises any one of the following:

[0017] B1), a nucleic acid molecule encoding the protein;

[0018] B2), an expression cassette containing the nucleic acid molecule described in B1),

[0019] B3), a recombinant vector containing the nucleic acid molecule described in B1) and / or a recombinant vector containing the expression cassette described in B2).

[0020] Furthermore, the nucleic acid molecule described in B1) comprises C1) or C2),

[0021] C1), a nucleic acid molecule whose coding sequence comprises SEQ ID NO: 2;

[0022] C2) A nucleic acid molecule that has more than 70% identity with the nucleic acid molecule described in C1).

[0023] Furthermore, the nucleic acid molecule in C1) may also be a nucleic acid molecule encoding the sequence of SEQ ID NO: 2.

[0024] The present application also provides an application, wherein the application comprises at least any one of the following:

[0025] D1), use of the protein in preparing glutamate;

[0026] D2), use of the protein in increasing glutamate production in microorganisms;

[0027] D3) Application of the biomaterial in constructing an engineered bacterium producing glutamate;

[0028] D4), use of the biomaterial in preparing glutamic acid;

[0029] D5) Use of the biomaterial in increasing the yield of glutamic acid in microorganisms;

[0030] D6) Use of the biomaterial in constructing a recombinant microorganism that produces high glutamate production. The recombinant microorganism that produces high glutamate production is obtained by modifying a target microorganism, and the recombinant microorganism has a higher glutamate production than the target microorganism.

[0031] Furthermore, the target microorganism described in D6) can be any of the following

[0032] G1) Microorganisms capable of producing glutamate;

[0033] G2), bacteria;

[0034] G3), Gram-positive bacteria;

[0035] G4), Corynebacterium bacteria;

[0036] G5), Corynebacterium glutamicum.

[0037] The present application also provides a recombinant microorganism, wherein the recombinant microorganism comprises at least any one of the following:

[0038] E1), a recombinant microorganism containing or expressing the protein;

[0039] E2), a recombinant microorganism containing the nucleic acid molecule described in B1);

[0040] E3), a recombinant microorganism containing the expression cassette and / or construct described in B2) above;

[0041] E4) A recombinant microorganism containing the recombinant vector described in B3) above.

[0042] The present application also provides a composition for preparing glutamic acid, which may contain the recombinant microorganism.

[0043] The active ingredient of the above composition may be the recombinant microorganism or / and the metabolite of the recombinant microorganism or / and the culture of the recombinant microorganism.

[0044] The above-mentioned 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).

[0045] In the above, the metabolite can 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 medium), removing the recombinant microorganism from the fermentation broth, and collecting the remaining components of the fermentation broth to obtain the metabolite of the recombinant microorganism.

[0046] The active ingredients of the above composition may further contain other biological components or non-biological components. Those skilled in the art may determine the other active ingredients of the above composition based on the effects of the composition.

[0047] The above composition may be the culture. The above composition may also be a bacterial agent.

[0048] The above-mentioned bacterial agent refers to a live bacterial preparation made by expanding the target microorganisms and using a carrier as an adsorbent to adsorb the fermentation liquid or solid fermentation product of the bacteria.

[0049] The above-mentioned microbial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0050] If necessary, the bacterial agent may further include a carrier, which may be a solid carrier or a liquid carrier.

[0051] The present application also provides a method for increasing glutamic acid production of a microorganism, the method comprising at least any one of the following:

[0052] F1), causing the target microorganism to express the protein;

[0053] F2) mutating the gene encoding succinate dehydrogenase in the target microorganism into the nucleic acid molecule described in B1) above, wherein the target microorganism contains the gene encoding succinate dehydrogenase;

[0054] F3), introducing the above-mentioned biological material into the target microorganism;

[0055] F4), increasing the expression level of the gene encoding the protein in the target microorganism or increasing the content of the protein in the target microorganism.

[0056] Furthermore, the target microorganism in F1) may or may not contain a gene encoding succinate dehydrogenase (wild type).

[0057] Furthermore, the mutation described in F2) can specifically involve mutating the deoxyribonucleotide at position 397 of the nucleotide sequence in SEQ ID NO: 1 in the target microorganism from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T). The coding sequence of the succinate dehydrogenase after the mutation (i.e., the succinate dehydrogenase mutant) is SEQ ID NO: 2. This mutation results in a mutation from arginine (R) to cysteine (C) at amino acid position 133 in the wild-type succinate dehydrogenase from Corynebacterium glutamicum in the target microorganism (the amino acid sequence of the wild-type succinate dehydrogenase is SEQ ID NO: 3). The amino acid sequence of the succinate dehydrogenase mutant is set forth in SEQ ID NO: 4.

[0058] In the present application, the coding sequence of the wild-type corresponding coding gene of the succinate dehydrogenase is SEQ ID NO: 1; the corresponding amino acid sequence is SEQ ID NO: 3. In the present application, the coding sequence of the mutant corresponding coding gene of the succinate dehydrogenase is SEQ ID NO: 2, which is also used in the present application. sdhA C397T The amino acid sequence of the succinate dehydrogenase mutant is SEQ ID NO: 4, which is also referred to as SdhA in this application. R133C express.

[0059] Furthermore, in the method described above, the target microorganism can be any one of the following G1) to G5): G1), a microorganism capable of producing glutamate; G2), bacteria; G3), Gram-positive bacteria; G4), bacteria of the genus Corynebacterium; G5), Corynebacterium glutamicum.

[0060] Furthermore, the Corynebacterium glutamicum contains the BBD29_05675 pseudoprotein (GenBank or NCBI reference sequence number: CP016335.1.2017.04).

[0061] The present application also provides a method for preparing glutamic acid, which at least comprises the step of preparing glutamic acid using the recombinant microorganism and / or the composition.

[0062] Among the recombinant microorganisms described in the present application, the recombinant microorganism obtained by using Corynebacterium glutamicum as the target microorganism is named recombinant Corynebacterium glutamicum, and 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_05675 pseudoprotein (GenBank or NCBI reference sequence number: CP016335.1.2017.04) site of the target Corynebacterium glutamicum.

[0063] The recombinant Corynebacterium glutamicum can also be obtained by mutating the deoxyribonucleotide at position 397 of the coding gene of the succinate dehydrogenase of the target Corynebacterium glutamicum (coding sequence is SEQ ID NO: 1) from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T) to obtain a mutant containing succinate dehydrogenase (SdhA R133C ) encoding gene (SEQ ID NO: 2).

[0064] The recombinant Corynebacterium glutamicum can also be obtained by transforming the target Corynebacterium glutamicum with the recombinant plasmid containing the expression cassette and / or construct of the encoding gene containing the succinate dehydrogenase mutant to obtain the encoding gene containing the succinate dehydrogenase mutant. sdhA C397T (SEQ ID NO: 2) and / or a recombinant bacterium expressing a succinate dehydrogenase mutant (SEQ ID NO: 4).

[0065] In some embodiments of the present application, the Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC29950.

[0066] The present application also provides use of the recombinant microorganism or the composition in preparing glutamic acid.

[0067] Furthermore, the method comprises 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 the present application may be L-glutamic acid.

[0068] In this application, "mutated succinate dehydrogenase", "succinate dehydrogenase mutant", "succinate dehydrogenase mutant (SdhA)" R133C )" and "SdhA R133C ” have the same meaning.

[0069] The beneficial technical effects achieved by this application are as follows:

[0070] The present application provides a succinate dehydrogenase mutant (SdhA) having the ability to increase glutamate production in a target microorganism. R133C ), the verification results of the recombinant bacteria showed that compared with the original target microorganism, the glutamate production of the recombinant Corynebacterium glutamicum expressing the succinate dehydrogenase mutant was significantly improved, achieving unexpected technical effects.

[0071] Preservation Instructions

[0072] Bacteria species name: Corynebacterium glutamicum;

[0073] Latin name: Corynebacterium glutamicum ;

[0074] Classification name: Corynebacterium glutamicum Corynebacterium glutamicum ;

[0075] Strain ID: YP077-2;

[0076] Name of depository institution: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0077] Abbreviation of depository unit: CGMCC;

[0078] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101;

[0079] Deposit date: March 7, 2024;

[0080] Registration number of the CGMCC Collection Center: CGMCC No. 29950. DETAILED DESCRIPTION

[0081] Terms used in this application:

[0082] Examples of resources describing many of the molecular biology-related terms used herein can be found in 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, Genes IX, Oxford University Press: New York, 2007.

[0083] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0084] To facilitate understanding of the present disclosure, several terms and abbreviations used herein are defined as follows:

[0085] In this application, "identity" refers to the identity of an amino acid sequence or nucleotide sequence. Amino acid sequence (or nucleotide sequence) identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, a search can be performed using blastp with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Perresidue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated to obtain a percent identity.

[0086] Specifically, the consistency of more than 70% may be more than 75% consistency. Specifically, the consistency of more than 75% may be more than 80% consistency. Specifically, the consistency of more than 80% may be more than 85% consistency. Specifically, the consistency of more than 85% may be more than 90% consistency. Specifically, the consistency of more than 90% may be more than 91% consistency, more than 92% consistency, more than 93% consistency, more than 94% consistency, more than 95% consistency, more than 96% consistency, more than 97% consistency, more than 98% consistency, or more than 99% consistency. More specifically, the consistency of more than 70% may 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. More specifically, the above 80% consistency can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% consistency.

[0087] 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.

[0088] As commonly understood in the art, the term "promoter" generally refers to DNA that contains an RNA polymerase binding site and / or a transcription start site and assists or promotes the transcription of transcribable DNA. The promoter sequence of prokaryotes is located at the 5' end of the transcription start site (TSS), covering a region of approximately 40 bp in length. Structurally, it generally includes the transcription start site (denoted as +1), the −35 region, the −10 region, and the spacer region between the −35 region and the −10 region. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising a combination of two or more heterologous sequences. Therefore, the promoters of the present application may include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences provided herein.

[0089] Promoters can be classified according to various criteria related to the expression pattern of the relevant coding or transcribable sequence or gene (including transgenic) operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. Promoters that drive expression in all or most tissues of the recipient are referred to as "constitutive" promoters. Promoters that drive expression at certain stages or phases of development are referred to as "developmental" promoters. "Inducible" promoters are promoters that initiate transcription in response to environmental stimuli (such as cold, drought, or light) or other stimuli (such as wounded or chemically applied). Promoters can also be classified according to their source, such as heterologous, homologous, chimeric, synthetic, etc.

[0090] The term "transcribable DNA" refers to DNA that can be transcribed into an RNA molecule.

[0091] The term "operably linked" may refer to a functional connection between a promoter and transcribable DNA, such that the promoter functions to initiate transcription of the transcribable DNA. The term "operably linked" may also refer to a functional connection between other regulatory elements and a gene of interest to regulate the transcription and / or expression of the gene of interest.

[0092] The term "construct" refers to any recombinant DNA molecule or recombinant RNA molecule. A recombinant DNA molecule can be a plasmid, a cosmid, a virus, a phage, or a linear or circular DNA. A construct typically includes one or more expression cassettes.

[0093] As used herein, an "expression cassette" refers to a composition comprising at least transcribable DNA operably linked to one or more regulatory elements, typically at least a promoter and a 3'UTR (eg, a terminator).

[0094] As used herein, the term "vector" means any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell, such as a plasmid, cosmid, virus, phage, or linear or circular DNA.

[0095] The term "microorganism capable of producing glutamate" refers to the ability of a microorganism to utilize external substances (such as culture medium) to produce and accumulate glutamate in vivo. This may further include the ability to secrete glutamate into the culture medium. Thus, glutamate can be collected while the microorganism is cultured in the culture medium.

[0096] The term "culture product" refers to any liquid or solid product (all materials within a culture vessel) containing a microbial population after artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of microorganisms or contain a certain amount of culture medium, metabolites, or other components produced during the culture process. It can also be a mixture containing a certain amount of culture medium and microbial metabolites, but without the microbial cells.

[0097] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0098] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0099] Corynebacterium glutamicum ATCC13869, also known as ATCC13869, was purchased from the American Type Culture Collection (ATCC) with the strain number 13869;

[0100] Corynebacterium glutamicum ( Corynebacterium glutamicum ) CGMCC No. 29950, also known as Corynebacterium glutamicum CGMCC No. 29950, CGMCC29950, or CGMCC NO. 29950. This strain was deposited under patent procedure on March 7, 2024, with the deposit number CGMCC NO. 29950. In the following examples, CGMCC29950 is used to represent this strain.

[0101] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0102] Example 1: Isolation and growth test of mutant strain S3

[0103] Based on Corynebacterium glutamicum CGMCC29950, random mutation was performed by UV mutagenesis and the growth advantage strain S3 was obtained. The genome sequencing analysis revealed that sdhA The arginine (R) at position 133 of the gene translation protein is mutated to cysteine (C), and the corresponding amino acid sequence of the succinate dehydrogenase SdhA mutant is shown in SEQ ID NO: 4.

[0104] The specific screening method of mutant strain S3 is as follows:

[0105] 1. Strain mutagenesis screening

[0106] The CGMCC29950 strain was used as the starting strain. After inoculation at 2.5% into a 24-well plate liquid culture medium, it was irradiated with ultraviolet light at a vertical distance of 15 cm for 45 seconds under a 15W ultraviolet lamp. The culture was then kept in the dark and the OD of the bacterial solution was measured regularly. 600 Select the fastest growing bacterial solution in the 24-well plate and dilute it by 10 5 , 10 6 , 10 7 Spread on the slant culture medium plate, select different strains S1 to S15 from the plate for growth test again. The specific method is: after culturing in 24-well plate for 12 hours, dilute the culture medium 2 times and measure the turbidity at 600 nm. The result is OD 600 Indicates cell mass, OD 600 The results were as follows: CGMCC29950: 0.387 ± 0.023; S1: 0.351 ± 0.027; S2: 0.252 ± 0.012; S3: 0.739 ± 0.030; S4: 0.377 ± 0.022; S5: 0.382 ± 0.032; S6: 0.619 ± 0.016; S7: 0.559 ± 0.030; S8: 0.587 ± 0.025; S9: 0.394 ± 0.041; S10: 0.345 ± 0.036; S11: 0.413 ± 0.034; S12: 0.577 ± 0.027; S13: 0.420 ± 0.035; S14: 0.344 ±0.007; S15: 0.496 ±0.029. The growth rate of the mutant strain S3 was significantly increased compared with the control strain CGMCC29950.

[0107] Liquid culture medium formula: sucrose 10 g / L, polypeptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, urea 2 g / L, sodium chloride 2.5 g / L, magnesium sulfate heptahydrate 0.1 g / L, thiamine 0.1 mg / L, biotin 0.2 mg / L, solvent is water, and the pH is adjusted to 7.0.

[0108] Slant culture medium: Add 20 g / L agar powder to the liquid culture medium.

[0109] 2. Genome sequencing

[0110] The genomes of the starting strain CGMCC29950 and mutant strain S3 were extracted using a genome extraction kit (Novozyme Biotechnology Co., Ltd.) and sequenced. Comparison of the sequencing results revealed that the succinate dehydrogenase gene in the tricarboxylic acid cycle sdhA (SEQ ID NO: 1) The 397th nucleotide was mutated from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T), resulting in sdhA C397T The mutant sequence of SdhA (SEQ ID NO: 2) was modified, resulting in the mutation of amino acid 133 from arginine (R) to cysteine (C) in the amino acid sequence of SdhA (SEQ ID NO: 3). The amino acid sequence after mutation is shown in SEQ ID NO. 4 and is named as succinate dehydrogenase mutant SdhA. R133C Known sdhA The encoded succinate dehydrogenase is one of the hubs connecting oxidative phosphorylation and electron transfer, and can be used as an indicator to evaluate the degree of operation of the tricarboxylic acid cycle. After mutation, the SdhA enzyme activity changes, which may cause changes in the tricarboxylic acid cycle flux, thereby leading to faster bacterial growth.

[0111] Example 2: Construction of a point mutation sdhA Recombinant vector of gene coding region

[0112] Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, two pairs of amplification sdhA The primers of the gene coding region were used to replace the allele in Corynebacterium glutamicum CGMCC29950. sdhA A point mutation was introduced into the gene coding region (SEQ ID NO: 1), wherein the point mutation sdhA The 397th cytosine deoxyribonucleotide (C) in the nucleotide sequence of the gene (SEQ ID NO: 1) was mutated to a thymine deoxyribonucleotide (T), resulting in a DNA molecule shown in SEQ ID NO: 2 (mutated sdhA Gene sequence, named sdhA C397T ).

[0113] The DNA molecule shown in SEQ ID NO: 1 encodes a protein having an amino acid sequence of SEQ ID NO: 3 (i.e., succinate dehydrogenase SdhA). The DNA molecule shown in SEQ ID NO: 2 encodes a succinate dehydrogenase mutant SdhA having an amino acid sequence of SEQ ID NO: 4. R133C The 133rd cysteine (C) in the amino acid sequence of the succinate dehydrogenase mutant (SEQ ID NO: 4) is mutated to arginine (R).

[0114] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen):

[0115] P1: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagCATTATCTTCCACATCCTCGACCTG-3' (SEQ ID NO: 10, the nucleotide sequence in lowercase format is the sequence on pK18);

[0116] P2: 5'-cagtcggactcgcggccacAgtagtcgccgcccttgacg-3' (SEQ ID NO: 11, the deoxynucleotide base in uppercase format (position 20) indicates the mutation site);

[0117] P3: 5'-cgtcaagggcggcgactacTgtggccgcgagtccgactg-3' (SEQ ID NO: 12, the deoxynucleotide base (position 20) in uppercase format indicates the mutation site);

[0118] P4: 5′-cagctatgaccatgattacgaattcgagctcggtacccGGAAGCAACGTCACGTGGGAC-3′ (SEQ ID NO: 13, the nucleotide sequence in lowercase format is the sequence on pK18).

[0119] Construction method: Using Corynebacterium glutamicum ATCC13869 as template, primers P1 and P2 were used for PCR amplification to obtain a 781 bp fragment with a mutation site. sdhA Gene fragment, named sdhA Up-1 (SEQ ID NO: 5 positions 1 to 781). Using Corynebacterium glutamicum ATCC13869 as a template, primers P3 and P4 were used for PCR amplification to obtain a 753 bp fragment with a mutation site. sdhA Gene fragment, named sdhA Down-1 (SEQ ID NO: 5 positions 743 to 1495).

[0120] 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.

[0121] PCR amplification program: pre-denaturation at 95°C for 5 min, (denaturation at 98°C for 20 s; annealing at 60°C for 15 s; extension at 72°C for 30 s; 30 cycles), over-extension at 72°C for 5 min.

[0122] The two DNA fragments ( sdhA Up-1 and sdhA Down-1) was separated and purified by agarose gel electrophoresis and then Xbal I and BamH The purified pK18mobsacB plasmid (purchased from Addgene) after digestion with I was ligated with NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation product was transformed into DH5α competent cells by heat shock and the resulting single clone was identified by PCR using primers P1 and P4. The one that could amplify a DNA fragment of 1495 bp (SEQ ID NO: 5) was a positive recombinant vector pK18- sdhA C397T The recombinant vector contains a kanamycin resistance marker. The vector was sent to a sequencing company for sequencing and the recombinant vector pK18- sdhA C397T Save for later use.

[0123] Recombinant vector pK18- sdhA C397T Contains a mutation site (C397T), which will lead to sdhA The 397th cytosine (C) in the gene coding region mutated to thymine (T), which ultimately led to the 133rd arginine (R) in the encoded protein SdhA mutated to cysteine (C).

[0124] The recombinant vector pK18- sdhA C397T The pK18mobsacB vector Xbal I and BamH The recombinant vector obtained by replacing the fragment (AGGATCCCC) between the enzyme recognition sites of I with the DNA fragment shown in SEQ ID NO: 5 and keeping the other sequences of the pK18mobsacB vector unchanged is described. sdhA C397T Containing the mutant gene shown in SEQ ID NO: 2 sdhA C397T The mutation site (C397T).

[0125] Example 3: Constructing a sdhA C397T Engineered strains with mutant genes

[0126] The allelic replacement plasmid (pK18- sdhA C397T ) was transformed into the Corynebacterium glutamicum strain CGMCC29950 by electroporation (sequencing confirmed that the wild type was retained on the chromosome of this strain). sdhA Gene coding sequence) and wild-type Corynebacterium glutamicum strain ATCC13869 were cultured on solid culture plates containing kanamycin (50 mg / L) (kanamycin was added to the slant culture medium in Example 1) for 40 hours. Single colonies produced by the culture were identified using primers P1 and P4 described in Example 2, respectively. Strains that amplified the DNA fragment with the nucleotide sequence set forth in SEQ ID NO: 5 were designated as positive strains. The positive strains were streaked on a medium containing 150 g / L sucrose (for plasmid elimination). After the strains grew, single colonies were cultured on media containing kanamycin and those not containing kanamycin. Strains that grew on the medium not containing kanamycin but did not grow on the medium containing kanamycin were selected for further PCR amplification using primers P1 and P4. The resulting multiple DNA fragments (1495 bp) were sequenced. Sequence alignment revealed that strains with a base sequence mutation (C397T) were positive strains with successful allelic substitution, i.e., strains that obtained the DNA fragment shown in SEQ ID NO: 5 by PCR amplification using primers P1 and P4 were positive strains. The positive strains obtained from Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum strain ATCC13869 were named YPG- sdhA -1. sdhA -1.

[0127] Recombinant bacteria YPG- sdhA -1. sdhA -1 all contain the mutant gene shown in SEQ ID NO: 2 sdhA C397T Sequence, capable of expressing the succinate dehydrogenase mutant SdhA shown in SEQ ID NO: 4 R133C . Recombinant bacteria YPG- sdhA -1 differs from Corynebacterium glutamicum CGMCC29950 only in that: YPG- sdhA -1 is the Corynebacterium glutamicum CGMCC29950 sdhA Sequence replaced by sdhA C397T A strain obtained by mutating a sequence while keeping other sequences unchanged; a recombinant strain sdhA -1 differs from wild-type Corynebacterium glutamicum ATCC13869 only in that: sdhA -1 is the wild type Corynebacterium glutamicum ATCC13869 sdhA Sequence replaced by sdhA C397T The strain is obtained by mutating one sequence and keeping other sequences unchanged.

[0128] Example 4: Constructing genome-wide overexpression sdhA C397T Engineered strains with mutant genes

[0129] Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, three pairs of upstream and downstream homology arm fragments were designed and synthesized. sdhA C397T The primers of the gene promoter region and coding region were inserted into Corynebacterium glutamicum CGMCC29950 and wild type Corynebacterium glutamicum ATCC13869 by homologous recombination. sdhA C397T Gene copies.

[0130] The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0131] P5: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagTGATCATCTGCGAAATACGGCC-3' (SEQ ID NO: 14, the nucleotide sequence in lowercase format is the sequence on pK18);

[0132] P6:5'-GGTCGAGGATGTGGAAGATAATGCAGATACAGTCTCATCAATTCTGTGTG-3' (SEQ IDNO: 15);

[0133] P7:5'-CACACAGAATTGATGAGACTGTATCTGCATTATCTTCCACATCCTCGACC-3' (SEQ IDNO: 16);

[0134] P8:5'-GGAAAAGCGAATCATCTCTCATTCCTTACTTGTAGTTCCTTGTCTGCAG-3' (SEQ ID NO: 17);

[0135] P9:5'-CTGCAGACAAGGAACTACAAGTAAGGAATGAGAGATGATTCGCTTTTCC-3' (SEQ ID NO: 18);

[0136] P10: 5′-cagctatgaccatgattacgaattcgagctcggtacccCGAGGAACCCACACCACACTC-3′ (SEQ ID NO: 19, the nucleotide sequence in lowercase format is the sequence on pK18).

[0137] The construction method is as follows:

[0138] Using genomic DNA from Corynebacterium glutamicum ATCC13869 as a template, PCR amplification was performed with primers P5 and P6 to obtain a DNA fragment having a nucleotide sequence of positions 1 to 554 of SEQ ID NO: 6. The DNA fragment contained a pK18 homology arm fragment, the sequence of which was shown in positions 1 to 36 of SEQ ID NO: 6; and a 495-bp upstream homology arm fragment corresponding to the BBD29_05675 pseudoprotein coding region and upstream spacer region on the genome of Corynebacterium glutamicum ATCC13869, the sequence of which was shown in positions 37 to 531 of SEQ ID NO: 6.

[0139] The genome of Corynebacterium glutamicum ATCC13869 was used as a template and PCR amplification was performed with primers P9 and P10 to obtain a DNA fragment having a nucleotide sequence of positions 2859-3467 in SEQ ID NO: 6. The DNA fragment includes a 547-bp downstream homology arm fragment corresponding to a partial coding region of the BBD29_05675 pseudoprotein, BBD29_05680, and a partial coding region of BBD29_05685 in the genome of Corynebacterium glutamicum ATCC13869, the sequence of which is shown at positions 2883-3429 in SEQ ID NO: 6. The DNA fragment also contains a pK18 homology arm fragment, the sequence of which is shown at positions 3430-3467 in SEQ ID NO: 6.

[0140] Plasmid pK18- sdhA C397T As a template, PCR amplification was performed with primers P7 and P8 to obtain a DNA fragment with the nucleotide sequence of SEQ ID NO: 6, positions 505-2907. sdhA C397T The gene promoter and coding region fragment is 2351bp, of which SEQ ID NO: 6 positions 532-860 are sdhA C397T The promoter region of the gene, positions 861-2882 are sdhA C397T The coding region of a gene.

[0141] After the PCR reaction, the three DNA fragments amplified above were recovered by electrophoresis using a column DNA gel recovery kit. The three recovered fragments were ligated with the pK18mobsacB plasmid (Addgene) purified after digestion with Xbal I and BamH I using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligation products were transformed into DH5α competent cells and the resulting single clones were identified by PCR using primers P5 and P10. The fragment with a size of 3467 bp was a positive integration plasmid (recombinant vector). The resulting recombinant vector was named pK18- sdhA C397T OE, the positive integration plasmid contains a kanamycin resistance marker, and recombinants with the plasmid integrated into the genome can be obtained through kanamycin screening.

[0142] The correctly sequenced integration plasmid pK18- sdhA C397T OE was electroporated into Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869, and then cultured on solid culture plates for 40 hours. The single colonies produced by the culture were identified by PCR using primers P11 and P12. The strains that amplified a fragment of 1132 bp (sequence shown in SEQ ID NO: 7) by PCR were positive, and the strains that did not amplify the fragment were the original bacteria. The positive strains were streaked and cultured on solid culture plates containing 150 g / L sucrose for 40 hours (for plasmid elimination). The single colonies produced by the culture were further identified by PCR using primers P13 and P14. The strains that amplified a fragment of 1436 bp (sequence shown in SEQ ID NO: 8) by PCR were positive. sdhA C397T The gene and its promoter were integrated into the BBD29_05675 pseudoprotein (GenBank or NCBI reference sequence number: CP016335.1.2017.04) site of the genome of Corynebacterium glutamicum CGMCC29950 or wild-type Corynebacterium glutamicum ATCC13869. sdhA C397T The bacteria were named YPG- sdhA -2. The wild type Corynebacterium glutamicum ATCC13869 was used as the starting strain, containing the point mutation gene sdhA C397T The bacteria is named sdhA -2.

[0143] Recombinant bacteria YPG- sdhA -2 and sdhA -2 all contain the mutation shown in SEQ ID NO: 2 sdhA C-397T Specifically, the recombinant YPG- sdhA -2 is inserted into the BBD29_05675 pseudoprotein site in the genome of Corynebacterium glutamicum CGMCC29950 sdhA C397T The recombinant bacteria obtained by removing the gene and its promoter sequence and keeping the other nucleotides of the genome of Corynebacterium glutamicum CGMCC29950 unchanged. sdhA -2 can express succinate dehydrogenase having an amino acid sequence of SEQ ID NO: 3 and a succinate dehydrogenase mutant SdhA having an amino acid sequence of SEQ ID NO: 4 R133C . Recombinant bacteria sdhA -2 is inserted into the BBD29_05675 pseudoprotein site in the wild-type Corynebacterium glutamicum ATCC13869 genome sdhA C397T The recombinant bacteria obtained by removing the gene and its promoter sequence and keeping the other nucleotides of the wild-type Corynebacterium glutamicum ATCC13869 genome unchanged. sdhA -2 can express succinate dehydrogenase having an amino acid sequence of SEQ ID NO: 3 and a succinate dehydrogenase mutant SdhA having an amino acid sequence of SEQ ID NO: 4 R133C . sdhA C397T The nucleotide sequence of the gene and its promoter sequence is 532-2882 in SEQ ID NO: 6, wherein 532-860 in SEQ ID NO: 6 is sdhA C397T The promoter region of the gene, positions 861-2882 are sdhA C397T The coding region of a gene.

[0144] PCR identification primers are as follows (synthesized by Shanghai Invitrogen):

[0145] P11: 5'-GCCTGGTTGGCGGACTTAGTC-3' (SEQ ID NO: 20, corresponding to the outside of the upstream homology arm),

[0146] P12: 5'-GTGGTAGGTGAACGCCTTGAC-3' (SEQ ID NO: 21, corresponding to sdhA within the gene),

[0147] P13: 5'-CCATGTACGAAGAGGCAATTGG-3' (SEQ ID NO: 22, corresponding to sdhA within the gene),

[0148] P14: 5'-ACCTCGAAGCCGATGGACAC-3' (SEQ ID NO: 23, corresponding to the outside of the downstream homology arm).

[0149] Example 5: Construction of plasmid for overexpression sdhA Engineered strains with mutant genes

[0150] Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, we designed and synthesized amplification sdhA C397T Primers for the gene coding region and promoter region were used to overexpress the gene in Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869 using expression vector pXMJ19 sdhA C397T Gene.

[0151] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen):

[0152] P15:5'-cagaataattaagcttgcatgcctgcaggtcgacCATTATCTTCCACATCCTCGACC-3' (SEQ ID NO: 24, the nucleotide sequence in lowercase format is the pXMJ19 homologous sequence);

[0153] P16:5′-ccaaaacagccaagctgaattcgagctcggtaccTTACTTGTAGTTCCTTGTCTGCAG-3′ (SEQ ID NO: 25, the nucleotide sequence expressed in lowercase format is the pXMJ19 homologous sequence).

[0154] Plasmid pK18- sdhA C397T OE was used as template and primers P15 and P16 were used for PCR amplification to obtain sdhA C397TThe gene promoter and coding region fragments were purified and then ligated with the expression vector pXMJ19 (purchased from TaKaRa, containing chloramphenicol resistance) recovered by double digestion with Xbal I and BamH I using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation product was transformed into DH5α competent cells and spread onto 2-YT agar plates containing chloramphenicol (34 mg / L) and cultured in a constant temperature incubator at 37°C for 12 h. The single clones grown in the culture were identified by PCR using primers P15 and P16. The PCR amplified a fragment of 2419 bp (sequence shown in SEQ ID NO: 9) containing sdhA C397T The positive transformant pXMJ19- sdhA C397T .

[0155] The correctly sequenced pXMJ19- sdhA C397T The plasmids were electroporated into Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869, respectively, and cultured on solid culture plates for 40 h. The single colonies produced by the culture were identified by PCR using primers P15 and P16. The strains that amplified the DNA fragment with the sequence shown in SEQ ID NO: 9 were positive. sdhA C397T The bacteria were named YPG- sdhA -3. Using wild-type Corynebacterium glutamicum ATCC13869 as the starting strain, the plasmid pXMJ19- sdhA C397T The bacteria is named sdhA -3. YPG- sdhA -3 and sdhA -3 contains sdhA C397T Gene promoter and coding region fragment, can express succinate dehydrogenase mutant SdhA R133C .

[0156] Example 6, L-glutamic acid fermentation experiment

[0157] The strains constructed in Examples 3-5, Corynebacterium glutamicum CGMCC29950, and wild-type Corynebacterium glutamicum ATCC13869 were fermented in 500 mL baffled shake flasks using the culture medium shown below and the control conditions shown below. After fermentation, L-glutamic acid production was detected using an SBA-biosensor analyzer (Institute of Biology, Shandong Academy of Sciences). Each strain was repeated three times, and the results are shown in Table 1.

[0158] Culture medium formula: glucose 5.0 g / L, phosphoric acid 0.38 g / L, magnesium sulfate 1.85 g / L, potassium chloride 1.6 g / L, biotin 550 μg / L, vitamin 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, p-aminobenzoic acid 0.75 mg / L, defoamer 0.0015 mL / dL, betaine 1.5 g / L, cane molasses 7 mL / L, corn steep liquor 77 mL / L, aspartic acid 1.7 g / L, hair powder 2 g / L. The solvent is water.

[0159] Fermentation control conditions: inoculation amount 10% (volume percentage), pH 7.0, culture temperature 32°C, control condition rotation speed 220 rpm, fermentation period 36 h.

[0160]

[0161] Note: P < 0.01 in the table indicates a highly significant difference compared to the starting strain. Data were processed using Excel software, and experimental results are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used for analysis of variance. P < 0.01 indicates a highly significant difference.

[0162] The fermentation results are shown in Table 1. The results show that: sdhA Gene coding region sdhA C397T Point mutation and overexpression sdhA C397T genes, can significantly increase the L-glutamic acid production of the strain, that is, sdhA The gene was mutated into C397T point mutation sdhA C397T Gene or introduce the above into the recipient bacteria sdhA C397T The gene can significantly increase the L-glutamate production of the strain. R133C The L-glutamic acid production of the strain can be significantly increased.

[0163] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art.

Claims

1. Protein, characterized in that: The protein comprises a protein obtained by mutating the 133rd amino acid of succinate dehydrogenase from arginine to cysteine; the protein has an amino acid sequence of SEQ ID NO:

4.

2. Biomaterial, characterized in that: The biomaterial comprises at least one of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette and / or construct containing the nucleic acid molecule described in B1), B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or a recombinant vector containing the expression cassette and / or construct described in B2).

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

2.

4. Application, characterized by: The application includes at least one of the following: D1) Use of the protein according to claim 1 in the preparation of glutamic acid; D2) Use of the protein according to claim 1 in increasing the glutamic acid production of Corynebacterium glutamicum; D3) Use of the biological material according to claim 2 or 3 in constructing a recombinant Corynebacterium glutamicum that produces glutamate; D4) Use of the biomaterial according to claim 2 or 3 in the preparation of glutamic acid; D5) Use of the biomaterial according to claim 2 or 3 in increasing the yield of glutamic acid in Corynebacterium glutamicum; D6) Use of the biomaterial according to claim 2 or 3 in constructing a recombinant Corynebacterium glutamicum with high glutamate production.

5. A recombinant microorganism, characterized in that: The recombinant microorganism comprises at least one of the following: E1), a recombinant microorganism containing or expressing the protein according to claim 1; E2), a recombinant microorganism containing the nucleic acid molecule according to B1) of claim 2 or 3; E3), a recombinant microorganism containing the expression cassette and / or construct described in B2) of claim 2 or 3; E4), a recombinant microorganism containing the recombinant vector described in B3) of claim 2 or 3; The microorganism is Corynebacterium glutamicum.

6. A composition for preparing glutamic acid, characterized in that: The composition contains the recombinant microorganism according to claim 5.

7. A method for increasing glutamic acid production of a microorganism, characterized in that: The method comprises at least one of the following: F1), causing the target microorganism to express the protein according to claim 1; F2), mutating the gene encoding succinate dehydrogenase in the target microorganism into the nucleic acid molecule according to claim 2 or 3, wherein the target microorganism contains the gene encoding succinate dehydrogenase; F3), introducing the biological material according to claim 2 or 3 into the target microorganism; F4), increasing the expression level of the gene encoding the protein according to claim 1 in the target microorganism or increasing the content of the protein according to claim 1 in the target microorganism; The microorganism is Corynebacterium glutamicum.

8. A method for preparing glutamic acid, characterized in that: The method comprises at least the step of producing glutamic acid using the recombinant microorganism according to claim 5 and / or the composition according to claim 6.

9. Use of the recombinant microorganism according to claim 5 or the composition according to claim 6 in the preparation of glutamic acid.

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