WO2022143763A1 - 具有增强的l-谷氨酸生产力的菌株及其构建方法与应用 - Google Patents

具有增强的l-谷氨酸生产力的菌株及其构建方法与应用 Download PDF

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WO2022143763A1
WO2022143763A1 PCT/CN2021/142440 CN2021142440W WO2022143763A1 WO 2022143763 A1 WO2022143763 A1 WO 2022143763A1 CN 2021142440 W CN2021142440 W CN 2021142440W WO 2022143763 A1 WO2022143763 A1 WO 2022143763A1
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seq
bbd29
amino acid
polynucleotide
glutamic acid
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PCT/CN2021/142440
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English (en)
French (fr)
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苏厚波
魏爱英
孟刚
杨立鹏
马风勇
贾慧萍
周晓群
赵春光
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宁夏伊品生物科技股份有限公司
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Priority to KR1020237025466A priority Critical patent/KR20230145055A/ko
Priority to US18/270,491 priority patent/US20240067998A1/en
Priority to JP2023540092A priority patent/JP2024505808A/ja
Priority to EP21914495.3A priority patent/EP4273228A1/en
Publication of WO2022143763A1 publication Critical patent/WO2022143763A1/zh
Priority to ZA2023/06650A priority patent/ZA202306650B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses

Definitions

  • the invention belongs to the field of genetic engineering and microorganism technology, and in particular relates to a strain with enhanced L-glutamic acid productivity and a construction method and application thereof.
  • L-glutamic acid the chemical name is L-2-aminoglutaric acid, the molecular formula is C 5 H 9 O 4 N, and the molecular weight is 147.13.
  • L-glutamic acid is a non-essential amino acid.
  • the glutamic acid that exists in biological organisms belongs to L-type glutamic acid.
  • L-glutamic acid is the precursor of monosodium glutamate. After monosodium glutamate is eaten, it can also be converted into glutamic acid in the stomach, and then participate in the synthesis of protein through digestion and absorption, and can synthesize other amino acids through transamination, which has high nutritional value. In addition to being widely used in food, it is also used in medicine, cosmetics and agricultural production.
  • glutamate is the only amino acid involved in the metabolism of the human brain and plays an important role in the maintenance of nervous system function. Therefore, for neurasthenia, increasing the intake of glutamate can improve the function of their nervous system.
  • glutamic acid can be used to synthesize sodium polyglutamate. Due to the strong hygroscopicity of this substance, it can be used as an emollient. Glutamic acid can also be combined with lauryl phthalochloride to form sodium lauryl phthalate glutamate, which is widely used in
  • Glutamate is mainly used in agriculture to produce fungicides, such as glutamate ketones.
  • glutamic acid can also be used as a carrier of fertilizers to promote the absorption of nitrogen, phosphorus and potassium by crops.
  • the present invention provides a polynucleotide and a recombinant strain containing the polynucleotide, and the use of the recombinant strain to improve the L-glutamic acid production capacity of bacteria.
  • the inventors of the present invention have found that the BBD29_00405 gene (GenBank: ANU32350.1) in the genome of Corynebacterium glutamicum ATCC13869 (GenBank: CP016335.1) with L-glutamic acid production ability can be modified by modifying The expression of the gene may be improved to obtain a recombinant strain with improved L-glutamic acid production. Compared with the unmodified wild-type strain, the recombinant strain has a stronger L-glutamic acid production ability.
  • the present invention adopts the following technical scheme to realize:
  • a first aspect of the present invention provides L-glutamic acid producing bacteria having improved expression of a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:3.
  • the improved expression is enhanced expression of the polynucleotide, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has a point mutation, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 Has point mutations and expression is enhanced.
  • the amino acid sequence of the SEQ ID NO: 3 is the protein encoded by the gene BBD29_00405.
  • the bacteria have enhanced L-glutamic acid production capacity compared to unmodified strains.
  • the term "bacteria having L-glutamic acid-producing ability" means the ability to produce and accumulate target L-glutamic acid in the medium and/or the cells of the bacteria to such an extent that when the bacteria are in Bacteria that can collect L-glutamic acid when cultured in the medium.
  • the bacterium having L-glutamic acid-producing ability may be a bacterium capable of accumulating the desired L-glutamic acid in the medium and/or the cells of the bacteria in a larger amount than that obtainable by the unmodified strain.
  • unmodified strain refers to a control strain that has not been modified in such a way that it has specific characteristics. That is, examples of unmodified strains include wild-type strains and parental strains.
  • L-glutamic acid refers to L-glutamic acid in free form, a salt thereof, or a mixture thereof.
  • the polynucleotide can encode about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more of the amino acid sequence of SEQ ID NO:3 Amino acid sequences of high, or about 99% or greater sequence homology.
  • sequence homology refers to the percent identity between two polynucleotides or two polypeptide modules. Sequence homology between one module and another can be determined by using methods known in the art. Such sequence homology can be determined, for example, by the BLAST algorithm.
  • Expression of a polynucleotide can be enhanced by substituting or mutating expression regulatory sequences, introducing mutations to the polynucleotide sequence, by increasing the copy number of the polynucleotide via chromosomal insertion or introduction of a vector, or a combination thereof, and the like.
  • the expression regulatory sequences of the polynucleotides can be modified. Expression regulatory sequences control the expression of polynucleotides to which they are operably linked, and can include, for example, promoters, terminators, enhancers, silencers, and the like. Polynucleotides can have changes in the initiation codon. Polynucleotides can be incorporated into chromosomes at specific sites, thereby increasing copy number. Herein, a specific site may include, for example, a transposon site or an intergenic site. Additionally, polynucleotides can be incorporated into expression vectors that are introduced into host cells to increase copy number.
  • the copy number is increased by incorporating a polynucleotide or a polynucleotide with a point mutation into a specific site in the chromosome of a microorganism.
  • the overexpression of the said nucleic acid sequence in one embodiment of the invention, the overexpression of the said nucleic acid sequence.
  • a polynucleotide or a polynucleotide having a point mutation is incorporated into an expression vector, and the expression vector is introduced into a host cell to increase the copy number.
  • a polynucleotide with a promoter sequence or a polynucleotide with a point mutation with a promoter sequence is incorporated into an expression vector, and the expression vector is introduced into a host cell, Thereby the amino acid sequence is overexpressed.
  • the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:1.
  • the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 has a point mutation such that methionine at position 199 of the amino acid sequence of SEQ ID NO:3 is replaced by a different amino acid replace.
  • the methionine at position 199 is replaced by isoleucine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 199th methionine is replaced by isoleucine is shown in SEQ ID NO:4.
  • the polynucleotide sequence with point mutation is formed by mutating the 597th base of the polynucleotide sequence shown in SEQ ID NO: 1.
  • the mutation includes the mutation of the 597th base of the polynucleotide sequence shown in SEQ ID NO: 1 from guanine (G) to adenine (A).
  • the polynucleotide sequence with point mutation comprises the polynucleotide sequence shown in SEQ ID NO:2.
  • operably linked refers to a functional linkage between a regulatory sequence and a polynucleotide sequence, whereby the regulatory sequence controls transcription and/or translation of the polynucleotide sequence.
  • the regulatory sequence can be a strong promoter capable of increasing the expression level of the polynucleotide.
  • Regulatory sequences may be promoters derived from microorganisms belonging to the genus Corynebacterium or may be promoters derived from other microorganisms.
  • the promoter can be a trc promoter, gap promoter, tac promoter, T7 promoter, lac promoter, trp promoter, araBAD promoter or cj7 promoter.
  • the promoter is the promoter of the polynucleotide (BBD29_00405) encoding the amino acid sequence of SEQ ID NO:3.
  • the term "vector” refers to a polynucleotide construct that contains a gene's regulatory and gene sequences and is configured to express a target gene in a suitable host cell.
  • a vector may in turn refer to a polynucleotide construct containing sequences useful for homologous recombination such that, as a result of the vector introduced into the host cell, the regulatory sequences of endogenous genes in the genome of the host cell may be altered, or may The expressed target gene is inserted into the host's genome at a specific site.
  • the vector used in the present invention may further comprise a selectable marker to determine the introduction of the vector into the host cell or the insertion of the vector into the chromosome of the host cell.
  • Selectable markers can include markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with such selective agents, transformed cells can be selected because only cells expressing the selectable marker can survive or display different phenotypic traits.
  • the vectors described herein are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (eg, lambda phage or M13 filamentous phage, etc.), cosmids (ie, cosmids), or viral vectors.
  • the vector used is the pK18mobsacB plasmid, the pXMJ19 plasmid.
  • the term "transformation” refers to the introduction of a polynucleotide into a host cell such that the polynucleotide is replicable as an extragenomic element or inserted into the genome of the host cell.
  • the method of transforming the vector used in the present invention may include a method of introducing nucleic acid into cells.
  • the electrical pulse method can be implemented according to the host cell.
  • the microorganism may be yeast, bacteria, algae or fungi.
  • the bacteria may be microorganisms belonging to the genus Corynebacterium, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum glutamicum), Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, Brevibacterium rose (Brevibacterium roseum), Brevibacterium thiogenitalis, etc.
  • Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum
  • Corynebacterium callunae Corynebacterium glutamicum glutamicum
  • Brevibacterium flavum Brevibacterium lactofermentum
  • Corynebacterium ammoniagenes Corynebacterium pekinense
  • Brevibacterium saccharolyticum Bre
  • the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC13869.
  • the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YPGLU001, which has been deposited in the General Microorganism Center of the China Microorganism Culture Collection Administration Committee on November 23, 2020, and the deposit address: Beijing No. 3, No. 1, Beichen West Road, Chaoyang District, 100101, the abbreviation of the depositary institution: CGMCC, and the biological deposit number is CGMCC No. 21220.
  • the second aspect of the present invention provides a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector comprising the polynucleotide sequence, and a recombinant strain containing the polynucleotide sequence.
  • the polynucleotide sequence includes a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, the 199th methionine of the sequence being substituted by a different amino acid.
  • the methionine at position 199 is replaced by isoleucine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 199th methionine is replaced by isoleucine is shown in SEQ ID NO:4.
  • the polynucleotide sequence encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO:3 contains the polynucleotide sequence shown in SEQ ID NO:1.
  • the polynucleotide sequence is formed by mutating the 597th base of the polynucleotide sequence shown in SEQ ID NO: 1.
  • the mutation refers to a change in the base/nucleotide of the site
  • the mutation method can be selected from at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination methods. kind.
  • PCR site-directed mutagenesis and/or homologous recombination are preferably used.
  • the mutation includes the mutation of guanine (G) at position 597 of the polynucleotide sequence shown in SEQ ID NO: 1 to adenine (A).
  • the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO:2.
  • the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:4.
  • the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.
  • the plasmid is the pK18mobsacB plasmid.
  • the plasmid is the pXMJ19 plasmid.
  • the polynucleotide sequence and the plasmid can be constructed into a recombinant vector through the NEBuider recombination system.
  • the recombinant strain contains the polynucleotide sequence.
  • the starting bacteria of the recombinant strain is Corynebacterium glutamicum YPGLU001, and the biological deposit number is CGMCC No. 21220.
  • the starting bacteria of the recombinant strain is ATCC 13869.
  • the third aspect of the present invention provides the above-mentioned polynucleotide sequence, the amino acid sequence encoded by the polynucleotide sequence, the recombinant vector comprising the polynucleotide sequence, and the recombinant strain containing the polynucleotide sequence in the production of L - Application of glutamic acid.
  • the fourth aspect of the present invention also provides a method for constructing a recombinant strain producing L-glutamic acid.
  • the construction method comprises the following steps:
  • the polynucleotide sequence of the wild-type BBD29_00405 gene as shown in SEQ ID NO: 1 in the host strain was modified, and the 597th base was mutated to obtain a recombinant strain comprising the mutated BBD29_00405 encoding gene.
  • the transformation includes at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to that the 597th base in SEQ ID NO: 1 is mutated from guanine (G) to adenine (A); specifically, the polynucleotide comprising the gene encoding the mutation BBD29_00405 The acid sequence is shown in SEQ ID NO:2.
  • construction method comprises the steps:
  • the step (1) comprises: constructing the BBD29_00405 gene with point mutation: according to the genome sequence of the unmodified strain, synthesizing two pairs of primers P1 and P2 and P3 and P4 for amplifying the BBD29_00405 gene fragment, by PCR
  • the point mutation was introduced into the wild-type BBD29_00405 gene SEQ ID NO: 1 by the site-directed mutagenesis method, and the nucleotide sequence of the point-mutated BBD29_00405 gene SEQ ID NO: 2 was obtained, which was denoted as BBD29_00405 G597A .
  • the genome of the unmodified strain can be derived from the ATCC13869 strain, whose genome sequence GenBank: CP016335.1 can be obtained from the NCBI website.
  • the primer is:
  • P2 5'AGACCGGCATCAAGTATGGTTCTGGGCA3' (SEQ ID NO:6)
  • the PCR amplification is performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 40s (30 cycles), overextension at 72°C 10min.
  • the overlapping PCR amplification is performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 100s (30 cycles), excess at 72°C Extend for 10min.
  • the step (2) includes the construction of recombinant plasmids, including: assembling the isolated and purified BBD29_00405 G597A and pK18mobsacB plasmids through the NEBuider recombination system to obtain the recombinant plasmids.
  • the step (3) includes the construction of a recombinant strain, and the recombinant plasmid is transformed into a host strain to obtain a recombinant strain.
  • the conversion of step (3) is an electroconversion method.
  • the host strain is ATCC 13869.
  • the host strain is Corynebacterium glutamicum YPGLU001, and the biological deposit number is CGMCC No. 21220.
  • the recombination is achieved by homologous recombination.
  • the fifth aspect of the present invention also provides a method for constructing a recombinant strain producing L-glutamic acid.
  • the construction method comprises the following steps:
  • BBD29_00405 the upstream and downstream homology arm fragments of BBD29_00405, the coding region of BBD29_00405 gene and its promoter region sequence, and introduce BBD29_00405 or BBD29_00405 G597A gene into the genome of the host strain by homologous recombination, to realize that the strain overexpresses BBD29_00405 or BBD29_00405 G597A gene.
  • the primer for amplifying the upstream homology arm fragment is:
  • the primers for amplifying the downstream homology arm fragments are:
  • the primers for amplifying the coding region of the gene and the sequence of its promoter region are:
  • the aforementioned P7/P12 is used as the primer, and the amplified upstream homology arm fragment, the downstream homology arm fragment, the gene coding region and its promoter region sequence fragment are mixed as The template is amplified to obtain integrated homology arm fragments.
  • the PCR system used 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5 mM each) 4 ⁇ L, Mg 2+ (25 mM) 4 ⁇ L, primers (10 ⁇ M) 2 ⁇ L each, Ex Taq (5U) PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 180s (30 cycles), and overextension at 72°C for 10 min.
  • the NEBuider recombination system is used to assemble the shuttle plasmid PK18mobsacB and the integrated homology arm fragment to obtain an integrated plasmid.
  • the integrated plasmid is transfected into a host strain, and the BBD29_00405 or BBD29_00405 G597A gene is introduced into the genome of the host strain by means of homologous recombination.
  • the host strain is Corynebacterium glutamicum YPGLU001
  • the biological deposit number is CGMCC No.21220.
  • the host strain is ATCC 13869.
  • the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 2.
  • the sixth aspect of the present invention also provides a method for constructing a recombinant strain for producing L-glutamic acid.
  • the construction method comprises the following steps:
  • BBD29_00405 gene coding region and the promoter region sequence or the BBD29_00405 G597A gene coding region and the promoter region sequence, construct an overexpression plasmid vector, and transfer the vector into a host strain to realize that the strain overexpresses BBD29_00405 or BBD29_00405 G597A gene.
  • the primers for amplifying the coding region of the gene and the sequence of its promoter region are:
  • P18 5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAGCCGGCGTAAGGATCCCGGAT3' (SEQ ID NO:22).
  • the PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5 mM each) 4 ⁇ L, Mg 2+ (25 mM) 4 ⁇ L, primers (10 ⁇ M) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L; the PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 100s (30 cycles), and overextension at 72°C for 10 min .
  • the NEBuider recombination system is used to assemble the shuttle plasmid pXMJ19 and the BBD29_00405 or BBD29_00405 G597A fragment with its own promoter to obtain an overexpression plasmid.
  • the host strain is Corynebacterium glutamicum YPGLU001
  • the biological deposit number is CGMCC No.21220.
  • the host strain is ATCC 13869.
  • the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO: 2.
  • the recombinant strain obtained by the present invention can be used alone in fermentation to produce L-glutamic acid, or can be mixed with other L-glutamic acid-producing bacteria to produce L-glutamic acid.
  • Another aspect of the present invention provides a method of producing L-glutamic acid, the method comprising culturing the bacteria; and obtaining L-glutamic acid from the culture.
  • Cultivation of bacteria can be carried out in a suitable medium under culture conditions known in the art.
  • the medium may contain: carbon sources, nitrogen sources, trace elements, and combinations thereof.
  • the pH of the culture can be adjusted.
  • the culturing may include preventing the generation of air bubbles, for example, by using an antifoaming agent.
  • culturing can include injecting a gas into the culture.
  • the gas can include any gas capable of maintaining aerobic conditions of the culture.
  • the temperature of the culture may be 20 to 45°C.
  • the resulting L-glutamic acid can be recovered from the culture by treating the culture with sulfuric acid or hydrochloric acid, etc., followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.
  • the present invention also provides a protein named as protein BBD29_00405M199I , which can be any of the following:
  • amino acid sequence is the protein of SEQ ID No.4;
  • A2 A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No. 4;
  • A3 A fusion protein with the same function obtained by linking a tag to the N-terminus and/or C-terminus of A1) or A2).
  • the present invention also provides a nucleic acid molecule named BBD29_00405 G597A , and the nucleic acid molecule BBD29_00405 G597A can be any of the following:
  • the coding sequence is the DNA molecule shown in SEQ ID No.2;
  • the nucleotide sequence is the DNA molecule shown in SEQ ID No.2.
  • the DNA molecule shown in SEQ ID No. 2 is the BBD29_00405 G597A gene described in the present invention.
  • the DNA molecule shown in SEQ ID No. 2 (BBD29_00405 G597A gene) encodes the protein BBD29_00405 M199I shown in SEQ ID No. 4.
  • the protein BBD29_00405 M199I amino acid sequence (SEQ ID No. 4) is obtained by changing the 199th methionine (M) in SEQ ID No. 3 to isoleucine (I).
  • the present invention also provides biological materials, and the biological materials can be any of the following:
  • C2 a recombinant vector containing the nucleic acid molecule BBD29_00405 G597A , or a recombinant vector containing the expression cassette described in C1);
  • C3 a recombinant microorganism containing the nucleic acid molecule BBD29_00405 G597A , or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2).
  • the present invention also provides any one of the following applications of any one of D1)-D8):
  • nucleotide sequence shown in SEQ ID No.1 is modified and/or one or several nucleotides are substituted and/or deleted and/or added to obtain the DNA molecule shown in SEQ ID No.1. DNA molecules that are more than 90% identical and have the same function;
  • D6 an expression cassette comprising the DNA molecule described in D4) or D5);
  • D7 a recombinant vector containing the DNA molecule described in D4) or D5), or a recombinant vector containing the expression cassette described in D6);
  • D8 a recombinant microorganism containing the DNA molecule described in D4) or D5), or a recombinant microorganism containing the expression cassette described in D6), or a recombinant microorganism containing the recombinant vector described in D7).
  • the DNA molecule shown in SEQ ID No.1 is the BBD29_00405 gene of the present invention.
  • the DNA molecule shown in SEQ ID No. 1 (BBD29_00405 gene) encodes the protein shown in SEQ ID No. 3.
  • identity refers to the identity of amino acid sequences or nucleotide sequences.
  • Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).
  • the identity of more than 80% may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the above 90% identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • Modulating the production of L-glutamic acid in a microorganism as described herein may increase or decrease the amount of L-glutamic acid accumulated in the microorganism (ie, promote or inhibit L-glutamic acid biosynthesis).
  • the present invention also provides a method for improving the output of L-glutamic acid in the microorganism, the method comprising any of the following:
  • E1 improve the expression amount or content of the nucleic acid molecule BBD29_00405 G597A in the target microorganism, obtain a microorganism whose output of L-glutamic acid is higher than the target microorganism;
  • E2 improve the expression amount or content of the DNA molecule described in D4) or D5) in the target microorganism, and obtain a microorganism whose output of L-glutamic acid is higher than that of the target microorganism;
  • the mutation may be a point mutation, that is, a mutation of a single nucleotide.
  • the point mutation may be to mutate the alanine residue at position 199 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to another amino acid residue.
  • the point mutation can be the mutation of alanine at position 199 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 to threonine to obtain a mutant protein whose amino acid sequence is SEQ ID No. 4 BBD29_00405 M199I .
  • the mutation refers to changing one or several bases in a gene through site-directed mutation, resulting in a change in the amino acid composition of the corresponding protein, resulting in a new protein or a new function of the original protein, that is, gene site-directed mutation.
  • Gene site-directed mutagenesis techniques such as oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis or cassette mutagenesis are well known to those skilled in the art.
  • the point mutation described herein can be a single-base substitution, a single-base insertion or a single-base deletion, specifically a single-base substitution.
  • the single base substitution can be an allelic substitution.
  • the point mutation can be nucleic acid modification of the 597th guanine (G) of the BBD29_00405 gene (SEQ ID No. 1).
  • the point mutation can be to change the 597th guanine (G) of the BBD29_00405 gene (SEQ ID No. 1) to adenine (A) to obtain the DNA molecule shown in SEQ ID No. 2.
  • the recombinant vector may specifically be a recombinant vector pK18- BBD29_00405G597A , PK18mobsacB-BBD29_00405, PK18mobsacB- BBD29_00405G597A , pXMJ19-BBD29_00405 or pXMJ19- BBD29_00405G597A .
  • the recombinant vector pK18-BBD29_00405 G597A contains the DNA molecule shown in positions 1-1473 of the mutated gene BBD29_00405 G597A shown in SEQ ID No. 2, specifically the DNA fragment BBD29_00405 G597A shown in SEQ ID No. 31-
  • the Up-Down fragment was inserted between the XbaI recognition sites of the pK18mobsacB vector, and other sequences of the pK18mobsacB vector were kept unchanged to obtain a recombinant vector.
  • the recombinant vector PK18mobsacB-BBD29_00405 is used to integrate the exogenous gene BBD29_00405 into the host chromosome, and to overexpress the wild-type BBD29_00405 gene in the production bacteria.
  • the recombinant vector PK18mobsacB- BBD29_00405G597A is used to integrate the exogenous gene BBD29_00405G597A into the host chromosome, and overexpress the mutant gene BBD29_00405G597A in the production bacteria.
  • the recombinant vector pXMJ19-BBD29_00405 is used to express the foreign gene BBD29_00405 extrachromosomally through a plasmid, and then overexpress the wild-type BBD29_00405 gene in the production bacteria.
  • the recombinant vector pXMJ19-BBD29_00405 G597A is used to express the exogenous gene BBD29_00405 G597A extrachromosomally through a plasmid, and then overexpress the mutant gene BBD29_00405 G597A in the production bacteria.
  • the recombinant vectors pK18- BBD29_00405G597A , PK18mobsacB-BBD29_00405, PK18mobsacB- BBD29_00405G597A , pXMJ19-BBD29_00405 and pXMJ19- BBD29_00405G597A are all within the protection scope of the present invention.
  • the recombinant microorganism may specifically be recombinant bacteria YPG-025, YPG-026, YPG-027, YPG-028 or YPG-029.
  • the recombinant bacteria YPG-025 is obtained by transforming the recombinant vector pK18-BBD29_00405 G597A into Corynebacterium glutamicum CGMCC No. 21220, and the recombinant bacteria YPG-025 contains SEQ ID No. 2
  • the mutated gene BBD29_00405G597A is shown .
  • the recombinant bacteria YPG-026 contains double copies of the BBD29_00405 gene shown in SEQ ID No. 1; the recombinant bacteria containing double copies of the BBD29_00405 gene can significantly and stably increase the expression of the BBD29_00405 gene.
  • the recombinant strain YPG-026 is an engineered strain that overexpresses the wild-type BBD29_00405 gene on its genome.
  • the recombinant strain YPG-027 contains the mutated BBD29_00405 G597A gene shown in SEQ ID No. 2; the recombinant strain YPG-027 is an engineering strain that overexpresses the mutant BBD29_00405 G597A gene on the genome.
  • Recombinant bacterium YPG-028 contains the BBD29_00405 gene shown in double-copy SEQ ID No.1; recombinant bacterium YPG-028 is an engineering bacterium that overexpresses wild-type BBD29_00405 gene on a plasmid, that is, extrachromosomally overexpressed by plasmid pXMJ19-BBD29_00405 .
  • Recombinant bacteria YPG-029 contains the mutant BBD29_00405 G597A gene shown in SEQ ID No. 2; recombinant bacteria YPG-029 is an engineering bacteria that overexpresses the mutant BBD29_00405 G597A gene on a plasmid, that is, extrachromosomally derived from plasmid pXMJ19-BBD29_00405 G597A overexpressed.
  • YPG-025, YPG-025, YPG-027, YPG-027 and YPG-029 are all within the protection scope of the present invention.
  • the present invention also provides a method for constructing the recombinant microorganism, the method comprising at least any of the following:
  • F3 Edit the DNA molecule shown in SEQ ID No.1 by using gene editing means (such as single-base gene editing), so that the target microorganism contains the DNA molecule shown in SEQ ID No.2.
  • the introduction can be by transforming the host bacteria with the vector carrying the DNA molecule of the present invention by any known transformation method such as chemical transformation method or electroporation transformation method.
  • the introduced DNA molecule can be single copy or multiple copies.
  • the introduction may be the integration of the exogenous gene into the host chromosome, or the extrachromosomal expression of the plasmid.
  • the present invention also provides a method for preparing L-glutamic acid, the method comprising utilizing any of the recombinant microorganisms described herein to produce L-glutamic acid.
  • the method can be fermentation method to prepare L-glutamic acid, and the recombinant microorganism can be Corynebacterium, specifically Corynebacterium glutamicum and its variants.
  • Corynebacterium glutamicum (Corynebacterium glutamicum), deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, zip code: 100101, preservation institution abbreviation: CGMCC, preservation The date is November 23, 2020, the biological deposit number is CGMCC No.21220, and the strain name is YPGLU001.
  • the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.
  • the experimental method of unreceipted specific conditions in the following examples usually according to conventional conditions such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer the proposed conditions.
  • the basal medium used for culturing the strains in the following examples has the same composition, and correspondingly required sucrose, kanamycin or chloramphenicol are added to the basal medium composition. If you need solid, just add 2% agar, pH 7.0, culture temperature 30 degrees.
  • the composition of the solutes in the basal medium is shown in Table 1 below, and the solutes shown in Table 1 are dissolved in water to obtain the basal medium:
  • Table 1 shows the composition of the basal medium
  • Corynebacterium glutamicum (Corynebacterium glutamicum) YPGLU001CGMCC No.21220 in the following examples has been deposited in the General Microorganism Center of China Microorganism Culture Collection Management Committee (abbreviated as CGMCC, address: Chaoyang District, Beijing, China on November 23, 2020) No. 3, No. 1 Yard, Beichen West Road, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC No.21220.
  • Corynebacterium glutamicum YPGLU001 also known as Corynebacterium glutamicum CGMCC No.21220.
  • Example 1 Construction of transformation vector pK18-BBD29_00405 G597A comprising the coding region of the BBD29_00405 gene of point mutation
  • the amino acid sequence corresponding to the encoded protein is SEQ ID NO: 3, the nucleotide of the BBD29_00405 gene
  • the 597th guanine (G) of the sequence is changed to adenine (A) (SEQ ID NO: 2: BBD29_00405 G597A ), and the 199th methionine (M) of the corresponding encoded protein amino acid sequence is changed to isoleucine ( I) (SEQ ID NO: 4: BBD29_00405M199I ).
  • the nucleotide sequence of the wild-type BBD29_00405 gene is SEQ ID NO:1, and the amino acid sequence of its encoding BBD29_00405 protein is SEQ ID NO:3;
  • the nucleotide sequence of the BBD29_00405 G597A mutant gene is SEQ ID NO:2
  • amino acid sequence of the BBD29_00405 M199I mutant protein is SEQ ID NO:4.
  • the primers are designed as follows (synthesized by Shanghai Invitrogen Company, wherein the underlined bases are mutated bases):
  • P2 5'AGACCGGCATCAAGTATGGTTCTGGGCA3' (SEQ ID NO:6)
  • Construction method Take Corynebacterium glutamicum ATCC13869 as the template, and carry out PCR amplification with primers P1 and P2, P3 and P4 respectively.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, template 1 ⁇ L, balance For water, the total volume is 50 ⁇ L.
  • PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 40s (30 cycles), and overextension at 72°C for 10min, two sizes of about 647bp and 927bp were obtained, respectively.
  • a DNA fragment containing the coding region of the BBD29_00405 gene (BBD29_00405 G597A -Up (SEQ ID No. 29) and BBD29_00405 G597A- Down (SEQ ID No. 30).
  • the Up-Down fragment the nucleotide sequence of which is SEQ ID No.31.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, template 1 ⁇ L, balance For water, the total volume is 50 ⁇ L.
  • the above PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 100s (30 cycles), and overextension at 72°C for 10 min.
  • This DNA fragment causes the guanine (G) at position 597 of the coding region of the BBD29_00405 gene in the mutagenic strain of ATCC13869 Corynebacterium glutamicum YPGLU001 (Biodeposit No. CGMCC No. 21220) to be changed to adenine (A), resulting in The 199th amino acid of the encoded protein was changed from methionine (M) to isoleucine (I).
  • the BBD29_00405 G597A -Up-Down and linearized pK18mobsacB plasmids were separated and purified by agarose gel electrophoresis, and then assembled by the NEBuider recombination system (NEB E5520S ) to obtain the vector pK18 -BBD29_00405 G597A , this plasmid contains the kanamycin resistance marker.
  • the vector pK18-BBD29_00405 G597A was sent to a sequencing company for sequencing identification, and the vector pK18-BBD29_00405 G597A containing the correct point mutation (GA) was saved for future use.
  • the recombinant vector pK18-BBD29_00405 G597A is a recombinant vector obtained by inserting the DNA fragment BBD29_00405 G597A -Up-Down fragment shown in SEQ ID No. 31 between the XbaI recognition sites of the pK18mobsacB vector, keeping other sequences of the pK18mobsacB vector unchanged.
  • Embodiment 2 Construction of engineering strain of BBD29_00405 G597A comprising point mutation
  • allelic replacement plasmid pK18-BBD29_00405 G597A was transformed into the high-yielding L-glutamic acid Corynebacterium glutamicum YPGLU001 (Biological Deposit No. CGMCC No. 21220) by electric shock, and it was confirmed by sequencing that the strain retained the wild type on the chromosome The BBD29_00405 gene coding region of BBD29_00405); the single colony produced by culture was identified by primer P1 and universal primer M13R, and the strain that could amplify a band of about 1554 bp (SEQ ID No. 32) was a positive strain.
  • the positive strains were cultured on the medium containing 15% sucrose; the single colonies produced by the culture were cultured on the medium containing kanamycin and without kanamycin, respectively, and the medium without kanamycin
  • the strains that did not grow on the kanamycin-containing medium were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen Company):
  • P6 5'ATCGGGTTGGAAATCGCAGA 3' (SEQ ID NO: 10);
  • the sequence of universal primer M13R is as follows: M13R: 5'CAG GAA ACA GCT ATG ACC3'
  • the above-mentioned PCR amplification product 261bp (SEQ ID No. 33) was subjected to sscp electrophoresis after high temperature denaturation and ice bath (with plasmid pK18-BBD29_00405 G597A amplified fragment as positive control, ATCC13869 amplified fragment as negative control, and water as blank control) , because the fragment structure is different and the electrophoresis position is different, the strain whose electrophoresis position is inconsistent with the negative control fragment and consistent with the positive control fragment is the strain with successful allelic replacement.
  • Recombinant bacteria YPG-025 introduced point mutation G597A in the coding region (SEQ ID No. 1) of BBD29_00405 gene of Corynebacterium glutamicum CGMCC No. 21220 by allele replacement, making the gene No. 597
  • the G mutation A of the gene is unchanged, and the genetic engineering bacteria YPG-025 containing the point mutation (G-A) is obtained.
  • Corynebacterium glutamicum YPG-025 the only difference of Corynebacterium glutamicum YPG-025 is: the BBD29_00405 gene shown in SEQ ID No.1 in the genome of Corynebacterium glutamicum CGMCC21220 is replaced with SEQ ID No.2 BBD29_00405 G597A gene shown. There is only one nucleotide difference between SEQ ID No. 1 and SEQ ID No. 2, located at position 597.
  • Table 2 shows the preparation of PAGE for sscp electrophoresis
  • Embodiment 3 the engineering strain that overexpresses BBD29_00405 or BBD29_00405 G597A gene on the construction genome
  • the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • P10 5'CAAACCAGAGTGCCCACGAACTAGCCGGCGTAAGGATCCC3'
  • BBD29_00405 G597A gene fragment (SEQ ID No.36) of about 1777bp and the downstream homology arm fragment of about 788bp (SEQ ID No.37), and then using P7/P12 as primers, mixed with the three amplified fragments above to be
  • the template was amplified to obtain a 3291 bp integrated homology arm fragment upstream-BBD29_00405-downstream (SEQ ID No. 38) or an integrated homology arm fragment upstream- BBD29_00405G597A -downstream (SEQ ID No. 39).
  • the amplified product was recovered by electrophoresis, and the required DNA fragment of about 3291 bp was recovered using a column-type DNA gel recovery kit.
  • the NEBuider recombination system was used to phase with the shuttle plasmid pk18mobsacB recovered by Xba I digestion.
  • the plasmid contains a kanamycin resistance marker, and the recombinant plasmid integrated into the genome can be obtained by kanamycin screening.
  • pk18mobsacB-BBD29_00405 is a recombinant vector obtained by inserting the integrated homology arm fragment upstream-BBD29_00405-downstream (SEQ ID No. 38) between the Xba I restriction sites of the shuttle plasmid pk18mobsacB.
  • pk18mobsacB-BBD29_00405 is a recombinant vector obtained by inserting the integrated homology arm fragment upstream- BBD29_00405G597A -downstream (SEQ ID No. 39) into the Xba I restriction site of the shuttle plasmid pk18mobsacB.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, template 1 ⁇ L, balance For water, the total volume is 50 ⁇ L.
  • the above PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 180s (30 cycles), and overextension at 72°C for 10 min.
  • the two integrated plasmids were electrotransformed into Corynebacterium glutamicum YPGLU001 (Biological Deposit No. CGMCC No. 21220) respectively, and the single colony produced by the culture was identified by PCR with the P13/P14 primers, and the PCR amplification contained a size of about 1970bp ( The fragment of SEQ ID No. 40) is the positive strain, and the fragment that cannot be amplified is the original strain. Positive strains were screened by 15% sucrose and cultured on media containing kanamycin and without kanamycin, respectively. The bacterial strains that do not grow on the base are further identified by PCR using the P15/P16 primers, and the bacteria with a size of about 1758bp (SEQ ID No.
  • BBD29_00405 or BBD29_00405 G597A gene is integrated into the bacterial strain Corynebacterium glutamicum YPGLU001 (Biological Deposit No. The strains on the genome of CGMCC No. 21220) were named as YPG-026 (without mutation points) and YPG-027 (with mutation points).
  • Recombinant bacterium YPG-026 integrates the upstream-BBD29_00405-downstream (SEQ ID No.38) of the integrated homology arm fragment into the genome of the strain Corynebacterium glutamicum YPGLU001 to obtain the BBD29_00405 gene shown in SEQ ID No.1 containing double copies
  • the recombinant bacteria containing double copies of the BBD29_00405 gene can significantly and stably increase the expression of the BBD29_00405 gene.
  • Recombinant bacteria YPG027 is to integrate the upstream of the homology arm fragment- BBD29_00405G597A -downstream (SEQ ID No. 39) into the genome of the strain Corynebacterium glutamicum YPGLU001 to obtain a recombination containing the BBD29_00405 G597A mutant gene shown in SEQ ID No. 2 bacteria.
  • Construction method take ATCC13869 or YPG-025 as template, carry out PCR amplification with primer P17/P18, obtain the DNA molecule (SEQ ID No. 42) containing BBD29_00405 or the DNA molecule (SEQ ID No. 43) containing BBD29_00405 G597A about 1807bp, the amplified product was recovered by electrophoresis, and the required 1807bp DNA fragment was recovered by a column DNA gel recovery kit. ) to obtain the overexpression plasmid pXMJ19-BBD29_00405 or pXMJ19- BBD29_00405G597A .
  • the plasmid contains a chloramphenicol resistance marker, and the plasmid can be transformed into the strain by chloramphenicol screening.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, template 1 ⁇ L, balance For water, the total volume is 50 ⁇ L.
  • the above PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 100s (30 cycles), and overextension at 72°C for 10 min.
  • Recombinant vector pXMJ19-BBD29_00405 The DNA molecule (SEQ ID No. 42) containing BBD29_00405 was inserted between the EcoR I restriction sites of shuttle plasmid pXMJ19 to obtain a recombinant vector.
  • Recombinant vector pXMJ19-BBD29_00405 G597A The DNA molecule (SEQ ID No. 43) containing BBD29_00405 G597A was inserted between the EcoR I restriction sites of the shuttle plasmid pXMJ19 to obtain a recombinant vector.
  • the plasmids were electro-transformed into Corynebacterium glutamicum YPGLU001 (Biological Deposit No. CGMCC No. 21220), and the single colony produced by the culture was identified by PCR with M13R (-48) and P18 primers, and the PCR amplification contained a size of about 1846bp.
  • the fragments (SEQ ID No. 44) of the transformed strains were named as YPG-028 (without mutation point) and YPG-029 (with mutation point).
  • the M13R(-48) sequence is as follows:
  • Recombinant bacterium YPG-028 contains the BBD29_00405 gene shown in double-copy SEQ ID No.1; recombinant bacterium YPG-028 is an engineering bacterium that overexpresses wild-type BBD29_00405 gene on a plasmid, that is, extrachromosomally overexpressed by plasmid pXMJ19-BBD29_00405 .
  • Recombinant bacteria YPG-029 contains the mutant BBD29_00405 G597A gene shown in SEQ ID No. 2; recombinant bacteria YPG-029 is an engineering bacteria that overexpresses the mutant BBD29_00405 G597A gene on a plasmid, that is, extrachromosomally derived from plasmid pXMJ19-BBD29_00405 G597A overexpressed.
  • the amplified product was recovered by electrophoresis, and the required 1405bp DNA fragment was recovered using a column-type DNA gel recovery kit, and the shuttle plasmid pk18mobsacB plasmid recovered by NEBuider recombination system and Xba I digestion ligated to obtain a knockout plasmid.
  • This plasmid contains a kanamycin resistance marker.
  • the knockout plasmid was electrotransformed into Corynebacterium glutamicum YPGLU001 (Biological Deposit No. CGMCC No. 21220), and the single colony produced by the culture was identified by PCR with the following primers (synthesized by Shanghai Yingjun Company):
  • the above-mentioned PCR amplified the strain of the band of about 1331bp (SEQ ID No. 49) and about 2804bp (SEQ ID No. 48) was the positive strain, and the strain that only amplified the 2804bp band was the starting strain.
  • Recombinant bacteria YPG-030 is the BBD29_00405 gene on the genome of Corynebacterium glutamicum CGMCC No. 21220 has been knocked out.
  • the strains YPG-025, YPG-026, YPG-027, YPG-028, YPG-029, YPG-030 and the original Corynebacterium glutamicum YPGLU001 constructed in Example 2-5 (the biological deposit number is CGMCC No.21220) Fermentation experiments were carried out in a BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium shown in Table 3 and the fermentation control process shown in Table 4, and the fermentation products were collected.
  • the bacterial concentration of the system was 15 g/L.
  • control the sugar content (residual sugar) of the system by adding 50-55% aqueous glucose solution.
  • Table 3 is the solute formula in the fermentation medium
  • Reagent name Proportion glucose 5.0g/L Phosphoric acid 0.38g/L Magnesium sulfate 1.85g/L Potassium chloride 1.6g/L Biotin 550 ⁇ g/L Vitamin B1 300 ⁇ g/L Ferrous sulfate 10mg/L Manganese sulfate 10g/dL KH 2 PO 4 2.8g/L Vitamin C 0.75mg/L Vitamin B12 2.5 ⁇ g/L p-aminobenzoic acid 0.75mg/L defoamer 0.0015mL/dL betaine 1.5g/L cane molasses 7mL/L corn syrup 77mL/L aspartic acid 1.7g/L hair powder 2g/L
  • the above-mentioned fermentation medium is a fermentation medium obtained by dissolving the solutes shown in Table 3 in water.
  • Table 4 is the fermentation control process
  • Table 5 is the result of L-glutamic acid fermentation experiment
  • the present invention finds that the product encoded by the gene has an impact on the L-glutamic acid production capacity by knocking out the BBD29_00405 gene, and a recombinant strain is obtained by introducing a point mutation in the coding sequence, or increasing the copy number or overexpression of the gene, so that the Compared with the unmodified strain, the obtained strain is favorable for producing high concentration of L-glutamic acid.
  • the inhibitor of CTD-2256P15.2 or its encoded micropeptide PACMP provided by the invention acts on tumor cells or tumor tissue , can significantly inhibit the growth of tumor cells, increase the apoptosis of tumor cells, reduce tumor volume, and has excellent anti-tumor effect.
  • novel anti-tumor drug combination scheme provided by the present invention, the combined use of CTD-2256P15.2 or its inhibitor of the micropeptide PACMP and other anti-tumor drugs can significantly enhance the killing effect of anti-tumor drugs on tumor cells and reduce tumor cells. chemotherapy resistance, thereby improving the clinical treatment effect of tumors.
  • CTD-2256P15.2 is highly expressed in chemotherapy-resistant tumor tissues and cell lines, and its high expression is significantly negatively correlated with progression-free survival and overall survival of tumor patients.
  • the application of the CTD2256P15.2 gene expression level provided by the invention can be used as a molecular index for predicting the sensitivity and prognosis of tumor patients to chemotherapy, and creates a new standard for effectively guiding the clinical chemotherapy drugs of tumor patients and evaluating the prognosis of treatment.
  • the present invention first introduced a point mutation in the BBD29_00405 gene coding region (SEQ ID No. 1) of Corynebacterium glutamicum CGMCC No. 21220 by allele replacement, and constructed a structure containing the point mutation ( G ⁇ A) genetically engineered bacteria YPG-025.
  • the exogenous genes were integrated into the host chromosome or expressed by a plasmid extrachromosomally.
  • Point mutation in the coding region of the BBD29_00405 gene or overexpression of the BBD29_00405 gene or its mutant gene BBD29_00405 G597A in the production strain is helpful for the improvement of L-glutamic acid production and transformation rate, while the knockout or weakening of the BBD29_00405 gene does not Conducive to the accumulation of L-glutamic acid.
  • BBD29_00405 gene and its variant (such as BBD29_00405 G597A gene) to construct the genetic engineering strain that produces L-glutamic acid, to promote L-glutamic acid output to improve, cultivate the high-yield, high-quality strain that meets industrialized production, It has wide application value and important economic significance for the industrial production of L-glutamic acid.

Abstract

公开了具有增强的L-谷氨酸生产力的菌株及其构建方法与应用。将谷氨酸棒杆菌中野生型BBD29_00405基因中引入点突变，使SEQ ID NO:1的第597位碱基由鸟嘌呤(G)突变为腺嘌呤(A)所得到的核苷酸序列。还提供将该多核苷酸序列导入产L-谷氨酸的谷氨酸棒杆菌中获得的重组菌株，该重组菌株中包括含有点突变的BBD29_00405基因，所获得的菌株与未改造的菌株相比，有利于生产高浓度的L-谷氨酸。

Description

具有增强的L-谷氨酸生产力的菌株及其构建方法与应用 技术领域
本发明属于基因工程和微生物技术领域，具体涉及具有增强的L-谷氨酸生产力的菌株及其构建方法与应用。
背景技术
L-谷氨酸，化学名称为L-2-氨基戊二酸,分子式为C 5H 9O 4N，分子量为147.13。L-谷氨酸是非必需氨基酸。在生物有机体中存在的谷氨酸，都属于L-型谷氨酸。且L-谷氨酸是味精的前体物质。味精被食用后，还可在胃中转化为谷氨酸，然后经消化吸收参与蛋白质的合成，并可通过转氨基作用合成其它氨基酸，营养价值较高。除了在食品方面的广泛应用外，还被应用于医药、化妆品及农业生产中。在人体的各大器官中，谷氨酸盐在大脑中的含量最高，同时谷氨酸是参与人脑代谢的唯一一种氨基酸，在神经系统功能的维护有重要作用。所以对神经衰弱者来说，加大谷氨酸摄入量可改善其神经系统的功能。在化妆品行业,谷氨酸可用于合成聚谷氨酸钠。由于该物质具有很强的吸湿性,可被用作润肤剂。谷氨酸也可以与月桂酞氯结合生成一月桂酞基谷氨酸钠，广泛应用于
化妆品行业。谷氨酸在农业上主要用于生产杀菌剂，如谷氨酸酮。在施肥的过程中,谷氨酸还可以作为肥料的载体，促进农作物对氮磷钾的吸收。
1956年,从自然界中分离到了一种谷氨酸产生菌,即谷氨酸棒杆菌,这是味精生产史上的重大变革。1957年开始了通过发酵法生产味精的时代。发酵法生产谷氨酸的成功,是整个发酵工业的伟大创举,同时也大大促进了其它发酵产品的研究与生产。对于工业发酵来说，决定发酵生产水平的因素主要有菌种性能、发酵工艺及下游提取工艺等。在这些因素中,菌种的产酸水平是内因,是决定发酵成败的关键。优良菌种的选育依然是高产L-谷氨酸的关键，随着高通量筛选技术的发展，结合传统诱变技术，有助于找到未知性状的高产L-谷氨酸突变株。这些突变株有可能改变L-谷氨酸代谢途径上某一点的活性或者整个途径的重组整合，从而来提高产酸量。
虽然已筛选到一些可以提高L-谷氨酸产量的菌株，但是为了满足日益增加的需求，仍需要找到更多高产L-谷氨酸的突变株。
发明公开
针对现有技术的不足，本发明提供一种多核苷酸及含有该多核苷酸的重组菌株,以及将重组菌株用于改善细菌的L-谷氨酸生产能力。为了实现上述目的，本发明的发明人研究发现，具有L-谷氨酸生产能力的谷氨酸棒杆菌ATCC13869基因组(GenBank：CP016335.1)中BBD29_00405基因(GenBank：ANU32350.1)，可以通过修饰该基因或改善其表达，获得L-谷氨酸产量提高的重组菌株，与未改造的野生型菌株相比，重组菌株的产L-谷氨酸能力更强。
本发明采用如下技术方案实现：
本发明的第一方面，提供了生成L-谷氨酸的细菌，其具有编码如SEQ ID NO:3所示的氨基酸序列的多核苷酸的改善的表达。根据本发明，所述改善的表达是所述多核苷酸表达增强，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变且表达是增强的。
所述SEQ ID NO:3的氨基酸序列是基因BBD29_00405编码的蛋白。
所述细菌与未修饰菌株相比具有增强的L-谷氨酸生产能力。
在本发明中，术语“具有L-谷氨酸生产能力的细菌”是指具有在培养基和/或细菌的细胞中以下述程度产生并累积目的L-谷氨酸的能力，使得当细菌在培养基中培养时可以收集L-谷氨酸的细菌。具有L-谷氨酸生产能力的细菌可以是能够以比未修饰菌株可获得的量更大的量在培养基和/或细菌的细胞中积累目的L-谷氨酸的细菌。
术语“未修饰菌株”是指尚未以使得具有特定特征的方式进行修饰的对照菌株。即，未修饰菌株的实例包括野生型菌株和亲本菌株。
在本发明中，除非另有说明，术语“L-谷氨酸”是指游离形式的L-谷氨酸、其盐或其混合物。
所述多核苷酸可以编码与SEQ ID NO:3的氨基酸序列具有约90％或更高、约92％或更高、约95％或更高、约97％或更高、约98％或更高、或约99％或更高的序列同源性的氨基酸序列。如本文中使用的，术语“同源性”指两种多核苷酸或两种多肽模块之间的百分比同一性。可以通过使用本领域中已知的方法测定一种模块和另一种模块之间的序列同源性。例如，可以通过BLAST算法测定此类序列同源性。
可以如下增强多核苷酸的表达：通过取代或突变表达调节序列、对多核苷酸序列引入突变、通过经由染色体插入或载体导入的多核苷酸拷贝数的增加、或其组合等。
可以修饰多核苷酸的表达调节序列。表达调节序列控制与其可操作连接的多核苷酸的表达，并且例如可以包括启动子、终止子、增强子、沉默子等。多核苷酸可以具有起始密码子的变化。可以将多核苷酸掺入染色体的特定位点中，从而增加拷贝数。在本文，特定的位点可以包括例如转座子位点或基因间位点。另外，可以将多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而增加拷贝数。
在本发明的一种实施方式中，通过将多核苷酸或者具有点突变的多核苷酸掺入微生物染色体的特定位点中，从而增加拷贝数。
在本发明的一种实施方式中，通过将带有启动子序列的多核苷酸或者带有启动子序列的具有点突变的多核苷酸掺入微生物染色体的特定位点中，从而过表达所述核酸序列。
在本发明的一种实施方式中，将多核苷酸或者具有点突变的多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而增加拷贝数。
在本发明的一种实施方式中，将带有启动子序列的多核苷酸或者带有启动子序列的具有点突变的多核苷酸掺入表达载体中，将所述表达载体导入宿主细胞中，从而过表达所述氨基酸序列。
在本发明的一个具体实施方式中，所述多核苷酸可以包含SEQ ID NO:1的核苷酸序列。
在本发明的一种实施方式中，编码SEQ ID NO:3的氨基酸序列的多核苷酸的具有点突变，使得SEQ ID NO:3的氨基酸序列的第199位甲硫氨酸被不同的氨基酸所取代。
根据本发明，优选第199位甲硫氨酸变为异亮氨酸所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第199位甲硫氨酸变为异亮氨酸所取代后的氨基酸序列如SEQ ID NO:4所示。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第597位碱基发生突变而形成的。
根据本发明，所述突变包括SEQ ID NO:1所示多核苷酸序列第597位碱基由鸟嘌呤(G)突变为腺嘌呤(A)。
在本发明的一个实施方式中，所述具有点突变的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
如本文中使用的，术语“可操作连接”指调节序列和多核苷酸序列之间的功能性连接，由此调节序列控制多核苷酸序列的转录和/或翻译。调节序列可以是能提高多核苷酸的表达水平的强启动子。调节序列可以是源自属于棒杆菌属的微生物的启动子或者可以是源自其它微生物的启动子。例如，启动子可以是trc启动子、gap启动子、tac启动子、T7启动子、lac启动子、trp启动子、araBAD启动子或cj7启动子。
在本发明的一个具体实施方式中，所述启动子是编码SEQ ID NO:3的氨基酸序列的多核苷酸(BBD29_00405)的启动子。
如本文中使用的，术语“载体”指含有基因的调节序列和基因序列并且配置为在合适的宿主细胞中表达靶基因的多核苷酸构建体。或者，载体又可以指多核苷酸构建体，其含有可用于同源重组的序列，从而由于对宿主细胞导入的载体，可以改变宿主细胞的基因组中的内源基因的调节序列，或者可以将可以表达的靶基因插入宿主的基因组的特定位点中。在这点上，本发明中使用的载体可以进一步包含选择标志物以确定载体对宿主细胞的导入或者载体对宿主细胞的染色体的插入。选择标志物可以包含赋予可选择表型，诸如药物抗性、营养缺陷型、针对细胞毒剂的抗性、或表面蛋白的表达的标志物。在用此类选择剂处理的环境中，由于仅表达选择标志物的细胞可以存活或者显示不同表型性状，可以选择经转化的细胞。本文所述载体是本领域技术人员公知的，包括但不限于：质粒、噬菌体(如λ噬菌体或M13丝状噬菌体等)、黏粒(即柯斯质粒)或病毒载体。
在本发明的一些具体实施方式中，使用的载体是pK18mobsacB质粒，pXMJ19质粒。
如本文中使用的，术语“转化”指将多核苷酸导入宿主细胞中，从而多核苷酸可以作为基因组外元件或者以插入宿主细胞的基因组中能复制。转化本发明中使用的载体的方法可以包括将核酸导入细胞的方法。另外，如相关技术中公开的，可以根据宿主细胞实施电脉冲方法。
本文中，所述微生物可为酵母、细菌、藻或真菌。
根据本发明，所述细菌可以是属于棒杆菌属的微生物，例如嗜乙酰棒杆菌(Corynebacterium acetoacidophilum)、醋谷棒杆菌(Corynebacterium acetoglutamicum)、美棒杆菌(Corynebacterium callunae)、谷氨酸棒杆菌(Corynebacterium glutamicum)、黄色短杆菌(Brevibacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)、产氨棒杆菌(Corynebacterium ammoniagenes)、北京棒杆菌(Corynebacterium pekinense)、解糖短杆菌(Brevibacterium saccharolyticum)、玫瑰色短杆菌(Brevibacterium roseum)、生硫短杆菌(Brevibacterium thiogenitalis)等。
在本发明的一个实施方案中，所述属于棒杆菌属的微生物是谷氨酸棒杆菌ATCC13869。
在本发明的一个实施方案中，所述属于棒杆菌属的微生物是谷氨酸棒杆菌YPGLU001,已于2020年11月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏机构简称：CGMCC，生物保藏编号为CGMCC No.21220。
本发明的第二个方面，提供一种多核苷酸序列，由该多核苷酸序列编码的氨基酸序列，包括所述多核苷酸序列的重组载体，含有所述多核苷酸序列的重组菌株。
根据本发明，所述多核苷酸序列包括编码含有如SEQ ID NO:3所示的氨基酸序列的多肽的多核苷酸，所述序列的第199位甲硫氨酸被不同的氨基酸所取代。
根据本发明，优选第199位甲硫氨酸变为异亮氨酸所取代。。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第199位甲硫氨酸变为异亮氨酸所取代后的氨基酸序列如SEQ ID NO:4所示。
根据本发明，优选所述编码含有SEQ ID NO:3所示的氨基酸序列的多肽的多核苷酸序列含有如SEQ ID NO:1所示的多核苷酸序列。
在本发明的一个实施方式中，所述多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第597位碱基发生突变而形成的。
根据本发明，所述突变是指所述位点的碱基/核苷酸发生变化，所述突变方法可以选自诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。在本发明中，优选采用PCR定点突变法和/或同源重组。
根据本发明，所述突变包括SEQ ID NO:1所示多核苷酸序列第597位鸟嘌呤(G)突变为腺嘌呤(A)。
在本发明的一个实施方式中，所述多核苷酸序列包括SEQ ID NO:2所示的多核 苷酸序列。
根据本发明，所述氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列。
根据本发明，所述重组载体是将所述多核苷酸序列导入质粒构建而成。
在本发明的一个实施方式中，所述质粒为pK18mobsacB质粒。
在本发明的另一个实施方式中，所述质粒为pXMJ19质粒。
具体地，可以将所述多核苷酸序列和所述质粒通过NEBuider重组系统构建成重组载体。
根据本发明，所述重组菌株含有所述的多核苷酸序列。
作为本发明的一个实施方案，所述重组菌株的出发菌为谷氨酸棒杆菌YPGLU001，生物保藏编号为CGMCC No.21220。
作为本发明的一个实施方案，所述重组菌株的出发菌为ATCC 13869。
本发明的第三个方面，提供上述多核苷酸序列、由该多核苷酸序列编码的氨基酸序列、包括所述多核苷酸序列的重组载体、含有所述多核苷酸序列的重组菌株在生产L-谷氨酸中的应用。
本发明的第四个方面，还提供一种生成L-谷氨酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
改造宿主菌株中如SEQ ID NO:1所示的野生型BBD29_00405基因的多核苷酸序列，使其第597位碱基发生突变，得到包含突变BBD29_00405编码基因的重组菌株。
根据本发明的构建方法，所述改造包括诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明的构建方法，所述突变是指SEQ ID NO:1中第597位碱基由鸟嘌呤(G)突变为腺嘌呤(A)；具体地，所述包含突变BBD29_00405编码基因的多核苷酸序列如SEQ ID NO:2所示。
进一步地，所述构建方法包括如下步骤：
(1)改造如SEQ ID NO:1所示的野生型BBD29_00405基因的核苷酸序列，使其第597位碱基发生突变，得到突变的BBD29_00405基因多核苷酸序列；
(2)将所述突变的多核酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到所述包含突变BBD29_00405编码基因的重组菌株。
根据本发明的构建方法，所述步骤(1)包括：点突变的BBD29_00405基因构建：根据未修饰菌株的基因组序列，合成两对扩增BBD29_00405基因片段的引物P1和P2及P3和P4，通过PCR定点突变法在野生型BBD29_00405基因SEQ ID NO:1中引入点突变，得到点突变的BBD29_00405基因核苷酸序列SEQ ID NO:2，记为BBD29_00405 G597A。
在本发明的一个实施方式中，所述未修饰菌株基因组可以来源于ATCC13869菌株，其基因组序列GenBank：CP016335.1可以从NCBI网站获取。
在本发明的一个实施方案中，所述步骤(1)中，所述引物为：
P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATGACTATTAATGTCTCCGA 3'(SEQ ID NO:5)
P2:5'AGACCGGCATCAAGTATGGTCTGGGCA3'(SEQ ID NO:6)
P3:5'TGCCCAGACCATACTTGATGCCGGTCT3'(SEQ ID NO:7)
P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTAGCCGGCGTAAGGATCCCGGAT 3'(SEQ ID NO:8)
在本发明的一个实施方案中，所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s，52℃退火30s，以及72℃延伸40s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方案中，所述重叠PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s，52℃退火30s，以及72℃延伸100s(30个循环)，72℃过度延伸10min。
根据本发明的构建方法，所述步骤(2)包括重组质粒的构建，包括：将分离纯化后的BBD29_00405 G597A和pK18mobsacB质粒，通过NEBuider重组系统组装，获得重组质粒。
根据本发明的构建方法，所述步骤(3)包括重组菌株的构建，将重组质粒转化至宿主菌株，得到重组菌株。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法。
在本发明的一个实施方式中，所述宿主菌株是ATCC 13869。
在本发明的一个实施方式中，所述宿主菌株为谷氨酸棒杆菌YPGLU001，生物保藏编号为CGMCC No.21220。
在本发明的一个实施方式中，所述重组是通过同源重组实现的。
本发明的第五个方面，还提供一种生成L-谷氨酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增BBD29_00405的上下游同源臂片段、BBD29_00405基因编码区及其启动子区序列，以同源重组的方式在宿主菌株的基因组中引入BBD29_00405或BBD29_00405 G597A基因，以实现所述菌株过表达BBD29_00405或BBD29_00405 G597A基因。
在本发明的一个实施方式中，扩增上游同源臂片段的引物是：
P7：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG 3'
P8：5'CCTACCACGA CGAGCACTAC ATCTACTCAT CTGAAGAATC 3'
在本发明的一个实施方式中，扩增下游同源臂片段的引物是：
P11：5'GGGATCCTTA CGCCGGCTAG TTCGTGGGCA CTCTGGTTTG 3'
P12：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCATAAGAAACAACCACTTCC 3'
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P9:5'GATTCTTCAG ATGAGTAGAT GTAGTGCTCG TCGTGGTAGG 3'
(SEQ ID NO:13)
P10:5'CAAACCAGAG TGCCCACGAA CTAGCCGGCG TAAGGATCCC 3'(SEQ ID NO:14)
在本发明的一个实施方式中，再以前述P7/P12为引物，以扩增的上游同源臂片段、下游同源臂片段、基因编码区及其启动子区序列片段的三个片段混合为模板进行扩增，获得整合同源臂片段。
在本发明的一个实施方式中，所采用的PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10μM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸180s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒PK18mobsacB和整合同源臂片段组装，获得整合质粒。
在本发明的一个实施方式中，将整合质粒转染宿主菌株，以同源重组的方式在宿主菌株的基因组中引入BBD29_00405或BBD29_00405 G597A基因。
在本发明的一个实施方式中，所述宿主菌株是谷氨酸棒杆菌YPGLU001，生物保藏编号为CGMCC No.21220。
在本发明的一个实施方式中，所述宿主菌株是ATCC 13869。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:2所示多核苷酸序列的菌株。
本发明的第六个方面，还提供一种生产L-谷氨酸的重组菌株的构建方法。
根据本发明，所述构建方法包括如下步骤：
扩增BBD29_00405基因编码区及启动子区序列，或BBD29_00405 G597A基因编码区及启动子区序列，构建过表达质粒载体，将所述载体转入宿主菌株中，以实现所述菌株过表达BBD29_00405或BBD29_00405 G597A基因。
在本发明的一个实施方式中，扩增所述基因编码区及其启动子区序列的引物是：
P17:5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC GTAG TGCTCG TCGTGGTAGG 3'(SEQ ID NO:21)
P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAGCCGGCG TAAGGATCCCGGAT 3'(SEQ ID NO:22)。
在本发明的一个实施方式中，所述PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10μM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL；所述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸100s(30个循环)，72℃过度延伸10min。
在本发明的一个实施方式中，采用NEBuider重组系统，将穿梭质粒pXMJ19和带有自身启动子的BBD29_00405或BBD29_00405 G597A片段组装，获得过表达质粒。
在本发明的一个实施方式中，所述宿主菌株是谷氨酸棒杆菌YPGLU001，生物保藏编号为CGMCC No.21220。
在本发明的一个实施方式中，所述宿主菌株是ATCC 13869。
在本发明的一个实施方式中，所述宿主菌株是携带有SEQ ID NO:2所示多核苷酸序列的菌株。
本发明获得重组菌株可以单独应用于发酵生产L-谷氨酸中，也可以和其他产L-谷氨酸的细菌混合发酵生产L-谷氨酸。
本发明的另一个方面提供了生产L-谷氨酸的方法，该方法包括培养所述细菌；并且从培养物中获得L-谷氨酸。
可以在本领域中已知的培养条件下在合适的培养基中进行细菌的培养。培养基可以包含：碳源、氮源、微量元素、及其组合。在培养中，可以调节培养物的pH。此外，培养时可以包括防止气泡产生，例如通过使用消泡剂进行气泡产生的防止。此外，培养时可以包括将气体注射入培养物中。气体可以包括能够维持培养物的需氧条件的任何气体。在培养中，培养物的温度可以是20至45℃。可以从培养物回收生成的L-谷氨酸，即用硫酸或氢氯酸等处理培养物，接着进行诸如阴离子交换层析、浓缩、结晶和等电点沉淀的方法的组合。
本发明还提供了蛋白质，名称为蛋白质BBD29_00405 M199I，所述蛋白质可为下述任一种：
A1)氨基酸序列是SEQ ID No.4的蛋白质；
A2)将SEQ ID No.4所示的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；
A3)在A1)或A2)的N端和/或C端连接标签得到的具有相同功能的融合蛋白质。
本发明还提供了核酸分子，名称为BBD29_00405 G597A，所述核酸分子BBD29_00405 G597A可为下述任一种：
B1)编码所述蛋白质BBD29_00405 M199I的核酸分子；
B2)编码序列是SEQ ID No.2所示的DNA分子；
B3)核苷酸序列是SEQ ID No.2所示的DNA分子。
SEQ ID No.2所示的DNA分子即为本发明所述BBD29_00405 G597A基因。
SEQ ID No.2所示的DNA分子(BBD29_00405 G597A基因)编码SEQ ID No.4所示的蛋白质BBD29_00405 M199I。
所述蛋白质BBD29_00405 M199I氨基酸序列(SEQ ID No.4)为将SEQ ID No.3中的第199位甲硫氨酸(M)变为异亮氨酸(I)而来。
本发明还提供了生物材料，所述生物材料可为下述任一种：
C1)含有所述核酸分子BBD29_00405 G597A的表达盒；
C2)含有所述核酸分子BBD29_00405 G597A的重组载体、或含有C1)所述表达盒的重组载体；
C3)含有所述核酸分子BBD29_00405 G597A的重组微生物、或含有C1)所述表达盒的重组微生物、或含有C2)所述重组载体的重组微生物。
本发明还提供了D1)-D8)中任一项的下述任一种应用：
F1)D1)-D8)中任一项在调控微生物的L-谷氨酸的产量中的应用；
F2)D1)-D8)中任一项在构建产L-谷氨酸的基因工程菌中的应用；
F3)D1)-D8)中任一项在制备L-谷氨酸中的应用；
其中，所述D1)-D8)为：
D1)所述蛋白质BBD29_00405 M199I；
D2)所述核酸分子BBD29_00405 G597A；
D3)所述生物材料；
D4)核苷酸序列为SEQ ID No.1的DNA分子；
D5)SEQ ID No.1所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与SEQ ID No.1所示的DNA分子具有90％以上的同一性，且具有相同功能的DNA分子；
D6)含有D4)或D5)中所述DNA分子的表达盒；
D7)含有D4)或D5)中所述DNA分子的重组载体、或含有D6)所述表达盒的重组载体；
D8)含有D4)或D5)中所述DNA分子的重组微生物、或含有D6)所述表达盒的重组微生物、或含有D7)所述重组载体的重组微生物。
SEQ ID No.1所示的DNA分子即为本发明所述BBD29_00405基因。
SEQ ID No.1所示的DNA分子(BBD29_00405基因)编码SEQ ID No.3所示的蛋白质。
本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
本文中，所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本文中，所述90％以上的同一性可为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本文所述调控微生物的L-谷氨酸的产量可为提高或降低微生物中L-谷氨酸的积累量(即促进或抑制L-谷氨酸的生物合成)。
本发明还提供了一种提高微生物中L-谷氨酸的产量的方法，所述方法包括下述任一种：
E1)提高目的微生物中的所述核酸分子BBD29_00405 G597A的表达量或含量，得到L-谷氨酸的产量高于所述目的微生物的微生物；
E2)提高目的微生物中的D4)或D5)所述DNA分子的表达量或含量，得到L-谷氨酸的产量高于所述目的微生物的微生物；
E3)对所述目的微生物中的核苷酸序列为SEQ ID No.1的DNA分子进行突变，得到L-谷氨酸的产量高于所述目的微生物的微生物。
上述方法中，所述突变可为点突变(point mutation)，即单个核苷酸的突变。
上述方法中，所述点突变可为将SEQ ID No.1所示DNA分子编码的氨基酸序列的第199位的丙氨酸残基突变为另一种氨基酸残基。
上述方法中，所述点突变可为将SEQ ID No.1所示DNA分子编码的氨基酸序列的第199位的丙氨酸突变为苏氨酸，得到氨基酸序列为SEQ ID No.4的突变蛋白质BBD29_00405 M199I。
所述突变是指通过定点突变改变基因中的某个或某几个碱基，导致对应的蛋白质氨基酸组成发生改变，产生新的蛋白质或使原蛋白质产生新的功能，即基因定点突变。基因的定点突变技术如寡核苷酸引物介导的定点突变、PCR介导的定点突变或盒式突变等是本领域技术人员所熟知的。
本文所述点突变可为单碱基置换、单碱基插入或单碱基缺失，具体地可为单碱基置换。所述单碱基置换可为等位基因置换。
所述点突变可为将BBD29_00405基因(SEQ ID No.1)的第597位鸟嘌呤(G)进行核酸改造。
具体地，所述点突变可为将BBD29_00405基因(SEQ ID No.1)的第597位鸟嘌呤(G)变为腺嘌呤(A)，得到SEQ ID No.2所示的DNA分子。
本文中，所述重组载体具体可为重组载体pK18‐BBD29_00405 G597A、PK18mobsacB-BBD29_00405、PK18mobsacB-BBD29_00405 G597A、pXMJ19-BBD29_00405或pXMJ19-BBD29_00405 G597A。
所述重组载体pK18‐BBD29_00405 G597A含有SEQ ID No.2所示的突变的基因BBD29_00405 G597A的第1-1473位所示的DNA分子，具体是将SEQ ID No.31所示 的DNA片段BBD29_00405 G597A-Up-Down片段插入pK18mobsacB载体的XbaI识别位点间，保持pK18mobsacB载体的其他序列不变，得到的重组载体。
所述重组载体PK18mobsacB-BBD29_00405用于将外源基因BBD29_00405整合到宿主染色体中，在生产菌中过表达野生型BBD29_00405基因。
所述重组载体PK18mobsacB-BBD29_00405 G597A用于将外源基因BBD29_00405 G597A整合到宿主染色体中，在生产菌中过表达突变型基因BBD29_00405 G597A。
所述重组载体pXMJ19-BBD29_00405用于将外源基因BBD29_00405通过质粒在染色体外表达，进而在生产菌中过表达野生型BBD29_00405基因。
所述重组载体pXMJ19-BBD29_00405 G597A用于将外源基因BBD29_00405 G597A通过质粒在染色体外表达，进而在生产菌中过表达突变型基因BBD29_00405 G597A。
所述重组载体pK18‐BBD29_00405 G597A、PK18mobsacB-BBD29_00405、PK18mobsacB-BBD29_00405 G597A、pXMJ19-BBD29_00405和pXMJ19-BBD29_00405 G597A均在本发明的保护范围内。
本文中，所述重组微生物具体可为重组菌YPG-025、YPG-026、YPG-027、YPG-028或YPG-029。
所述重组菌YPG-025是将所述重组载体pK18‐BBD29_00405 G597A转化入谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220中得到的重组菌，所述重组菌YPG-025含有SEQ ID No.2所示的突变的基因BBD29_00405 G597A。
所述重组菌YPG-026含有双拷贝的SEQ ID No.1所示的BBD29_00405基因；含有双拷贝BBD29_00405基因的重组菌可以显著和稳定地提高BBD29_00405基因的表达量。重组菌YPG-026为在基因组上过表达野生型BBD29_00405基因的工程菌。
所述重组菌YPG-027含有SEQ ID No.2所示的突变的BBD29_00405 G597A基因；重组菌YPG-027为在基因组上过表达突变型BBD29_00405 G597A基因的工程菌。
重组菌YPG-028含有双拷贝SEQ ID No.1所示的BBD29_00405基因；重组菌YPG-028为在质粒上过表达野生型BBD29_00405基因的工程菌，即由质粒pXMJ19-BBD29_00405在染色体外进行过表达。
重组菌YPG-029含有SEQ ID No.2所示的突变的BBD29_00405 G597A基因；重组菌YPG-029为在质粒上过表达突变型BBD29_00405 G597A基因的工程菌，即由质粒pXMJ19-BBD29_00405 G597A在染色体外进行过表达。
所述重组菌YPG-025、YPG-025、YPG-027、YPG-027和YPG-029均在本发明的保护范围内。
本发明还提供了一种构建所述重组微生物的方法，所述方法包括至少下述任一种：
F1)将所述核酸分子BBD29_00405 G597A导入目的微生物，得到所述重组微生物；
F2)将SEQ ID No.1所示的DNA分子导入目的微生物，得到所述重组微生物；
F3)利用基因编辑手段(如单碱基基因编辑)对SEQ ID No.1所示的DNA分子进行编辑，使目的微生物中含有SEQ ID No.2所示的DNA分子。
所述导入可为通过化学转化法或电击转化法等任何已知的转化方法将携带本发明DNA分子的载体转化宿主菌。导入的DNA分子可以是单拷贝也可以是多拷贝。所述导入可以是将外源基因整合到宿主染色体中，也可以是由质粒在染色体外表达。
本发明还提供了一种制备L-谷氨酸的方法，所述方法包括利用本文中任一所述重组微生物生产L-谷氨酸。
上述方法中，所述方法可为发酵法制备L-谷氨酸，所述重组微生物可为棒杆菌属(Corynebacterium)，具体可为谷氨酸棒杆菌(Corynebacterium glutamicum)及其变体。
生物保藏说明
谷氨酸棒杆菌(Corynebacterium glutamicum)，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏机构简称：CGMCC，保藏日期为2020年11月23日，生物保藏编号为CGMCC No.21220，菌株命名：YPGLU001。
实施发明的最佳方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
以下实施例中培养所述菌株使用的基础培养基组成相同，在此基础培养基组成上添加相应需要的蔗糖、卡那霉素或氯霉素等。如果需要固体的，只需要添加2％的琼脂即可，pH7.0，培养温度30度。基础培养基中的溶质组成如下表1所示，将表1所示的溶质溶于水中，得到基础培养基：
表1为基础培养基的组成
试剂名称 配比 发酵罐
葡萄糖 g/L 5.0
磷酸 g/L 0.38
硫酸镁 g/L 1.85
氯化钾 g/L 1.6
生物素 μg/L 550
维生物素B1 μg/L 300
硫酸亚铁 mg/L 10
硫酸锰 g/dl 10
KH 2PO 4 g/L 2.8
维生素C mg/L 0.75
维生素B12 μg/L 2.5
对氨基苯甲酸 mg/L 0.75
消泡剂 ml/dl 0.0015
甜菜碱 g/L 1.5
甘蔗糖蜜 ml/L 7
玉米浆 ml/L 77
天冬氨酸 g/L 1.7
毛发粉 g/L 2
下述实施例中的谷氨酸棒杆菌(Corynebacterium glutamicum)YPGLU001CGMCC No.21220已于2020年11月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏登记号为CGMCC No.21220。谷氨酸棒杆菌YPGLU001，又称为谷氨酸棒杆菌CGMCC No.21220。
实施例1、构建包含点突变的BBD29_00405基因编码区的转化载体pK18-BBD29_00405 G597A
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组(GenBank：CP016335.1)序列，设计并合成两对扩增BBD29_00405基因(GenBank：AKF27993.1)编码区序列的引物，以等位基因置换的方式在菌株ATCC13869和高产L-谷氨酸的谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中引入点突变，对应编码蛋白的氨基酸序列为SEQ ID NO:3，BBD29_00405基因的核苷酸序列第597位鸟嘌呤(G)变为腺嘌呤(A)(SEQ ID NO：2：BBD29_00405 G597A)，对应编码蛋白的氨基酸序列第199位甲硫氨酸(M)变为异亮氨酸(I)(SEQ ID NO：4：BBD29_00405 M199I)。
野生型BBD29_00405基因的核苷酸序列为SEQ ID NO:1，其编码BBD29_00405蛋白的氨基酸序列为SEQ ID NO:3；
BBD29_00405 G597A突变基因的核苷酸序列为SEQ ID NO:2，其编码BBD29_00405 M199I突变蛋白的氨基酸序列为SEQ ID NO:4。
引物设计如下(上海invitrogen公司合成，其中下划线为突变碱基)：
P1:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATGACTATTAATGTCTCCGA 3'(SEQ ID NO:5)
P2:5'AGACCGGCATCAAGTATGGTCTGGGCA3'(SEQ ID NO:6)
P3:5'TGCCCAGACCATACTTGATGCCGGTCT3'(SEQ ID NO:7)
P4:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTAGCCGGCGTAAGGATCCCGGAT 3'(SEQ ID NO:8)
构建方法：以谷氨酸棒杆菌ATCC13869为模板，分别以引物P1和P2，P3和P4，进行PCR扩增。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，模板为1μL，余量为水，总体积50μL。
上述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸40s(30个循环)，72℃过度延伸10min，获得两条大小分别约647bp和927bp，含有BBD29_00405基因编码区的DNA片段(BBD29_00405 G597A-Up(SEQ ID No.29)和BBD29_00405 G597A-Down(SEQ ID No.30)。
将BBD29_00405 G597A-Up和BBD29_00405 G597A-Down经琼脂糖凝胶电泳分离纯化后；再以上述两条DNA片段为模板，以P1和P4为引物，通过重叠PCR扩增出长约1547bp的BBD29_00405 G597A-Up-Down片段，其核苷酸序列为SEQ ID No.31。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，模板为1μL，余量为水，总体积50μL。
上述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸100s(30个循环)，72℃过度延伸10min。
此DNA片段导致ATCC13869的诱变菌株谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中BBD29_00405基因编码区的第597位的鸟嘌呤(G)变为腺嘌呤(A)，最终导致编码蛋白的第199位氨基酸由甲硫氨酸(M)变为异亮氨酸(I)。
将pK18mobsacB质粒(购自Addgene公司)用XbaI酶切后，用琼脂糖凝胶电泳分离纯化BBD29_00405 G597A-Up-Down和线性化的pK18mobsacB质粒，再通过NEBuider重组系统(NEB E5520S)组装，获得载体pK18-BBD29_00405 G597A，该质粒上含有卡那霉素抗性标记。并将载体pK18-BBD29_00405 G597A送测序公司测序鉴定，将含有正确点突变(G-A)的载体pK18-BBD29_00405 G597A保存备用。
重组载体pK18-BBD29_00405 G597A是将SEQ ID No.31所示的DNA片段BBD29_00405 G597A-Up-Down片段插入pK18mobsacB载体的XbaI识别位点间，保持pK18mobsacB载体的其他序列不变，得到的重组载体。
实施例2、构建包含点突变的BBD29_00405 G597A的工程菌株
构建方法：将等位替换质粒pK18-BBD29_00405 G597A通过电击转化入高产L-谷氨酸的谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220，经测序确认该菌株染色体上保留有野生型的BBD29_00405基因编码区)中；对培养产生的单菌落分别通过引物P1和通用引物M13R进行鉴定，能扩增出大小约1554bp(SEQ ID No.32)条带的菌株为阳性菌株。将阳性菌株在含15％蔗糖的培养基上培养；对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用如下引物(上海invitrogen公司合成)进行PCR鉴定：
P5:5'AGAAGGCAACCTGCGCATGA 3'(SEQ ID NO:9)
P6:5'ATCGGGTTGGAAATCGCAGA 3'(SEQ ID NO:10)；
通用引物M13R序列如下：M13R:5’CAG GAA ACA GCT ATG ACC3’
上述PCR扩增产物261bp(SEQ ID No.33)通过高温变性、冰浴后进行sscp电泳(以质粒pK18-BBD29_00405 G597A扩增片段为阳性对照，ATCC13869扩增片段为阴性对照，水作为空白对照)，由于片段结构不同，电泳位置不同，因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。
以引物P5和P6再次通过PCR扩增等位替换成功的菌株目的片段，并连接到PMD19-T载体测序，通过比对碱基序列确定是否替换成功；并将来自高产谷氨酸的谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)替换成功的突变株命名为YPG-025。
重组菌YPG-025为以等位基因置换的方式在谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220的BBD29_00405基因编码区(SEQ ID No.1)中引入点突变G597A，使该基因第597的G突变A，该基因其他序列不变，得到包含点突变(G‐A)的基因工程菌YPG-025。
与谷氨酸棒杆菌CGMCC21220相比，谷氨酸棒杆菌YPG-025的差异仅在于：将谷氨酸棒杆菌CGMCC21220基因组中的SEQ ID No.1所示的BBD29_00405基因取代为SEQ ID No.2所示的BBD29_00405 G597A基因。SEQ ID No.1和SEQ ID No.2仅存在一个核苷酸差异，位于第597位。
SSCP电泳PAGE的制备及条件如下表2：
表2为sscp电泳的PAGE的制备
Figure PCTCN2021142440-appb-000001
Figure PCTCN2021142440-appb-000002
实施例3、构建基因组上过表达BBD29_00405或BBD29_00405 G597A基因的工程菌株
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组(GenBank：CP016335.1)序列，设计并合成三对扩增上下游同源臂片段及BBD29_00405基因编码区及启动子区序列的引物，以同源重组的方式在菌株谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中引入BBD29_00405或BBD29_00405 G597A基因。
引物设计如下(上海invitrogen公司合成)：
P7：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG 3'
P8：5'CCTACCACGA CGAGCACTAC ATCTACTCAT CTGAAGAATC 3'
P9：5'GATTCTTCAG ATGAGTAGAT GTAGTGCTCG TCGTGGTAGG 3'
P10：5'CAAACCAGAG TGCCCACGAA CTAGCCGGCG TAAGGATCCC 3'
P11：5'GGGATCCTTA CGCCGGCTAG TTCGTGGGCA CTCTGGTTTG 3'
P12：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCATAAGAAACAACCACTTCC 3'
构建方法：以谷氨酸棒杆菌ATCC13869或YPI019为模板，分别以引物P7/P8，P9/P10，P11/P12，进行PCR扩增，获得上游同源臂片段约806bp，BBD29_00405(SEQ ID No.34)或BBD29_00405 G597A基因片段(SEQ ID No.36)约1777bp及下游同源臂片段约788bp(SEQ ID No.37)，再以P7/P12为引物，用以上扩增的三个片段混合为模板进行扩增，获得3291bp整合同源臂片段上游-BBD29_00405-下游(SEQ ID No.38)或整合同源臂片段上游-BBD29_00405 G597A-下游(SEQ ID No.39)。PCR反应结束后，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒进行回收所需要的约3291bp的DNA片段，采用NEBuider重组系统与经Xba I酶切回收的穿梭质粒pk18mobsacB相连接，获得整合质粒(即重组载体)pk18mobsacB-BBD29_00405或pk18mobsacB-BBD29_00405 G597A，质粒上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得质粒整合到基因组上的重组子。
pk18mobsacB-BBD29_00405为将整合同源臂片段上游-BBD29_00405-下游(SEQ ID No.38)插入穿梭质粒pk18mobsacB的Xba I酶切位点间，得到的重组载体。
pk18mobsacB-BBD29_00405为将整合同源臂片段上游-BBD29_00405 G597A-下游(SEQ ID No.39)插入穿梭质粒pk18mobsacB的Xba I酶切位点间，得到的重组载体。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，模板为1μL，余量为水，总体积50μL。
上述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸180s(30个循环)，72℃过度延伸10min。
将2个整合质粒分别电转化入谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中，对培养产生的单菌落通过P13/P14引物进行PCR鉴定，PCR扩增出含有大小约1970bp(SEQ ID No.40)的片段的为阳性菌株，扩增不到片段的为原菌。阳性菌株经15％蔗糖筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P15/P16引物进行PCR鉴定，扩增出大小约1758bp(SEQ ID No.41)的菌为BBD29_00405或BBD29_00405 G597A基因整合到菌株谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)基因组上的菌株，其被命名为YPG-026(不含突变点)和YPG-027(含突变点)。
P13:5'GTCCAAGGTGACGGCCGCAC 3'
P14:5'AGCTTCGCCGATGTTGCGCA 3'
P15:5'AGGTTGCACCCGCCATCGCTGCA3'
P16:5'ATATTCGGCCCAGCAGCAGC 3'
重组菌YPG-026为将整合同源臂片段上游-BBD29_00405-下游(SEQ ID No.38)整合到菌株谷氨酸棒杆菌YPGLU001基因组，得到含有双拷贝的SEQ ID No.1所示的BBD29_00405基因的重组菌，含有双拷贝BBD29_00405基因的重组菌可以显著和稳定地提高BBD29_00405基因的表达量。
重组菌YPG027为将整合同源臂片段上游-BBD29_00405 G597A-下游(SEQ ID No.39)整合到菌株谷氨酸棒杆菌YPGLU001基因组，得到含有SEQ ID No.2所示的BBD29_00405 G597A突变基因的重组菌。
实施例4、构建质粒上过表达BBD29_00405或BBD29_00405 G597A基因的工程菌株
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组(GenBank：CP016335.1)序列，设计并合成一对扩增BBD29_00405基因编码区及启动子区序列的引物，引物设计如下(上海invitrogen公司合成)：
P17：5'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGTAGTGCTCGTCGTGGTAGG3'
P18：5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAGCCGGCGTAAGGATCCCGGAT 3'
构建方法：以ATCC13869或YPG-025为模板，以引物P17/P18进行PCR扩增，获得含有BBD29_00405的DNA分子(SEQ ID No.42)或含有BBD29_00405 G597A的DNA分子(SEQ ID No.43)约1807bp，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒进行回收所需要的1807bp的DNA片段，采用NEBuider重组系统与经EcoR I酶切、回收的穿梭质粒pXMJ19(BioVector NTCC BiovectorpXMJ19)相连接，获得过表达质粒pXMJ19-BBD29_00405或pXMJ19-BBD29_00405 G597A。质粒上含有氯霉素抗性标记，可以通过氯霉素筛选获得质粒转化到菌株中。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，模板为1μL，余量为水，总体积50μL。
所述上述PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸100s(30个循环)，72℃过度延伸10min。
重组载体pXMJ19-BBD29_00405将含有BBD29_00405的DNA分子(SEQ ID No.42)插入穿梭质粒pXMJ19的EcoR I酶切位点间，得到的重组载体。
重组载体pXMJ19-BBD29_00405 G597A将含有BBD29_00405 G597A的DNA分子(SEQ ID No.43)插入穿梭质粒pXMJ19的EcoR I酶切位点间，得到的重组载体。
将质粒分别电转化入谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中，对培养产生的单菌落通过M13R(-48)和P18引物进行PCR鉴定，PCR扩增出含有大小约1846bp的片段(SEQ ID No.44)的为转入菌株，其被命名为YPG-028(不含突变点)和YPG-029(含突变点)。
M13R(-48)序列如下：
5‘AGCGGATAAC AATTTCACAC AGGA3’
重组菌YPG-028含有双拷贝SEQ ID No.1所示的BBD29_00405基因；重组菌YPG-028为在质粒上过表达野生型BBD29_00405基因的工程菌，即由质粒pXMJ19-BBD29_00405在染色体外进行过表达。
重组菌YPG-029含有SEQ ID No.2所示的突变的BBD29_00405 G597A基因；重组菌YPG-029为在质粒上过表达突变型BBD29_00405 G597A基因的工程菌，即由质粒pXMJ19-BBD29_00405 G597A在染色体外进行过表达。
实施例5 构建基因组上缺失BBD29_00405基因的工程菌株
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组(GenBank：CP016335.1)序列，合成两对扩增BBD29_00405基因编码区两端片段的引物，作为上下游同源臂片段。引物设计如下(上海英俊公司合成)：
P19：5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTCTGGGGGTGAGCGCGGAT 3'
P20：AGGAAAATAACGCATCCATCTGCCCCTTTACAAATCCACCGCAAACACTGGGAT 3'
P21：TGGATTTGTAAAGGGGCAGATGGATGCGTTATTTTCCTTCACTTTTCGTATCCA 3'
P22：5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTCTGGCGCATCGAACAGGTCGAAGGA 3'
以谷氨酸棒杆菌ATCC13869为模板，分别以引物P19/P20及P21/P22，进行PCR扩增，获得上游同源臂片段709bp(SEQ ID No.45)及下游同源臂片段734bp(SEQ ID No.46)。再用引物P19/P22进行OVER PCR得到整个同源臂片段1405bp(SEQ ID No.47)。PCR反应结束后，对扩增的产物进行电泳回收，采用柱式DNA凝胶回收试剂盒进行回收所需要的1405bp的DNA片段，并通过NEBuider重组系统与经Xba I酶切回收的穿梭质粒pk18mobsacB质粒相连接，获得敲除质粒。该质粒上含有卡那霉素抗性标记。
将敲除质粒电转化入谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中，对培养产生的单菌落分别通过如下引物(上海英俊公司合成)进行PCR鉴定：
P23:5'GTCTGGGGGTGAGCGCGGAT3'
P24:5'CTCTGGCGCATCGAACAGGTCGAAGGA 3'
上述PCR扩增出大小约1331bp(SEQ ID No.49)及约2804bp(SEQ ID No.48)的条带的菌株为阳性菌株，只扩增出2804bp条带的菌株为出发菌。阳性菌株在15％蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P23/P24引物进行PCR鉴定，扩增出大小为1331bp条带的菌株为BBD29_00405基因编码区被敲除的基因工程菌株，其被命名为YPG-030。
重组菌YPG-030为将谷氨酸棒杆菌CGMCC No.21220上的基因组上的BBD29_00405基因被敲除。
实施例6 L-谷氨酸发酵实验
将实施例2-5构建的菌株YPG-025、YPG-026、YPG-027、YPG-028、YPG-029、 YPG-030和原始谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表3所示的培养基和表4所示发酵控制工艺进行发酵实验，收集发酵产物。
完成接种的初始时刻，体系菌浓度为15g/L。发酵过程中：通过补加50-55％葡萄糖水溶液控制体系含糖量(残糖)。
每个菌株重复三次，结果如表5所示。
表3为发酵培养基中溶质配方
试剂名称 配比
葡萄糖 5.0g/L
磷酸 0.38g/L
硫酸镁 1.85g/L
氯化钾 1.6g/L
生物素 550μg/L
维生物素B1 300μg/L
硫酸亚铁 10mg/L
硫酸锰 10g/dL
KH 2PO 4 2.8g/L
维生素C 0.75mg/L
维生素B12 2.5μg/L
对氨基苯甲酸 0.75mg/L
消泡剂 0.0015mL/dL
甜菜碱 1.5g/L
甘蔗糖蜜 7mL/L
玉米浆 77mL/L
天冬氨酸 1.7g/L
毛发粉 2g/L
上述发酵培养基为将表3所示的溶质溶于水中，得到的发酵培养基。
表4为发酵控制工艺
Figure PCTCN2021142440-appb-000003
表5为L-谷氨酸发酵实验结果
Figure PCTCN2021142440-appb-000004
结果如表5所示，在产L-谷氨酸的工程菌谷氨酸棒杆菌YPGLU001(生物保藏编号为CGMCC No.21220)中，对BBD29_00405基因编码区进行点突变BBD29_00405 G597A都有助于L-谷氨酸产量的提高；过表达BBD29_00405、BBD29_00405 G597A有助提高L-谷氨酸的产量，而对BBD29_00405基因敲除，不利于L-谷氨酸的积累。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业应用
本发明通过对BBD29_00405基因的敲除，发现该基因编码的产物对L-谷氨酸生产能力产生影响，通过在编码序列引入点突变，或者增加该基因的拷贝数或过表达获得重组菌株，所获得的菌株与未改造的菌株相比，有利于生产高浓度的L-谷氨酸本发明提供的CTD-2256P15.2或其编码的微肽PACMP的抑制剂在作用于肿瘤细胞或肿瘤组织时，能显著抑制肿瘤细胞的生长，增加肿瘤细胞的凋亡，缩小肿瘤体积，具有优异的抗肿瘤效果。本发明提供的新型抗肿瘤药物组合方案，将CTD-2256P15.2或其编码微肽PACMP的抑制剂和其他抗肿瘤药物联合使用，能显著增强抗肿瘤药物对肿瘤细胞的杀伤作用，降低肿瘤细胞的化疗耐药性，从而改善肿瘤临床治疗效果。CTD-2256P15.2在化疗耐药的肿瘤组织和细胞系中高表达，且其高表达与肿瘤病人的无疾病进展生存期和总体生存期显著负相关。本发明提供的CTD2256P15.2基因表达水平可作为预测肿瘤病人对化疗敏感性及预后的分子指标的应用，为有效指导肿瘤病人的临床化疗用药，评价治疗预后开创了新标准。
具体地，本发明首先以等位基因置换的方式在谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220的BBD29_00405基因编码区 (SEQ ID No.1)中引入点突变，构建了包含点突变(G‐A)的基因工程菌YPG-025。为进一步研究验证在生产菌中过表达野生型BBD29_00405基因或其突变基因BBD29_00405 G597A可以增加L-谷氨酸的产量，分别将外源基因整合到宿主染色体中或由质粒在染色体外表达，构建了基因组上和质粒上过表达BBD29_00405基因或BBD29_00405 G597A基因的工程菌YPG-026、YPG-027、YPG-028和YPG-029。实验表明，BBD29_00405基因及其变体参与了L-谷氨酸的生物合成，通过对BBD29_00405基因进行过表达或敲除、或定点突变(如点突变)可以调控L-谷氨酸在微生物内的积累量。对BBD29_00405基因编码区进行点突变或在生产菌中过表达BBD29_00405基因或其突变基因BBD29_00405 G597A，有助于L-谷氨酸产量及转化率的提高，而对BBD29_00405基因进行敲除或弱化，不利于L-谷氨酸的积累。可利用BBD29_00405基因及其变体(如BBD29_00405 G597A基因)来构建生产L-谷氨酸的基因工程菌种，以促进L-谷氨酸产量提高，培育符合工业化生产的高产、高质量菌种，对L-谷氨酸的工业化生产具有广泛的应用价值和重要的经济意义。

Claims (20)

  1. 一种生成L-谷氨酸的细菌，其特征在于，具有编码SEQ ID NO:3的氨基酸序列的多核苷酸的改善的表达；
    优选，所述改善的表达是编码SEQ ID NO:3的氨基酸序列的多核苷酸的表达增强，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变，或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变且表达是增强的。
  2. 如权利要求1所述的细菌，其特征在于，编码SEQ ID NO:3的氨基酸序列的多核苷酸的点突变，使得SEQ ID NO:3的氨基酸序列的第199位甲硫氨酸被不同的氨基酸所取代；优选第199位甲硫氨酸被异亮氨酸所取代。
  3. 如权利要求1或2所述的细菌，其特征在于，编码SEQ ID NO:3的氨基酸序列的多核苷酸包含SEQ ID NO:1的核苷酸序列。
  4. 如权利要求1-3任一项所述的细菌，其特征在于，所述具有点突变的多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第597位碱基发生突变而形成的；
    优选，所述突变包括SEQ ID NO:1所示多核苷酸序列第597位碱基由鸟嘌呤(G)突变为腺嘌呤(A)；
    优选，所述具有点突变的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
  5. 如权利要求1-4任一项所述的细菌，其特征在于，所述细菌是棒杆菌属细菌，优选嗜乙酰棒杆菌(Corynebacterium acetoacidophilum)、醋谷棒杆菌(Corynebacterium acetoglutamicum)、美棒杆菌(Corynebacterium callunae)、谷氨酸棒杆菌(Corynebacterium glutamicum)、黄色短杆菌(Brevibacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)、产氨棒杆菌(Corynebacterium ammoniagenes)、北京棒杆菌(Corynebacterium pekinense)、解糖短杆菌(Brevibacterium saccharolyticum)、玫瑰色短杆菌(Brevibacterium roseum)、生硫短杆菌(Brevibacterium thiogenitalis)；优选为谷氨酸棒杆菌YPGLU001，生物保藏编号为CGMCC No.21220，或谷氨酸棒杆菌ATCC 13869。
  6. 一种生产L-谷氨酸的方法，所述方法包括：培养权利要求1-5任一项所述的细菌，并从所述培养物中回收L-谷氨酸。
  7. 一种多核苷酸，其特征在于，包括编码含有SEQ ID NO:3所示的氨基酸序列的多核苷酸，其中第199位甲硫氨酸被不同的氨基酸所取代；优选第199位甲硫氨酸被异亮氨酸所取代；
    优选所述多核苷酸包括编码含有SEQ ID NO:4所示的氨基酸序列的多核苷酸；
    优选所述多核苷酸是由SEQ ID NO:1所示多核苷酸序列第597位碱基发生突变而形成的；优选所述突变是SEQ ID NO:1所示多核苷酸序列第597位碱基由鸟 嘌呤(G)突变为腺嘌呤(A)；
    优选所述多核苷酸包括SEQ ID NO:2所示的多核苷酸序列。
  8. 一种蛋白，其特征在于，所述蛋白的氨基酸序列如SEQ ID NO:4所示。
  9. 含有权利要求7所述的多核苷酸和/或权利要求8所述的蛋白的重组载体、表达盒、转基因细胞系和/重组菌。
  10. 权利要求7所述的多核苷酸、权利要求8所述的蛋白、权利要求9所述的重组载体、表达盒、转基因细胞系和/重组菌在生产L-谷氨酸中的应用。
  11. 蛋白质，其特征在于，所述蛋白质为下述任一种：
    A1)氨基酸序列是SEQ ID No.4的蛋白质；
    A2)将SEQ ID No.4所示的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；
    A3)在A1)或A2)的N端和/或C端连接标签得到的具有相同功能的融合蛋白质。
  12. 核酸分子，其特征在于，所述核酸分子为下述任一种：
    B1)编码权利要求11所述蛋白质的核酸分子；
    B2)编码序列是SEQ ID No.2所示的DNA分子；
    B3)核苷酸序列是SEQ ID No.2所示的DNA分子。
  13. 生物材料，其特征在于，所述生物材料为下述任一种：
    C1)含有权利要求12所述核酸分子的表达盒；
    C2)含有权利要求12所述核酸分子的重组载体、或含有C1)所述表达盒的重组载体；
    C3)含有权利要求12所述核酸分子的重组微生物、或含有C1)所述表达盒的重组微生物、或含有C2)所述重组载体的重组微生物。
  14. D1)-D8)中任一项的下述任一种应用：
    F1)D1)-D8)中任一项在调控微生物的L-谷氨酸的产量中的应用；
    F2)D1)-D8)中任一项在构建产L-谷氨酸的基因工程菌中的应用；
    F3)D1)-D8)中任一项在制备L-谷氨酸中的应用；
    其中，所述D1)-D8)为：
    D1)权利要求11所述的蛋白质；
    D2)权利要求12所述的核酸分子；
    D3)权利要求13所述的生物材料；
    D4)核苷酸序列为SEQ ID No.1的DNA分子；
    D5)SEQ ID No.1所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与SEQ ID No.1所示的DNA分子具有90％以上的同一性，且具有相同功能的DNA分子；
    D6)含有D4)或D5)中所述DNA分子的表达盒；
    D7)含有D4)或D5)中所述DNA分子的重组载体、或含有D6)所述表达盒的重组载体；
    D8)含有D4)或D5)中所述DNA分子的重组微生物、或含有D6)所述表达盒的重组微生物、或含有D7)所述重组载体的重组微生物。
  15. 一种提高微生物中L-谷氨酸的产量的方法，其特征在于，所述方法包括下述任一种：
    E1)提高目的微生物中的权利要求12所述核酸分子的表达量或含量，得到L-谷氨酸的产量高于所述目的微生物的微生物；
    E2)提高目的微生物中的权利要求14中D4)或D5)所述DNA分子的表达量或含量，得到L-谷氨酸的产量高于所述目的微生物的微生物；
    E3)对所述目的微生物中的核苷酸序列为SEQ ID No.1的DNA分子进行突变，得到L-谷氨酸的产量高于所述目的微生物的微生物。
  16. 根据权利要求15所述的方法，其特征在于，所述突变为点突变。
  17. 根据权利要求16所述的方法，其特征在于，所述点突变为将SEQ ID No.1所示DNA分子编码的氨基酸序列的第199位的甲硫氨酸残基突变为另一种氨基酸残基。
  18. 根据权利要求16或17所述的方法，其特征在于，所述点突变为将SEQ ID No.1所示DNA分子编码的氨基酸序列的第199位的丙氨酸突变为异亮氨酸，得到氨基酸序列为SEQ ID No.4的突变蛋白质。
  19. 一种构建权利要求13或14中所述重组微生物的方法，其特征在于，所述方法包括至少下述任一种：
    F1)将权利要求12所述的核酸分子导入目的微生物，得到所述重组微生物；
    F2)将SEQ ID No.1所示的DNA分子导入目的微生物，得到所述重组微生物；
    F3)利用基因编辑手段对SEQ ID No.1所示的DNA分子进行编辑，使目的微生物中含有SEQ ID No.2所示的DNA分子。
  20. 一种制备L-谷氨酸的方法，其特征在于，所述方法包括利用权利要求13或14中所述的重组微生物生产L-谷氨酸。
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