Succinate dehydrogenase mutant and application thereof in increasing yield of glutamic acid

Abstract

The invention discloses a succinate dehydrogenase mutant and application thereof in increasing the yield of glutamic acid, and belongs to the technical field of microorganisms. The technical problem to be solved by the invention is how to improve the glutamic acid yield of microorganisms. In order to solve the technical problem, the protein provided by the invention 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 an amino acid sequence of SEQ ID NO: 4. The invention provides the succinate dehydrogenase mutant (SdhAR133C) capable of improving the glutamic acid yield in a target microorganism, and a recombinant bacterium verification result shows that compared with an original target microorganism, the glutamic acid yield of recombinant corynebacterium glutamicum expressing the succinate dehydrogenase mutant (SdhAR133C) is remarkably improved, and an unexpected technical effect is achieved.

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 in the molecule and its chemical name is α-aminoglutaric acid. As an important nutrient for human growth, glutamate not only has special physiological effects, but also has unique functions in the food industry.

[0003] At present, the most commonly used production method of glutamic acid is fermentation method, mainly using Corynebacterium glutamicum as the production bacteria to ferment and produce glutamic acid.Corynebacterium glutamicum is a heterotrophic aerobic type, is a Gram-positive bacteria, has the characteristics of fast growth rate, non-pathogenicity, weak degradation ability to its own metabolites.Fermentation method has the advantages of wide sources of raw materials, low production cost, controllable product quality, single product, etc.But the fermentation performance of the strain of producing glutamic acid is still poor at present, and the glutamic acid conversion rate is undesirable, and glutamic acid is the main raw material of producing monosodium glutamate, and the industrial demand for glutamic acid is extremely high, and existing strains can not meet the demand of large-scale industrial production at all.Further improving the bacterial strain, improving the production strain growth, and improving the glutamic acid conversion rate are still problems that need to be solved at present. Summary of the invention

[0004] The technical problem to be solved by the present application is: how to increase the glutamic acid production of microorganisms. The technical problem to be solved by the present application is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] To solve the above technical problems, 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) (i.e., a succinate dehydrogenase mutant); The protein comprises any of the following: A1), a protein comprising the amino acid sequence of SEQ ID NO: 4; A2), a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1), which has an amino acid sequence identity of more than 70% with the protein of A1), and has the function of succinate dehydrogenase; A3) is a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1) or A2).

[0006] 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.

[0007] The protein of the present application is a succinate dehydrogenase mutant of Corynebacterium glutamicum (wild type, amino acid sequence is SEQ ID NO: 3) in which the 133rd amino acid in SEQ ID NO: 3 is mutated from arginine (R) to cysteine ​​(C). The amino acid sequence of the obtained succinate dehydrogenase mutant is SEQ ID NO: 4.

[0008] In the present application, SEQ ID NO: 4 consists of 673 amino acid residues.

[0009] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0010] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0011] Further, the connection described in A3) can be a peptide bond connection formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in A1) or A2). Or, the connection described in A3) can be a peptide bond connection formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in A1) or A2). A connecting peptide can also be included between the tag and the protein. The connecting peptide is dehydrated and condensed with the N-terminus of the tag and the C-terminus of the protein to form a peptide bond. Or the connecting peptide is dehydrated and condensed with the C-terminus of the tag and the N-terminus of the protein to form a peptide bond.

[0012] The present application also provides a biological material related to the protein, wherein the biological material comprises any of the following: B1), a nucleic acid molecule encoding the protein; B2), an expression cassette 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 described in B2).

[0013] Furthermore, the nucleic acid molecule described in B1) comprises C1) or C2), C1), a nucleic acid molecule whose coding sequence comprises SEQ ID NO: 2; C2), a nucleic acid molecule having more than 70% identity with the nucleic acid molecule described in C1).

[0014] Furthermore, the nucleic acid molecule in C1) may also be a nucleic acid molecule having a coding sequence of SEQ ID NO: 2.

[0015] The present application also provides an application, wherein the application comprises at least any one of the following: D1), use of the protein in preparing glutamate; D2), use of the protein in increasing glutamate production of microorganisms; D3) Application of the biomaterial in constructing an engineered bacterium producing glutamate; D4), application of the biomaterial in the preparation of glutamic acid; D5) Application of the biomaterial in increasing the yield of glutamic acid of microorganisms; D6) Use of the biomaterial in constructing a recombinant microorganism with high glutamic acid production. The recombinant microorganism with high glutamic acid production is obtained by transforming a target microorganism, and the glutamic acid production of the recombinant microorganism is higher than that of the target microorganism.

[0016] Furthermore, the target microorganism described in D6) can be any of the following G1) Microorganisms capable of producing glutamate; G2), bacteria; G3), Gram-positive bacteria; G4), Corynebacterium; G5), Corynebacterium glutamicum.

[0017] The present application also provides a recombinant microorganism, wherein the recombinant microorganism comprises at least any one of the following: E1), a recombinant microorganism containing or expressing the protein; E2), a recombinant microorganism containing the nucleic acid molecule described in B1); E3), a recombinant microorganism containing the expression cassette and / or construct described in B2) above; E4), a recombinant microorganism containing the recombinant vector described in B3) above.

[0018] The present application also provides a composition for preparing glutamate, which may contain the recombinant microorganism.

[0019] 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.

[0020] The above-mentioned culture may be a substance obtained by culturing the recombinant microorganism in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the recombinant microorganism and a substance secreted into a liquid culture medium, or a solid fermentation product containing the recombinant microorganism and a substance secreted into a solid culture medium).

[0021] In the above, the metabolite may be a product obtained by removing the recombinant microorganism from the culture, such as culturing the recombinant microorganism in a liquid fermentation medium, collecting the fermentation broth (containing the recombinant microorganism and substances secreted into the liquid culture medium), removing the recombinant microorganism from the fermentation broth, and collecting the remaining components of the fermentation broth to obtain the metabolite of the recombinant microorganism.

[0022] The active ingredients of the above composition may also contain other biological ingredients or non-biological ingredients. Those skilled in the art may determine the other active ingredients of the above composition according to the effects of the composition.

[0023] The above composition may be the culture. The above composition may also be a bacterial agent.

[0024] 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.

[0025] The above-mentioned bacterial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0026] The bacterial agent may further include a carrier as required, and the carrier may be a solid carrier or a liquid carrier.

[0027] The present application also provides a method for increasing the glutamate production of a microorganism, the method comprising at least any one of the following: F1), causing the target microorganism to express the protein; 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; F3), introducing the above-mentioned biological materials into the target microorganism; 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.

[0028] Furthermore, the target microorganism in F1) may contain a gene encoding succinate dehydrogenase (wild type) or may not contain a gene encoding succinate dehydrogenase (wild type).

[0029] Further, the mutation described in F2) may specifically be a mutation of the deoxyribonucleotide at position 397 of the nucleotide sequence in the target microorganism at SEQ ID NO: 1 from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T), and the coding sequence of the succinate dehydrogenase (i.e., the succinate dehydrogenase mutant) after the mutation is SEQ ID NO: 2. The mutation causes the 133rd amino acid in the wild-type succinate dehydrogenase of Corynebacterium glutamicum in the target microorganism (the amino acid sequence of the wild-type succinate dehydrogenase is SEQ ID NO: 3) to mutate from arginine (R) to cysteine ​​(C), and the amino acid sequence of the succinate dehydrogenase mutant is shown in SEQ ID NO: 4.

[0030] 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 corresponding coding gene of the succinate dehydrogenase mutant is SEQ ID NO: 2, which is also used in the present application. sdhA C397T The amino acid sequence corresponding to the succinate dehydrogenase mutant is SEQ ID NO: 4, which is also referred to as SdhA in this application. R133C express.

[0031] Furthermore, in the method described above, the target microorganism may 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.

[0032] Furthermore, the Corynebacterium glutamicum contains BBD29_05675 pseudoprotein (GenBank or NCBI reference sequence number: CP016335.1.2017.04).

[0033] 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.

[0034] Among the recombinant microorganisms described in the present application, the recombinant microorganism obtained by using Corynebacterium glutamicum as the target microorganism is named as 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.

[0035] The recombinant Corynebacterium glutamicum can also be obtained by mutating the deoxyribonucleotide at position 397 of SEQ ID NO: 1 of the coding gene of 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).

[0036] The recombinant Corynebacterium glutamicum can also be obtained by transforming the target Corynebacterium glutamicum with a recombinant plasmid containing an expression cassette and / or a construct of a gene encoding a gene containing a succinate dehydrogenase mutant to obtain a gene encoding a gene containing a succinate dehydrogenase mutant. sdhA C397T (SEQ ID NO: 2) and / or a recombinant bacterium expressing a succinate dehydrogenase mutant (SEQ ID NO: 4).

[0037] In some embodiments of the present application, the Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC29950.

[0038] The present application also provides the use of the recombinant microorganism or the composition in preparing glutamic acid.

[0039] Furthermore, the method comprises the steps of culturing the recombinant microorganism or composition with a culture medium, collecting the culture product, and obtaining glutamic acid. The glutamic acid described in the present application may be L-glutamic acid.

[0040] In the present application, "mutated succinate dehydrogenase", "succinate dehydrogenase mutant", "succinate dehydrogenase mutant (SdhA R133C )" and "SdhA R133C ” have the same meaning.

[0041] The beneficial technical effects achieved by this application are as follows: The present application provides a succinate dehydrogenase mutant (SdhA) having 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 glutamate production of the recombinant Corynebacterium glutamicum expressing the succinate dehydrogenase mutant was significantly improved, achieving unexpected technical effects.

[0042] Collection Instructions Bacterial species name: Corynebacterium glutamicum; Latin name: Corynebacterium glutamicum ; Classification name: Corynebacterium glutamicum Corynebacterium glutamicum ; Strain ID: YP077-2; Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration; Abbreviation of depository unit: CGMCC; Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; Deposit date: March 7, 2024; The registration number of the Collection Center is: CGMCC No. 29950. DETAILED DESCRIPTION

[0043] Terms used in this application: 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.

[0044] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

[0045] To facilitate understanding of the present disclosure, several terms and abbreviations used herein are defined as follows: In this application, "consistency" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence (or nucleotide sequence) can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for a pair of amino acid sequences to calculate the identity, and then the identity value (%) can be obtained.

[0046] Specifically, the consistency of more than 70% may be more than 75%. Specifically, the consistency of more than 75% may be more than 80%. Specifically, the consistency of more than 80% may be more than 85%. Specifically, the consistency of more than 85% may be more than 90%. Specifically, the consistency of more than 90% may be more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%. 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%. 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.

[0047] 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.

[0048] As generally understood in the art, the term "promoter" may generally refer to a DNA containing an RNA polymerase binding site and / or a transcription start site and assisting or promoting transcription of a transcribable DNA. The promoter sequence of a prokaryotic organism is located at the 5' end of the transcription start site (TSS), covering a region of about 40 bp in length, and structurally generally includes a transcription start site (denoted as +1), a -35 region, a -10 region, and a spacer region between the -35 region and the -10 region. The promoter may be produced, altered, or derived from a known or naturally occurring promoter by artificial synthesis. The promoter may also include a chimeric promoter comprising a combination of two or more heterologous sequences. Therefore, the promoter of the present application may include a variant of a promoter sequence that is similar in composition but not identical to other promoter sequences provided herein.

[0049] Promoters can be classified according to various standards related to the expression pattern of the relevant coding or transcribable sequence or gene (including transgenic) operably connected to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. The promoter that drives expression in all or most tissues of the receptor is called "constitutive" promoter. The promoter that drives expression in certain periods or stages of development is called "developmental" promoter. "Inducible" promoter is a promoter that starts 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.

[0050] The term "transcribable DNA" refers to DNA that can be transcribed into an RNA molecule.

[0051] The term "operably linked" may refer to a functional connection between a promoter and a transcribable DNA, so 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 target gene to regulate the transcription and / or expression of the target gene.

[0052] The term "construct" means any recombinant DNA molecule or recombinant RNA molecule. A recombinant DNA molecule may be a plasmid, a cosmid, a virus, a phage, or a linear or circular DNA. A construct typically includes one or more expression cassettes.

[0053] As used herein, an "expression cassette" refers to a composition comprising at least transcribable DNA operably linked to one or more regulatory elements, usually at least a promoter and a 3'UTR (eg, a terminator).

[0054] As used herein, the term "vector" means any construct that can be used for transformation purposes, ie, to introduce heterologous DNA into a host cell, such as a plasmid, cosmid, virus, phage, or linear or circular DNA.

[0055] The term "microorganism capable of producing glutamate" means that the microorganism has the following ability: the ability to produce and accumulate glutamate in vivo using external substances (such as culture medium), and further includes the ability to secrete glutamate into the culture system. Thus, glutamate can be collected when the microorganism is cultured in the culture medium.

[0056] The term "culture product" refers to a liquid or solid product (all materials in the culture container) that has a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which 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 without the microbial cells.

[0057] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] Corynebacterium glutamicum ATCC13869, also known as ATCC13869, was purchased from the American Type Culture Collection (ATCC) with a strain number of 13869; Corynebacterium glutamicum ( Corynebacterium glutamicum ) CGMCC No. 29950, also known as Corynebacterium glutamicum CGMCC No. 29950, CGMCC29950 or CGMCC NO. 29950, the strain was deposited under patent procedure on March 7, 2024, and the deposit number is CGMCC NO. 29950. In the following embodiments, CGMCC29950 is used to represent the strain.

[0060] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0061] Example 1. Isolation and growth test of mutant strain S3 Based on Corynebacterium glutamicum CGMCC29950, the growth advantage strain S3 was obtained by random mutation using ultraviolet mutagenesis. The genome of S3 was sequenced and analyzed, and it was found that sdhA The arginine (R) at position 133 of the gene translation protein is mutated to cysteine ​​(C), and the amino acid sequence of the corresponding succinate dehydrogenase SdhA mutant is shown in SEQ ID NO:4.

[0062] The specific screening method of mutant strain S3 is as follows: 1. Strain mutagenesis screening The CGMCC29950 strain was used as the starting strain. After inoculation with 2.5% of the inoculum into the 24-well plate liquid culture medium, it was irradiated with ultraviolet light at a vertical distance of 15 cm for 45 s under a 15W ultraviolet lamp. The culture was then kept away from light 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 on the 24-well plate for 12 hours, dilute the culture solution twice 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 mutant strain S3 was significantly increased compared with the control strain CGMCC29950.

[0063] 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, adjust pH to 7.0.

[0064] Slant culture medium: Add 20 g / L agar powder to the liquid culture medium.

[0065] 2. Genome sequencing The genome of the original strain CGMCC29950 and the mutant strain S3 was extracted and sequenced using a genome extraction kit (Novozyme Biotechnology Co., Ltd.). By comparing the sequencing results, it was found that the succinate dehydrogenase gene in the tricarboxylic acid cycle sdhA (SEQ ID NO: 1) The nucleotide at position 397 is mutated from cytosine deoxyribonucleotide (C) to thymine deoxyribonucleotide (T), resulting in sdhA C397T The mutant sequence (SEQ ID NO: 2) of SdhA resulted in the amino acid at position 133 in the amino acid sequence (SEQ ID NO: 3) of SdhA being mutated from arginine (R) to cysteine ​​(C). The amino acid sequence after the 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.

[0066] Example 2: Construction of a sdhA Recombinant vector of gene coding region Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, two pairs of amplification sdhA The primers for the gene coding region were used to replace the allele in the C. 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) is mutated to a thymine deoxyribonucleotide (T), thereby obtaining a DNA molecule shown in SEQ ID NO: 2 (mutated sdhA Gene sequence, named sdhA C397T ).

[0067] 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 (SEQ ID NO: 4) of the succinate dehydrogenase mutant is mutated from arginine (R).

[0068] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen): P1: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagCATTATCTTCCACATCCTCGACCTG-3' (SEQ ID NO: 10, the nucleotide sequence in lowercase format is the sequence on pK18); P2: 5'-cagtcggactcgcggccacAgtagtcgccgcccttgacg-3' (SEQ ID NO: 11, the deoxynucleotide base (position 20) indicated by capitalization is the mutation position); P3: 5'-cgtcaagggcggcgactacTgtggccgcgagtccgactg-3' (SEQ ID NO: 12, the deoxynucleotide base (position 20) indicated by capitalization is the mutation position); P4: 5′-cagctatgaccatgattacgaattcgagctcggtacccGGAAGCAACGTCACGTGGGAC-3′ (SEQ ID NO: 13, the nucleotide sequence in lowercase format is the sequence on pK18).

[0069] 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 and primers P3 and P4, a 753 bp fragment with a mutation site was obtained. sdhA Gene fragment, named sdhA Down-1 (SEQ ID NO: 5 positions 743 to 1495).

[0070] 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.

[0071] 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.

[0072] 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) was ligated with NEBuilder (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 with primers P1 and P4. The one that could amplify a DNA fragment of 1495 bp (SEQ ID NO: 5) was the 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 identification, and the recombinant vector pK18- sdhA C397T Save for later use.

[0073] 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 mutation of the 133rd arginine (R) in the encoded protein SdhA to cysteine ​​(C).

[0074] The recombinant vector pK18- sdhA C397T The pK18mobsacB vector Xbal I and BamH The recombinant vector obtained by replacing the fragment (AGGATCCCC) between the restriction endonuclease I recognition sites with the DNA fragment shown in SEQ ID NO: 5, and keeping the other sequences of the pK18mobsacB vector unchanged, the recombinant vector pK18- sdhA C397T Containing the mutant gene shown in SEQ ID NO: 2 sdhA C397T The mutation site (C397T).

[0075] Example 3: Constructing a sdhA C397T Mutant gene engineered strains The allelic replacement plasmid (pK18- sdhA C397T ) was transformed into the Corynebacterium glutamicum strain CGMCC29950 by electroporation (sequencing confirmed that the wild-type sdhA Gene coding sequence) and wild-type Corynebacterium glutamicum strain ATCC13869 were cultured on a solid culture plate containing kanamycin (50 mg / L) (kanamycin was added to the slant culture medium in Example 1) for 40 h. The single colonies produced by the culture were identified by primers P1 and P4 in Example 2, respectively, and the strains that could amplify the nucleotide sequence of the DNA fragment shown in SEQ ID NO: 5 were positive strains. The positive strain was streaked and cultured on a medium containing 150 g / L sucrose (for plasmid elimination). After the strain grew, the single colony was cultured on a medium containing kanamycin and one without kanamycin, respectively. The strain that grew on the medium without kanamycin but did not grow on the medium containing kanamycin was selected for further PCR amplification using primers P1 and P4. The obtained multiple DNA fragments (1495 bp) were sequenced. Through sequence alignment, the strain with a mutation in the base sequence (C397T) was a positive strain with successful allelic substitution, that is, the strain that obtained the DNA fragment shown in SEQ ID NO: 5 by PCR amplification using primers P1 and P4 was a positive strain. The positive strains obtained from Corynebacterium glutamicum CGMCC29950 and the wild-type Corynebacterium glutamicum strain ATCC13869 were named YPG- sdhA -1. sdhA -1.

[0076] 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 The sequence is replaced by sdhA C397T A strain obtained by mutating a sequence and keeping other sequences unchanged; a recombinant bacterium sdhA -1 differs from wild-type Corynebacterium glutamicum ATCC13869 only in that: sdhA -1 is the wild type Corynebacterium glutamicum ATCC13869 sdhA The sequence is replaced by sdhA C397T The strain obtained by mutating the sequence and keeping other sequences unchanged.

[0077] Example 4: Construction of overexpression on genome sdhA C397T Mutant gene engineered strains 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 the coding region were inserted into Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869 by homologous recombination. sdhA C397T Gene copy.

[0078] Primers were designed as follows (synthesized by Shanghai Invitrogen): P5: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagTGATCATCTGCGAAATACGGCC-3' (SEQ ID NO: 14, the nucleotide sequence in lowercase format is the sequence on pK18); P6:5'-GGTCGAGGATGTGGAAGATAATGCAGATACAGTCTCATCAATTCTGTGTG-3' (SEQ IDNO: 15); P7:5'-CACACAGAATTGATGAGACTGTATCTGCATTATCTTCCACATCCTCGACC-3' (SEQ IDNO: 16); P8:5'-GGAAAAGCGAATCATCTCTCATTCCTTACTTGTAGTTCCTTGTCTGCAG-3' (SEQ ID NO: 17); P9:5'-CTGCAGACAAGGAACTACAAGTAAGGAATGAGAGATGATTCGCTTTTCC-3' (SEQ ID NO: 18); P10: 5′-cagctatgaccatgattacgaattcgagctcggtacccCGAGGAACCCACACCACACTC-3′ (SEQ ID NO: 19, the nucleotide sequence in lowercase format is the sequence on pK18).

[0079] The construction method is as follows: The genomic DNA of Corynebacterium glutamicum ATCC13869 is used as a template, and PCR amplification is performed with primers P5 and P6 to obtain a DNA fragment whose nucleotide sequence is the 1st to 554th positions of SEQ ID NO: 6. The DNA fragment contains a pK18 homology arm fragment, whose sequence is the 1st to 36th positions of SEQ ID NO: 6; and contains a 495bp upstream homology arm fragment corresponding to the BBD29_05675 pseudoprotein coding region and the upstream spacer region on the genome of Corynebacterium glutamicum ATCC13869, whose sequence is the 37th to 531st positions of SEQ ID NO: 6.

[0080] The genome of Corynebacterium glutamicum ATCC13869 is used as a template, and PCR amplification is performed with primers P9 and P10 to obtain a DNA fragment whose nucleotide sequence is 2859-3467 in SEQ ID NO: 6. The DNA fragment includes a downstream homology arm fragment of 547 bp corresponding to the BBD29_05675 pseudoprotein partial coding region, BBD29_05680, and BBD29_05685 partial coding regions of the genome of Corynebacterium glutamicum ATCC13869, and its sequence is shown in 2883-3429 in SEQ ID NO: 6. The DNA fragment also contains a pK18 homology arm fragment, and its sequence is shown in 3430-3467 in SEQ ID NO: 6.

[0081] Plasmid pK18- sdhA C397T As a template, PCR amplification was performed with primers P7 and P8 to obtain a DNA fragment with a nucleotide sequence of SEQ ID NO: 6, positions 505-2907. The DNA fragment contains 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.

[0082] After the PCR reaction, the three DNA fragments obtained by the above amplification were electrophoretically recovered using a column DNA gel recovery kit. The three recovered fragments were connected to the pK18mobsacB plasmid (Addgene) purified after digestion with Xbal I and BamH I using NEBuilder enzyme (NEB) at 50 °C for 30 min. The single clones grown after the ligation product was transformed into DH5α competent cells were identified by PCR using primers P5 and P10. The fragment size of 3467 bp was obtained as a positive integration plasmid (recombinant vector). The obtained recombinant vector was named pK18- sdhA C397T OE, this positive integration plasmid contains a kanamycin resistance marker, and recombinants with the plasmid integrated into the genome can be obtained through kanamycin screening.

[0083] The correctly sequenced integration plasmid pK18- sdhA C397T OE was transformed into Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869 by electroporation, 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 positive strains were amplified by PCR containing a fragment of 1132 bp (sequence as shown in SEQ ID NO: 7), and the strains that could 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), and the single colonies produced by the culture were further identified by PCR using primers P13 and P14. The bacteria amplified by PCR containing a fragment of 1436 bp (sequence as shown in SEQ ID NO: 8) were sdhA C397T The gene and its promoter are 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 a point mutation gene sdhA C397T The bacteria is named sdhA -2.

[0084] Recombinant bacteria YPG- sdhA -2 and sdhA -2 all contain the mutation shown in SEQ ID NO: 2 sdhA C-397T Specifically, the recombinant bacteria 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 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 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.

[0085] The PCR identification primers are as follows (synthesized by Shanghai Invitrogen): P11: 5'-GCCTGGTTGGCGGACTTAGTC-3' (SEQ ID NO: 20, corresponding to the outside of the upstream homology arm), P12: 5'-GTGGTAGGTGAACGCCTTGAC-3' (SEQ ID NO: 21, corresponding to sdhA inside the gene), P13: 5'-CCATGTACGAAGAGGCAATTGG-3' (SEQ ID NO: 22, corresponding to sdhA inside the gene), P14: 5'-ACCTCGAAGCCGATGGACAC-3' (SEQ ID NO: 23, corresponding to the outer side of the downstream homology arm).

[0086] Example 5: Construction of overexpression on plasmid sdhA Mutant gene engineered strains 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.

[0087] The recombinant vector was constructed using NEBuilder assembly technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen): P15: 5′-cagaataattaagcttgcatgcctgcaggtcgacCATTATCTTCCACATCCTCGACC-3′ (SEQ ID NO: 24, the nucleotide sequence in lowercase format is the pXMJ19 homologous sequence); P16: 5′-ccaaaacagccaagctgaattcgagctcggtaccTTACTTGTAGTTCCTTGTCTGCAG-3′ (SEQ ID NO: 25, the nucleotide sequence in lowercase format is the pXMJ19 homologous sequence).

[0088] Plasmid pK18- sdhA C397T OE was used as template, and primers P15 and P16 were used for PCR amplification to obtain sdhA C397T The gene promoter and coding region fragments were purified and then connected with the expression vector pXMJ19 (purchased from TaKaRa, containing chloramphenicol resistance) recovered by double digestion with Xbal I and BamH I through NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation product was transformed into DH5α competent cells and spread on 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 fragment with a size of 2419 bp (sequence as shown in SEQ ID NO: 9) amplified by PCR was the one containing sdhA C397TThe positive transformant pXMJ19- sdhA C397T .

[0089] The sequenced pXMJ19- sdhA C397T The plasmids were electrotransformed into Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869, respectively, and cultured on solid culture plates for 40 hours. The single colonies produced by the culture were identified by PCR using primers P15 and P16. The DNA fragment with the sequence shown in SEQ ID NO: 9 was a positive strain. sdhA C397T The bacteria were named YPG- sdhA -3. The wild-type Corynebacterium glutamicum ATCC13869 was used as the starting strain, containing 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 .

[0090] Example 6, L-glutamic acid fermentation experiment The strains constructed in Examples 3-5, Corynebacterium glutamicum CGMCC29950 and wild-type Corynebacterium glutamicum ATCC13869 were fermented in 500 mL baffled shake flasks with the culture medium and control conditions shown below. After the fermentation, the 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.

[0091] 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, defoaming agent 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.

[0092] 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.

[0093]

[0094] Note: P<0.01 in the table indicates that there is a very significant difference compared with the starting strain. The data were processed using Excel software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used for the test, and P<0.01 indicated a very significant difference.

[0095] The fermentation results are shown in Table 1. The results show that: sdhA Gene coding region sdhA C397T Point mutation and overexpression sdhA C397T gene, 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 genes can significantly increase the L-glutamate production of the strain. R133C The L-glutamic acid production of the strain can be significantly increased.

[0096] 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 experimental conditions, the present application can be implemented in a wider 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 a word, 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 departing from the disclosed scope in the present application and changes made with 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 comprises at least one of the following: A1), a protein comprising the amino acid sequence of SEQ ID NO: 4; A2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1).

2. Biomaterial, characterized in that: The biological material 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 in that: 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 glutamate production of microorganisms; D3) Use of the biomaterial according to claim 2 or 3 in constructing an engineered bacterium producing 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 production of glutamic acid by microorganisms; D6) Use of the biomaterial according to claim 2 or 3 in constructing a recombinant microorganism 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 described in 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 claim 2 or 3 B3).

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 the 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 described in 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.

8. The method according to claim 7, characterized in that: The target microorganism is Corynebacterium glutamicum.

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

10. 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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