WO2022143762A1 - 一种改造基因bbd29_14900的重组菌株及其构建方法与应用 - Google Patents

一种改造基因bbd29_14900的重组菌株及其构建方法与应用 Download PDF

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WO2022143762A1
WO2022143762A1 PCT/CN2021/142439 CN2021142439W WO2022143762A1 WO 2022143762 A1 WO2022143762 A1 WO 2022143762A1 CN 2021142439 W CN2021142439 W CN 2021142439W WO 2022143762 A1 WO2022143762 A1 WO 2022143762A1
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bbd29
gene
protein
seq
bacteria
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PCT/CN2021/142439
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English (en)
French (fr)
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马风勇
魏爱英
孟刚
赵春光
贾慧萍
苏厚波
杨立鹏
郭小炜
田斌
周晓群
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宁夏伊品生物科技股份有限公司
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Priority to EP21914494.6A priority Critical patent/EP4273246A1/en
Priority to JP2023540090A priority patent/JP2024505807A/ja
Priority to KR1020237025464A priority patent/KR20230145054A/ko
Publication of WO2022143762A1 publication Critical patent/WO2022143762A1/zh
Priority to US18/270,493 priority patent/US20240076701A1/en

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    • 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
    • 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
    • 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
    • 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
    • C12P13/18—Glutamic acid; Glutamine using biotin or its derivatives
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • C12R2001/13—Brevibacterium
    • 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

Definitions

  • the invention belongs to the technical field of genetic engineering and microorganisms, and in particular relates to a recombinant strain of modified gene BBD29_14900 and a construction method and application thereof, and the specific application is to produce L-glutamic acid.
  • the daily seasoning monosodium glutamate is L-glutamic acid monosodium salt. Since the invention and industrial production of monosodium glutamate in Japan in 1909, it has developed into a worldwide amino acid fermentation industry with the theme of glutamic acid fermentation after several changes. The fermentation to produce amino acids such as MSG glutamic acid is different from traditional wine-making and antibiotic fermentation. It is a metabolically controlled fermentation that changes the metabolism of microorganisms.
  • the strains used by manufacturers for glucose fermentation are mainly some mutant strains, and the production strains are mainly obtained through the breeding of microorganisms that are mainly mutagenic.
  • classical microbial breeding techniques such as mutagenesis, cell fusion and other means under the existing acid-producing capacity, in order to greatly improve the acid production rate and sugar-acid conversion rate. Therefore, it is the most effective method to use modern genetic engineering methods to transform strains to improve the acid production rate and sugar-acid conversion rate.
  • the production strains can be transformed by means of genetic engineering, the acid production rate of glutamic acid and the conversion rate of sugar and acid will have a great breakthrough.
  • the present invention provides a recombinant strain of modified gene BBD29_14900 and a construction method and application thereof.
  • An object of the present invention is to develop a new technique for improving the L-glutamic acid production capacity of bacteria, thereby providing a method for efficiently producing L-glutamic acid.
  • the inventors of the present invention have found through research that the gene BBD29_14900 or its homologous gene can obtain bacteria with enhanced L-glutamic acid production capacity by modifying the gene or improving its expression. Based on these findings, the present invention has been completed.
  • the present invention provides L-glutamic acid-producing bacteria in which the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or a homologous sequence thereof is improved.
  • the present invention also provides a method for producing L-glutamic acid by using the microorganism.
  • a first aspect of the present invention provides an L-glutamic acid producing bacterium having improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or a homologous sequence thereof.
  • the improved expression is enhanced expression of the polynucleotide, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 or a homologous sequence thereof has a point mutation, or the amino acid encoding SEQ ID NO: 3
  • the polynucleotide of the sequence or its homologous sequence has point mutations and expression is enhanced.
  • amino acid sequence of the SEQ ID NO: 3 or its homologous sequence is the protein encoded by the gene BBD29_14900 or its homologous gene.
  • 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.
  • the bacteria having L-glutamic acid-producing ability may be bacteria capable of accumulating target L-glutamic acid in a medium in an amount of preferably 0.5 g/L or more, more preferably 1.0 g/L or more.
  • 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 polynucleotide 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 before mutation 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 the aspartic acid (D) at position 372 of the amino acid sequence of SEQ ID NO:3 is different replaced by amino acids.
  • the aspartic acid at position 372 is replaced by asparagine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 372nd aspartic acid is replaced by asparagine is shown in SEQ ID NO:4.
  • the polynucleotide sequence with point mutation is formed by mutating the 1114th base of the polynucleotide sequence shown in SEQ ID NO: 1.
  • the mutation includes the mutation of the 1114th 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_14900 gene) 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.
  • transformed cells can be selected because only cells expressing the selectable marker can survive or display different phenotypic traits.
  • 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 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 is highly glutamic acid-producing, and the preservation information is as follows: strain name: Corynebacterium glutamicum; Latin name: Corynebacterium glutamicum; Strain number: YPGLU001; Preservation institution: General Microbiology Center of China Microorganism Culture Collection; Abbreviation of Preservation Institution: CGMCC; Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation Date: 2020 November 23, 2009; the registration number of the collection center: CGMCC No.21220.
  • the bacteria may also have other improvements related to increased L-glutamic acid production, for example, glutamate dehydrogenase, glutamine synthase, glutamine-ketoglutarate aminotransferase, etc. Enzyme activity or increased or decreased expression of a gene, or a gene can be replaced by a foreign gene.
  • 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, and the aspartic acid at position 372 of the sequence is substituted with a different amino acid.
  • the aspartic acid at position 372 is replaced by asparagine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 372nd aspartic acid is replaced by asparagine 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 1114th 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 employed.
  • the mutation includes the mutation of the 1114th base of the polynucleotide sequence shown in SEQ ID NO: 1 from guanine (G) 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 by the NEBuider recombination system.
  • the recombinant strain contains the polynucleotide sequence.
  • the starting bacteria of the recombinant strain is Corynebacterium glutamicum CGMCC No.21220.
  • the starting bacteria of the recombinant strain is ATCC 13869.
  • the third 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_14900 gene as shown in SEQ ID NO: 1 in the host strain was transformed, and the 1114th base was mutated to obtain a recombinant strain comprising the mutated BBD29_14900 encoding gene.
  • the transformation includes at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to that the 1114th base in SEQ ID NO: 1 is changed from guanine (G) to adenine (A); specifically, the polynucleotide comprising the gene encoding the mutation BBD29_14900 The acid sequence is shown in SEQ ID NO:2.
  • construction method comprises the steps:
  • the step (1) comprises: constructing the BBD29_14900 gene of the 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_14900 gene fragment, by PCR
  • a point mutation was introduced into the wild-type BBD29_14900 gene SEQ ID NO: 1 by site-directed mutagenesis, and the nucleotide sequence of the point-mutated BBD29_14900 gene SEQ ID NO: 2 was obtained, which was denoted as BBD29_14900 G1114A .
  • the genome of the unmodified strain can be derived from the ATCC13869 strain, the genome sequence of which can be obtained from the NCBI website.
  • the primer is:
  • P2 (SEQ ID NO: 6): 5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3'
  • P3 (SEQ ID NO: 7): 5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3'
  • 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, and overextension at 72°C for 10 min .
  • the overlapping PCR amplification is carried out 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 90s, 30 cycles, overextension at 72°C 10min.
  • the step (2) includes the construction of recombinant plasmids, including: assembling the isolated and purified BBD29_14900 G1114A 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 CGMCC No.21220.
  • the recombination is achieved by homologous recombination.
  • 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:
  • BBD29_14900 Amplify the upstream and downstream homology arm fragments of BBD29_14900, the BBD29_14900 gene coding region and its promoter region sequence, and introduce the BBD29_14900 or BBD29_14900 G1114A gene into the genome of the host strain by homologous recombination to achieve the strain overexpressing BBD29_14900 or BBD29_14900 G1114A gene.
  • the primer for amplifying the upstream homology arm fragment is:
  • P8 (SEQ ID NO: 12): 5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-3'
  • the primers for amplifying the downstream homology arm fragments are:
  • P11 SEQ ID NO: 15: 5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3'
  • the primers for amplifying the coding region of the gene and the sequence of its promoter region are:
  • P9 SEQ ID NO: 13: 5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3'
  • P10 SEQ ID NO: 14: 5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3'
  • the aforementioned P7/P12 is used as a primer, and the amplified upstream homology arm fragment, the downstream homology arm fragment, the gene coding region and the three fragment mixtures of the promoter region sequence fragments are used as The template is amplified to obtain integrated homology arm fragments.
  • the PCR system used 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) 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 120s (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_14900 or BBD29_14900 G1114A gene is introduced into the genome of the host strain by means of homologous recombination.
  • the host strain is Corynebacterium glutamicum 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 fifth 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:
  • the primers for amplifying the coding region of the gene and the sequence of its promoter region are:
  • 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 pM) 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 90s (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_14900 or BBD29_14900 G1114A fragment with its own promoter to obtain an overexpression plasmid.
  • the host strain is Corynebacterium glutamicum 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 invention provides a protein (mutant protein, named BBD29_14900 D372N protein), which is obtained by mutating the 372nd amino acid residue of BBD29_14900 protein from aspartic acid to asparagine;
  • the BBD29_14900 protein is as follows (a1) or (a2) or (a3):
  • (a2) a protein derived from bacteria and having more than 95% identity with (a1) and related to bacterial glutamic acid production;
  • (a3) A protein derived from (a1) which is obtained by subjecting the protein shown in (a1) to substitution and/or deletion and/or addition of one or several amino acid residues and is related to bacterial glutamic acid production.
  • identity refers to sequence similarity to the native amino acid sequence. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the identity of more than 95% may specifically be more than 96% identity or more than 97% identity or more than 98% identity or more than 99% identity.
  • BBD29_14900 D372N protein is shown in SEQ ID NO:4.
  • the BBD29_14900 gene is the gene encoding the BBD29_14900 protein.
  • the BBD29_14900 gene is as follows (b1) or (b2) or (b3):
  • (b2) a DNA molecule that is derived from bacteria and has more than 95% identity with (b1) and encodes the protein;
  • (b3) a DNA molecule that hybridizes to (b1) under stringent conditions and encodes the protein.
  • identity refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the identity of more than 95% may specifically be more than 96% identity or more than 97% identity or more than 98% identity or more than 99% identity.
  • the stringent conditions can be hybridization and membrane washing in a solution of 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS at 65°C.
  • the gene encoding the BBD29_14900 D372N protein (named as BBD29_14900 G1114A gene) also belongs to the protection scope of the present invention.
  • the BBD29_14900 G1114A gene is as follows (c1) or (c2) or (c3):
  • (c2) a DNA molecule that is derived from bacteria and has more than 95% identity with (c1) and encodes the protein
  • identity refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the identity of more than 95% may specifically be more than 96% identity or more than 97% identity or more than 98% identity or more than 99% identity.
  • the stringent conditions can be hybridization and membrane washing in a solution of 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS at 65°C.
  • DNA molecules with BBD29_14900 G1114A gene expression cassettes with BBD29_14900 G1114A gene or recombinant vectors with BBD29_14900 G1114A gene or recombinant bacteria with BBD29_14900 G1114A gene all belong to the protection scope of the present invention.
  • the DNA molecule having the BBD29_14900 G1114A gene can be the DNA molecule shown in SEQ ID NO:30 or the DNA molecule shown in SEQ ID NO:31.
  • the recombinant vector having the BBD29_14900 G1114A gene can be a plasmid having the DNA molecule shown in SEQ ID NO:30 or a plasmid having the DNA molecule shown in SEQ ID NO:31.
  • the recombinant vector with the BBD29_14900G1114A gene can be the integration plasmid PK18mobsacB- BBD29_14900G1114A or the overexpression plasmid pXMJ19-BBD29_14900G1114A in the Examples .
  • the recombinant bacteria can be specifically recombinant bacteria.
  • the recombinant bacteria with the BBD29_14900 G1114A gene may be the recombinant bacteria with the DNA molecule shown in SEQ ID NO:30 or the recombinant bacteria with the DNA molecule shown in SEQ ID NO:31 or the recombinant bacteria with the DNA molecule shown in SEQ ID NO:2. Recombinant bacteria with the DNA molecules shown.
  • the recombinant bacteria with the BBD29_14900 G1114A gene can be specifically prepared in the following manner: the BBD29_14900 gene in the bacterial genome is replaced with the BBD29_14900 G1114A gene.
  • the implementation of replacing the BBD29_14900 gene in the bacterial genome with the BBD29_14900 G1114A gene is as follows: the DNA molecule shown in SEQ ID NO: 29 is introduced into the bacteria.
  • the implementation of replacing the BBD29_14900 gene in the bacterial genome with the BBD29_14900 G1114A gene is as follows: the vector pK18-BBD29_14900 G1114A in the example is introduced into the bacteria.
  • the BBD29_14900 G1114A gene can be integrated into the genomic DNA for expression or expressed in a plasmid.
  • the recombinant bacteria with the BBD29_14900 G1114A gene can be specifically prepared by the following method: introducing the DNA molecule shown in SEQ ID NO: 30 or the DNA molecule shown in SEQ ID NO: 31 into the bacteria.
  • the recombinant bacteria with the BBD29_14900 G1114A gene can be specifically prepared in the following manner: the plasmid with the DNA molecule shown in SEQ ID NO:30 or the plasmid with the DNA molecule shown in SEQ ID NO:31 is introduced into the bacteria.
  • the recombinant bacteria with the BBD29_14900 G1114A gene can be prepared in the following manner: The integration plasmid PK18mobsacB-BBD29_14900 G1114A or the overexpression plasmid pXMJ19-BBD29_14900 G1114A in the example is introduced into the bacteria.
  • the present invention also protects the application of BBD29_14900 D372N protein, BBD29_14900 G1114A gene, expression cassette with BBD29_14900 G1114A gene or recombinant vector with BBD29_14900 G1114A gene or recombinant bacteria with BBD29_14900 G1114A gene;
  • the present invention also protects the application of specific substances
  • the substance for improving the expression of the BBD29_14900 G1114A gene may specifically be the BBD29_14900 G1114A gene or a recombinant plasmid having the BBD29_14900 G1114A gene.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB- BBD29_14900G1114A or the overexpression plasmid pXMJ19-BBD29_14900G1114A in the Examples .
  • the substance for increasing the expression of the BBD29_14900 gene may specifically be the BBD29_14900 gene or a recombinant plasmid having the BBD29_14900 gene.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB-BBD29_14900 in the Examples or the overexpression plasmid pXMJ19-BBD29_14900.
  • the present invention also provides a recombinant bacteria obtained by overexpressing BBD29_14900 G1114A gene or BBD29_14900 gene in bacteria.
  • the implementation of overexpressing the BBD29_14900 G1114A gene is as follows: introducing the BBD29_14900 G1114A gene or a recombinant plasmid having the BBD29_14900 G1114A gene into bacteria.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB- BBD29_14900G1114A or the overexpression plasmid pXMJ19-BBD29_14900G1114A in the Examples .
  • the overexpression of the BBD29_14900 gene is achieved as follows: the BBD29_14900 gene or a recombinant plasmid having the BBD29_14900 gene is introduced into bacteria.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB-BBD29_14900 in the Examples or the overexpression plasmid pXMJ19-BBD29_14900.
  • the invention also protects the application of the recombinant bacteria in the preparation of glutamic acid.
  • the present invention also protects a method for improving the glutamic acid production of bacteria, comprising the following steps: replacing the BBD29_14900 gene in the bacterial genome with the BBD29_14900 G1114A gene.
  • the implementation of replacing the BBD29_14900 gene in the bacterial genome with the BBD29_14900 G1114A gene is as follows: the vector pK18-BBD29_14900 G1114A in the example is introduced into the bacteria.
  • the present invention also provides a method for increasing the glutamic acid production of bacteria, comprising the steps of: overexpressing BBD29_14900 G1114A gene in bacteria or overexpressing BBD29_14900 gene in bacteria or increasing the abundance of BBD29_14900 D372N protein in bacteria or improving bacteria
  • the abundance of BBD29_14900 protein in bacteria may increase the activity of BBD29_14900 D372N protein in bacteria or increase the activity of BBD29_14900 protein in bacteria.
  • the implementation of overexpressing the BBD29_14900 G1114A gene is as follows: introducing the BBD29_14900 G1114A gene or a recombinant plasmid having the BBD29_14900 G1114A gene into bacteria.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB- BBD29_14900G1114A or the overexpression plasmid pXMJ19-BBD29_14900G1114A in the Examples .
  • the overexpression of the BBD29_14900 gene is achieved as follows: the BBD29_14900 gene or a recombinant plasmid having the BBD29_14900 gene is introduced into bacteria.
  • the recombinant plasmid can be the integration plasmid PK18mobsacB-BBD29_14900 in the Examples or the overexpression plasmid pXMJ19-BBD29_14900.
  • the present invention also protects the application of BBD29_14900 D372N protein or BBD29_14900 protein in regulating the glutamate production of bacteria.
  • the regulation is positive regulation, that is, the BBD29_14900 D372N protein content increases, and the glutamate production increases.
  • the regulation is positive regulation, that is, the BBD29_14900 D372N protein content is reduced, and the glutamate production is reduced.
  • the regulation is positive regulation, that is, the protein content of BBD29_14900 increases, and the glutamic acid production increases.
  • the regulation is positive regulation, that is, the BBD29_14900 protein content is reduced, and the glutamate production is reduced.
  • the specific method includes the following steps: fermenting the recombinant bacteria.
  • Bacterial fermentation can be carried out in a suitable medium under fermentation 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 20-45°C.
  • the method may further comprise the step of obtaining glutamate from the culture.
  • Obtaining glutamate from the culture can be accomplished by various means including, but not limited to, treatment of the culture with sulfuric acid or hydrochloric acid, etc., followed by methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation combination.
  • the formula of the exemplary fermentation medium is shown in Table 3, and the balance is water.
  • the bacterial concentration in the system is 15 g/L.
  • the sugar content (residual sugar) of the system is controlled by adding an aqueous glucose solution.
  • any of the bacteria described above include but are not limited to the following: Corynebacterium, preferably Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum (Corynebacterium acetoglutamicum) Corynebacterium glutamicum), Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, Brevibacterium rosacea Brevibacterium roseum, Brevibacterium thiogenitalis.
  • Any of the above-mentioned bacteria are bacteria having the ability to produce glutamic acid.
  • Bacteria having the ability to produce glutamic acid means that the bacteria have the ability to produce and accumulate glutamic acid in the culture medium and/or cells of the bacteria. Thus, glutamate can be collected when the bacteria are grown in the medium.
  • the bacteria can be wild-type bacteria collected naturally or modified bacteria.
  • Modified bacteria refers to modified bacteria obtained by artificially mutating and/or mutagenizing naturally collected wild-type bacteria.
  • Corynebacterium glutamicum can be Corynebacterium glutamicum CGMCC21260.
  • Corynebacterium glutamicum CGMCC21220 the strain name is YPGLU001, abbreviated as Corynebacterium glutamicum CGMCC21220, has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee (referred to as CGMCC, address) on November 23, 2020 It is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and the deposit registration number is CGMCC 21220.
  • glutamic acid means glutamic acid in a broad sense, including free form glutamic acid, salts of glutamic acid, or a mixture of the two.
  • the glutamic acid is L-glutamic acid.
  • the BBD29_14900 protein in Corynebacterium glutamicum is shown in SEQ ID NO:3, and its encoding gene is shown in SEQ ID NO:1.
  • the BBD29_14900 D372N protein shown in SEQ ID NO:4 is obtained, and the encoding gene of the BBD29_14900 D372N protein is shown in SEQ ID NO:2.
  • the difference of the BBD29_14900 G1114A gene is that the 1114th position is changed from guanine deoxyribonucleotide (G) to adenine deoxyribonucleotide (A).
  • the difference of BBD29_14900 D372N protein is that the 372nd amino acid residue is changed from aspartic acid (D) to asparagine (N).
  • the present invention finds that the BBD29_14900 protein positively regulates the glutamic acid production of bacteria, that is, the BBD29_14900 protein content increases and the glutamic acid production increases, while the BBD29_14900 protein content decreases and the glutamic acid production decreases. Inhibition of BBD29_14900 gene expression can reduce glutamate production, while overexpression of BBD29_14900 gene increases glutamate production. Further, the present invention obtains BBD29_14900 D372N protein through point mutation, and its function is better than that of BBD29_14900 protein. The invention has great application value for the industrial production of glutamic acid.
  • the medium used for culturing the strains in the following examples is obtained by adding other components on the basis of the basal medium.
  • Other ingredients are sucrose, kanamycin or chloramphenicol.
  • Solid medium contains agarose.
  • the composition of the basal medium is shown in Table 1. Unless otherwise specified, the culture temperature of the strains in the examples is all 32°C.
  • Electrophoresis conditions put the electrophoresis tank in ice, use 1 ⁇ TBE buffer, voltage 120V, and electrophoresis time 10h.
  • Corynebacterium glutamicum CGMCC21220 the strain name is YPGLU001, abbreviated as Corynebacterium glutamicum CGMCC21220, has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee (referred to as CGMCC, address) on November 23, 2020 It is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and the deposit registration number is CGMCC 21220.
  • Corynebacterium glutamicum ATCC 13869 namely Corynebacterium glutamicum CICC 20216; the full name of CICC is China Industrial Microorganism Culture Collection and Management Center.
  • Example 1 Construction of transformation vector pK18-BBD29_14900 G1114A comprising the coding region of the BBD29_14900 gene of point mutation
  • the BBD29_14900 gene coding region on the chromosome of Corynebacterium CGMCC21220 is identical to the coding region of the BBD29_14900 gene on the chromosome of Corynebacterium glutamicum ATCC13869) and a point mutation was introduced.
  • the amino acid sequence of the corresponding encoded protein before the mutation is SEQ ID NO:3, and the nucleotide sequence of the BBD29_14900 gene before the mutation is SEQ ID NO:1.
  • a single point mutation was introduced into the gene, that is, the 1114th position of the coding region was changed from guanine deoxyribonucleotide (G) to adenine deoxyribonucleotide (A).
  • a single point mutation of the protein was caused, that is, the amino acid residue at position 372 was changed from aspartic acid (D) to asparagine (N).
  • the mutated gene is called the BBD29_14900 G1114A gene, as shown in SEQ ID NO:2.
  • the mutated protein was designated as BBD29_14900 D372N protein, as shown in SEQ ID NO:4.
  • the primers are as follows (synthesized by Shanghai Invitrogen Company):
  • P2 (SEQ ID NO: 6): 5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3'
  • P3 (SEQ ID NO: 7): 5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3'
  • PCR system 50 ⁇ L: Template, 5 ⁇ L of 10 ⁇ Ex Taq Buffer, 4 ⁇ L of dNTP Mixture (2.5 mM each), 4 ⁇ L of Mg 2+ (25 mM), 2 ⁇ L of each primer (10 ⁇ M), 0.25 ⁇ L of Ex Taq (5U/ ⁇ L), the rest The amount is water.
  • PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 40s, and overextension at 72°C for 10 min.
  • step 2 The amplification products of the two PCR systems in step 1 are recovered respectively, and at the same time, as a template, a primer pair composed of P1 and P4 is used for PCR amplification.
  • PCR system 50 ⁇ L: Template, 5 ⁇ L of 10 ⁇ Ex Taq Buffer, 4 ⁇ L of dNTP Mixture (2.5 mM each), 4 ⁇ L of Mg 2+ (25 mM), 2 ⁇ L of each primer (10 ⁇ M), 0.25 ⁇ L of Ex Taq (5U/ ⁇ L), the rest The amount is water.
  • PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s, and overextension at 72°C for 10 min.
  • the BBD29_14900 G1114A fragment can be integrated into Corynebacterium glutamicum CGMCC21220 by homologous recombination, resulting in the mutation of the 1114th nucleotide in the coding region of the BBD29_14900 gene from guanine deoxyribonucleotide (G) to adenine deoxyribonucleoside acid (A), eventually resulting in the mutation of amino acid residue 372 of the encoded protein from aspartic acid (D) to asparagine (N).
  • the pK18mobsacB plasmid (product of Addgene) was digested with restriction endonuclease Xba I, and the linearized plasmid was recovered.
  • the vector pK18-BBD29_14900 G1114A constructed in Example 1 was introduced into Corynebacterium glutamicum CGMCC21220 by electroporation, and the single colonies produced by the culture were activated by primer P1 and universal primer M13R (M13R: 5'-CAGGAAACAGCTATGACC-3').
  • M13R 5'-CAGGAAACAGCTATGACC-3'
  • the strains that can amplify a band of about 1260bp are positive strains.
  • Positive strains were cultured on medium plates containing 15% sucrose, and single colonies produced by culture were cultured on plates containing kanamycin and without The strains that grow on and do not grow on the medium containing kanamycin were further identified by PCR using a primer pair composed of P5 and P6 (synthesized by Shanghai Invitrogen Company).
  • P5 (SEQ ID NO: 9): 5'-GCAGCCAAGGCCAAGAAGAT-3';
  • P6 (SEQ ID NO: 10): 5'-TCCCTGTTTAAGACTGCATT-3'.
  • Corynebacterium glutamicum YPG-019 the only difference of Corynebacterium glutamicum YPG-019 is that the BBD29_14900 gene shown in Sequence 1 of the Sequence Listing in the genome of Corynebacterium glutamicum CGMCC21220 is replaced by Sequence 2 of the Sequence Listing BBD29_14900 G1114A gene shown. There is only one nucleotide difference between sequence 1 and sequence 2, which is located at position 1114.
  • the primers are as follows (synthesized by Shanghai Invitrogen Company):
  • P8 (SEQ ID NO: 12): 5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-3'
  • P9 SEQ ID NO: 13: 5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3'
  • P10 SEQ ID NO: 14: 5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3'
  • P11 SEQ ID NO: 15: 5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3'
  • a primer pair consisting of P7/P8 a primer pair consisting of P9/P10, and a primer pair consisting of P11/P12 were used for PCR amplification, and the upstream homology arm fragment was obtained.
  • 806bp, BBD29_14900 G1114A gene fragment is about 1494bp, and the downstream homology arm fragment is about 788bp, and then a primer pair composed of P7/P12 is used, and the three amplified fragments above are mixed as templates for amplification, and an integrated homology arm of about 3008bp is obtained. Fragment (sequenced as shown in SEQ ID NO: 30).
  • the integrated homology arm fragment was ligated with the shuttle plasmid PK18mobsacB recovered by Xba I digestion to obtain the integrated plasmid PK18mobsacB-BBD29_14900 G1114A .
  • the integrated plasmid contains a kanamycin resistance marker, and the recombinant plasmid integrated into the genome can be obtained by kanamycin selection.
  • PCR system 50 ⁇ L: Template, 5 ⁇ L of 10 ⁇ Ex Taq Buffer, 4 ⁇ L of dNTP Mixture (2.5mM each), 4 ⁇ L of Mg 2+ (25mM), 2 ⁇ L of primers (10 ⁇ M), 0.25 ⁇ L of Ex Taq (5U/ ⁇ L), the rest The amount is water.
  • 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 120s, 30 cycles, and overextension at 72°C for 10 min.
  • the integrated plasmid was electro-transformed into Corynebacterium glutamicum CGMCC21220, and the single colony produced by the culture was identified by PCR with the primer pair composed of P13/P14.
  • the PCR amplified fragments containing a fragment of about 1827 bp were the positive strains that were successfully transformed.
  • the strain that cannot increase the fragment is the untransformed strain.
  • P14 (SEQ ID NO: 18): 5'-GCAGCCTTAACTGGGGAAAG-3'
  • P15 (SEQ ID NO: 19): 5'-GGAGCGCCGCCTCATCGAGC-3'
  • P16 (SEQ ID NO: 20): 5'-ATATTCGGCCCAGCAGCAGC-3'
  • the integrated plasmid PK18mobsacB-BBD29_14900 was subjected to the above steps, and the obtained recombinant bacteria was Corynebacterium glutamicum YPG-020.
  • Corynebacterium glutamicum YPG-020 is a recombinant bacterium that overexpresses the BBD29_14900 gene in its genomic DNA.
  • the integrated plasmid PK18mobsacB-BBD29_14900 G1114A was subjected to the above steps, and the obtained recombinant bacteria was Corynebacterium glutamicum YPG-021.
  • Corynebacterium glutamicum YPG-021 is a recombinant bacterium that overexpresses BBD29_14900 G1114A gene in genomic DNA.
  • BBD29_14900 G1114A gene fragment of 1524bp (through sequencing, BBD29_14900 G1114A gene fragment is as shown in SEQ ID NO:31) .
  • BBD29_14900 G1114A gene fragment was ligated with the shuttle plasmid pXMJ19 recovered by EcoR I digestion to obtain the overexpression plasmid pXMJ19-BBD29_14900 G1114A .
  • the overexpression plasmid contains a chloramphenicol resistance marker, and the plasmid can be transformed into the strain by screening with chloramphenicol.
  • PCR system 50 ⁇ L: Template, 5 ⁇ L of 10 ⁇ Ex Taq Buffer, 4 ⁇ L of dNTP Mixture (2.5mM each), 4 ⁇ L of Mg 2+ (25mM), 2 ⁇ L of primers (10pM), 0.25 ⁇ L of Ex Taq (5U/ ⁇ L), the rest The amount is water.
  • PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s, and overextension at 72°C for 10 min.
  • the overexpression plasmid was electrotransformed into Corynebacterium glutamicum CGMCC21220, and the single colony produced by the culture was identified by PCR using the primer pair composed of M13R(-48) and P18.
  • the recombinant bacteria were amplified by PCR with a fragment of about 1563 bp.
  • the overexpression plasmid pXMJ19-BBD29_14900 was subjected to the above steps, and the obtained recombinant bacteria was Corynebacterium glutamicum YPG-022.
  • Corynebacterium glutamicum YPG-022 is a recombinant bacterium that overexpresses the BBD29_14900 gene.
  • the overexpression plasmid pXMJ19-BBD29_14900 G1114A was subjected to the above steps, and the obtained recombinant bacteria was Corynebacterium glutamicum YPG-023.
  • Corynebacterium glutamicum YPG-023 is a recombinant bacterium that overexpresses the BBD29_14900 G1114A gene.
  • Embodiment 5 the engineering strain that deletes BBD29_14900 gene on the construction genome
  • the primers are as follows (synthesized by Shanghai handsome company):
  • P20 (SEQ ID NO: 24): 5'-AGGCGTCGATAAGCAAATTTATCATTTAGCCTTGTTAATC-3'
  • P21 (SEQ ID NO: 25): 5'-GATTAACAAGGCTAAATGATAAATTTGCTTATCGACGCCT-3'
  • the primer pair composed of P19/P20 or the primer pair composed of P21/P22 was used for PCR amplification, respectively, to obtain the upstream homology arm fragment (about 804bp) and the downstream homology arm fragment ( about 791bp).
  • the upstream homology arm fragment and the downstream homology arm fragment were used as templates, and the primer pair composed of primers P19/P22 was used for PCR amplification to obtain the entire homology arm fragment (about 1555bp, sequenced as shown in SEQ ID NO:32) ).
  • the entire homology arm fragment was ligated with the shuttle plasmid pk18mobsacB plasmid recovered by digestion with Xba I through the NEBuider recombination system to obtain a knockout plasmid.
  • the kanamycin resistance marker is contained on the knockout plasmid.
  • the knockout plasmid was electrotransformed into Corynebacterium glutamicum CGMCC21220, and the single colonies produced by culture were identified by PCR with primer pairs composed of P23 and P24 (synthesized by Shanghai Yingjun Company).
  • the strains with 1481bp and 2660bp bands amplified by PCR are positive strains successfully transformed, and the strains with only 1481bp bands amplified are untransformed strains. Positive strains were screened on 15% sucrose medium and cultured on kanamycin-containing and kanamycin-free medium plates, respectively.
  • strains that do not grow on the culture medium of mycin were further identified by PCR using primer pairs composed of P23 and P24, and the strain with only one amplification product and 1481bp was a genetically engineered strain whose BBD29_14900 gene coding region was knocked out, and it was named It is Corynebacterium glutamicum YPG-024.
  • Embodiment 6 L-glutamic acid fermentation experiment
  • the recombinant bacteria constructed in the above examples and Corynebacterium glutamicum CGMCC21220 were fermented in a BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.).
  • the fermentation medium formula is shown in Table 3 (the remainder is water), and the meaning of dl is 0.1L.
  • control process is shown in Table 4. At the initial moment of completing the inoculation, the bacterial concentration in the system was 15 g/L.
  • 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
  • the invention disclosed in the present invention has the following effects: the present invention finds that the product encoded by the gene has an impact on the L-glutamic acid production capacity by weakening or knocking out the BBD29_14900 gene, by introducing point mutations in the coding sequence, or increasing the gene Compared with the unmodified strain, the obtained strain is favorable for producing high concentration of glutamate.

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Abstract

提供了一种改造基因BBD29_14900的重组菌株及其构建方法与应用，具体应用为产L-谷氨酸。还提供了一种对棒杆菌中BBD29_14900基因编码序列引入点突变或改进其表达的方法，该方法可以使带有突变的菌株提高谷氨酸的发酵产量。该点突变是使BBD29_14900基因序列第1114位碱基由鸟嘌呤(G)突变为腺嘌呤(A)，使编码的相应氨基酸序列第372位天冬氨酸被天冬酰胺取代。

Description

一种改造基因BBD29_14900的重组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，具体涉及一种改造基因BBD29_14900的重组菌株及其构建方法与应用，具体应用为产L-谷氨酸。
背景技术
众所周知，日常所用调味料味精就是L-谷氨酸单钠盐。自1909年日本发明并工业化生产味精以来，几经变迁，已发展成为以谷氨酸发酵为主题的世界性氨基酸发酵工业。生产味精谷氨酸之类氨基酸的发酵，区别于传统的酿酒和抗菌素发酵，是一种改变微生物代谢的代谢控制发酵。
目前，厂家用葡萄糖发酵所使用的菌种主要为一些突变株，所述生产菌种主要是通过诱变为主的微生物育种而获得。但如果在现有产酸能力下，继续采用经典微生物育种技术，例如诱变、细胞融合等手段，以期达到大幅度提高产酸率和糖酸转化率已相当困难。因此，采用现代基因工程手段改造菌种，从而提高产酸率和糖酸转化率，不失为目前最有效的方法。
如果可以利用基因工程手段改造生产菌种，谷氨酸的产酸率和糖酸转化率将会有较大的突破。
发明公开
本发明提供了一种改造基因BBD29_14900的重组菌株及其构建方法与应用。
本发明的目的是开发用于改善细菌的L-谷氨酸生产能力的新技术，从而提供一种有效生产L-谷氨酸的方法。
为了实现上述目的，本发明的发明人通过研究发现，基因BBD29_14900或其同源基因，可以通过修饰所述基因或改善其表达，能够得到具有增强的L-谷氨酸生产能力的细菌。基于这些发现，完成了本发明。
本发明提供了生成L-谷氨酸的细菌，其中编码SEQ ID NO:3的氨基酸序列或其同源序列的多核苷酸的表达改善。本发明还提供了通过使用所述微生物生产L-谷氨酸的方法。
本发明的第一个方面提供了生成L-谷氨酸的细菌，其具有编码SEQ ID NO:3的氨基酸序列或其同源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的氨基酸序列或其同源序列的多核苷酸具有点突变，或者编码SEQ ID NO:3的氨基酸序列或其同源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的氨基酸序列或其同源序列是基因BBD29_14900或其同源基因编码的蛋白。
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。
具有L-谷氨酸生产能力的细菌可以是能够在培养基中以优选0.5g/L以上，更优选1.0g/L以上的量积累目标L-谷氨酸的细菌。
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，突变前的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372位天冬氨酸(D)被不同的氨基酸所取代。
根据本发明，优选第372位天冬氨酸被天冬酰胺所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第372位天冬氨酸被天冬酰胺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1114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1114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_14900基因)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。
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。
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(Corynebacterium glutamicum)YPGLU001，该菌高产谷氨酸，保藏信息如下：菌种名称：谷氨酸棒杆菌；拉丁名：Corynebacterium glutamicum；菌株编号：YPGLU001；保藏机构：中国微生物菌种保藏管理委员会普通微生物中心；保藏机构简称：CGMCC；地址：北京市朝阳区北辰西路1号院3号；保藏日期：2020年11月23日；保藏中心登记入册编号：CGMCC No.21220。
根据本发明，所述细菌还可以具有与提高L-谷氨酸产量有关的其他改进，例如，谷氨酸脱氢酶、谷氨酰胺合成酶、谷氨酰胺-酮戊二酸氨基转移酶等酶的活性或基因的增强或降低的表达，或者可以使基因被外来基因取代。
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SEQ ID NO:3所示的氨基酸序列的多肽的多核苷酸，且所述序列的第372位天冬氨酸被不同的氨基酸所取代。
根据本发明，优选第372位天冬氨酸被天冬酰胺所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第372位天冬氨酸被天冬酰胺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1114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1114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，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_14900基因的多核苷酸序列，使其第1114位碱基发生突变，得到包含突变BBD29_14900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1114位碱基由鸟嘌呤(G)变为腺嘌呤(A)；具体地，所述包含突变BBD29_14900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_14900基因的核苷酸序列，使其第1114位碱基发生突变，得到突变的BBD29_14900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_14900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_14900基因构建：根据未修饰菌株的基因组序列，合成两对扩增BBD29_14900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_14900基因SEQ ID NO:1中引入点突变，得到点突变的BBD29_14900基因核苷酸序列SEQ ID NO:2，记为BBD29_14900 G1114A。
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(SEQ ID NO:5):
5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGCTGTGGTTATCCTCGCTG-3'
P2(SEQ ID NO:6):5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3'
P3(SEQ ID NO:7):5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3'
P4(SEQ ID NO:8):
5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGTGTCACTAGGCTAGTC-3'
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90s，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_14900 G1114A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，所述宿主菌株是谷氨酸棒杆菌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：
扩增BBD29_14900的上下游同源臂片段、BBD29_14900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_14900或BBD29_14900 G1114A基因，以实现所述菌株过表达BBD29_14900或BBD29_14900 G1114A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(SEQ ID NO:11):
5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG-3'
P8(SEQ ID NO:12):5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-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(SEQ ID NO:15):5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3'
P12(SEQ ID NO:16):
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(SEQ ID NO:13):5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3'
P10(SEQ ID NO:14):5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3'
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物为模板进行扩增，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(10pM)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120s(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_14900或BBD29_14900 G1114A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。
本发明的第五个方面，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_14900基因编码区及启动子区序列，或BBD29_14900 G1114A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_14900或BBD29_14900 G1114A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(SEQ ID NO:21):
5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCCCAAATTCAGCCCACT-3'
P18(SEQ ID NO:22):
5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAAGCGTTTTGCGCTTCGG-3'。
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(10pM)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90s(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_14900或BBD29_14900 G1114A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。
本发明提供了一种蛋白质(突变蛋白，命名为BBD29_14900 D372N蛋白)，是将BBD29_14900蛋白的第372位氨基酸残基由天冬氨酸突变为天冬酰胺得到的；
所述BBD29_14900蛋白为如下(a1)或(a2)或(a3)：
(a1)SEQ ID NO:3所示的蛋白质；
(a2)来源于细菌且与(a1)具有95％以上同一性且与细菌产谷氨酸相关的蛋白质；
(a3)将(a1)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且与细菌产谷氨酸相关的由(a1)衍生的蛋白质。
这里使用的术语“同一性”指与天然氨基酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述95％以上同一性具体可为96％以上同一性或97％以上同一性或98％以上同一性或99％以上同一性。
具体的，所述BBD29_14900 D372N蛋白如SEQ ID NO:4所示。
BBD29_14900基因为编码所述BBD29_14900蛋白的基因。
具体的，所述BBD29_14900基因为如下(b1)或(b2)或(b3)：
(b1)编码区如SEQ ID NO:1所示的DNA分子；
(b2)来源于细菌且与(b1)具有95％以上同一性且编码所述蛋白质的DNA分子；
(b3)在严格条件下与(b1)杂交且编码所述蛋白质的DNA分子。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述95％以上同一性具体可为96％以上同一性或97％以上同一性或98％以上同一性或99％以上同一性。
所述严格条件可为在0.1×SSPE(或0.1×SSC)，0.1％SDS的溶液中，在65℃条件下杂交并洗膜。
所述BBD29_14900 D372N蛋白的编码基因(命名为BBD29_14900 G1114A基因)也属于本发明的保护范围。
具体的，BBD29_14900 G1114A基因为如下(c1)或(c2)或(c3)：
(c1)编码区如SEQ ID NO:2所示的DNA分子；
(c2)来源于细菌且与(c1)具有95％以上同一性且编码所述蛋白质的DNA分子；
(c3)在严格条件下与(c1)杂交且编码所述蛋白质的DNA分子。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述95％以上同一性具体可为96％以上同一性或97％以上同一性或98％以上同一性或99％以上同一性。
所述严格条件可为在0.1×SSPE(或0.1×SSC)，0.1％SDS的溶液中，在65℃条件下杂交并洗膜。
具有BBD29_14900 G1114A基因的DNA分子、具有BBD29_14900 G1114A基因的表达盒或具有BBD29_14900 G1114A基因的重组载体或具有BBD29_14900 G1114A基因的重组菌均属于本发明的保护范围。
示例性的，具有BBD29_14900 G1114A基因的DNA分子可为SEQ ID NO:30所示的DNA分子或SEQ ID NO:31所示的DNA分子。
示例性的，具有BBD29_14900 G1114A基因的重组载体可为具有SEQ ID NO:30所示的DNA分子的质粒或具有SEQ ID NO:31所示的DNA分子的质粒。
示例性的，具有BBD29_14900 G1114A基因的重组载体可为实施例中的整合质粒PK18mobsacB-BBD29_14900 G1114A或过表达质粒pXMJ19-BBD29_14900 G1114A。
所述重组菌具体可为重组细菌。
示例性的，具有BBD29_14900 G1114A基因的重组菌可为具有SEQ ID NO:30所示的DNA分子的重组菌或具有SEQ ID NO:31所示的DNA分子的重组菌或具有SEQ ID NO:2所示的DNA分子的重组菌。
具有BBD29_14900 G1114A基因的重组菌具体可采用如下方式制备：将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因。
示例性的，将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因的实现方式如下：将SEQ ID NO:29所示的DNA分子导入细菌。
示例性的，将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因的实现方式如下：将实施例中的载体pK18-BBD29_14900 G1114A导入细菌。
所述重组菌中，BBD29_14900 G1114A基因可以整合至基因组DNA中进行表达也可以在质粒中进行表达。
示例性的，具有BBD29_14900 G1114A基因的重组菌具体可采用如下方式制备：将SEQ ID NO:30所示的DNA分子或SEQ ID NO:31所示的DNA分子导入细菌。
示例性的，具有BBD29_14900 G1114A基因的重组菌具体可采用如下方式制备：将具有SEQ ID NO:30所示的DNA分子的质粒或具有SEQ ID NO:31所示的DNA分子的质粒导入细菌。
示例性的，具有BBD29_14900 G1114A基因的重组菌具体可采用如下方式制备：将实施例中的整合质粒PK18mobsacB-BBD29_14900 G1114A或过表达质粒pXMJ19-BBD29_14900 G1114A导入细菌。
本发明还保护BBD29_14900 D372N蛋白、BBD29_14900 G1114A基因、具有BBD29_14900 G1114A基因的表达盒或具有BBD29_14900 G1114A基因的重组载体或具有BBD29_14900 G1114A基因的重组菌的应用；
所述应用为如下(Ⅰ)或(Ⅱ)：
(Ⅰ)在提高细菌谷氨酸产量中的应用；
(Ⅱ)在生产谷氨酸中的应用。
本发明还保护特定物质的应用；
所述应用为如下(Ⅰ)或(Ⅱ)：
(Ⅰ)在提高细菌谷氨酸产量中的应用；
(Ⅱ)在生产谷氨酸中的应用。
所述特定物质为如下(d1)、(d2)、(d3)、(cd4)、(d5)或(d6)：
(d1)用于提高BBD29_14900 G1114A基因表达的物质；
(d2)用于提高BBD29_14900 D372N蛋白丰度的物质；
(d3)用于提高BBD29_14900 D372N蛋白活性的物质；
(d4)用于提高BBD29_14900基因表达的物质；
(d5)用于提高BBD29_14900蛋白丰度的物质；
(d6)用于提高BBD29_14900蛋白活性的物质。
示例性的，所述用于提高BBD29_14900 G1114A基因表达的物质具体可为BBD29_14900 G1114A基因或具有BBD29_14900 G1114A基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900 G1114A或过表达质粒pXMJ19-BBD29_14900 G1114A。
示例性的，所述用于提高BBD29_14900基因表达的物质具体可为BBD29_14900基因或具有BBD29_14900基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900或过表达质粒pXMJ19-BBD29_14900。
本发明还提供了一种重组菌，是在细菌中过表达BBD29_14900 G1114A基因或BBD29_14900基因得到的。
示例性的，过表达BBD29_14900 G1114A基因的实现方式如下：在细菌中导入BBD29_14900 G1114A基因或具有BBD29_14900 G1114A基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900 G1114A或过表达质粒pXMJ19-BBD29_14900 G1114A。
示例性的，过表达BBD29_14900基因的实现方式如下：在细菌中导入BBD29_14900基因或具有BBD29_14900基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900或过表达质粒pXMJ19-BBD29_14900。
本发明还保护所述重组菌在制备谷氨酸中的应用。
本发明还保护一种提高细菌的谷氨酸产量的方法，包括如下步骤：将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因。
示例性的，将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因的实现方式如下：将实施例中的载体pK18-BBD29_14900 G1114A导入细菌。
本发明还提供了一种提高细菌的谷氨酸产量的方法，包括如下步骤：在细菌中过表达BBD29_14900 G1114A基因或在细菌中过表达BBD29_14900基因或提高细菌中 BBD29_14900 D372N蛋白的丰度或提高细菌中BBD29_14900蛋白的丰度或提高细菌中BBD29_14900 D372N蛋白的活性或提高细菌中BBD29_14900蛋白的活性。
示例性的，过表达BBD29_14900 G1114A基因的实现方式如下：在细菌中导入BBD29_14900 G1114A基因或具有BBD29_14900 G1114A基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900 G1114A或过表达质粒pXMJ19-BBD29_14900 G1114A。
示例性的，过表达BBD29_14900基因的实现方式如下：在细菌中导入BBD29_14900基因或具有BBD29_14900基因的重组质粒。示例性的，所述重组质粒可为实施例中的整合质粒PK18mobsacB-BBD29_14900或过表达质粒pXMJ19-BBD29_14900。
本发明还保护BBD29_14900 D372N蛋白或BBD29_14900蛋白在调控细菌的谷氨酸产量中的应用。
所述调控为正调控，即BBD29_14900 D372N蛋白含量增高，谷氨酸产量增高。
所述调控为正调控，即BBD29_14900 D372N蛋白含量降低，谷氨酸产量降低。
所述调控为正调控，即BBD29_14900蛋白含量增高，谷氨酸产量增高。
所述调控为正调控，即BBD29_14900蛋白含量降低，谷氨酸产量降低。
应用所述重组菌制备谷氨酸时，具体方法包括如下步骤：发酵所述重组菌。
本领域技术人员可以采用现有技术中的发酵方法进行发酵。也可通过常规试验进行发酵方法的优化和改进。可以在本领域中已知的发酵条件下在合适的培养基中进行细菌的发酵。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20-45℃。
所述方法还可包括如下步骤：从培养物中获得谷氨酸。从培养物中获得谷氨酸可通过各种方式实现，包括但不限于：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。
所述发酵中，示例性的发酵培养基的配方见表3，余量为水。
所述发酵中，示例性的发酵控制工艺见表4。
示例性的，所述发酵中，体系中的菌浓度为15g/L。
示例性的，所述发酵的发酵过程中：通过补加葡萄糖水溶液控制体系含糖量(残糖)。
以上任一所述细菌包括但不限于如下：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)。
以上任一所述细菌为具有生产谷氨酸的能力的细菌。
“具有生产谷氨酸的能力的细菌”是指细菌具有以下能力：在培养基和/或细菌的细胞中产生并累积谷氨酸的能力。从而，当细菌在培养基中培养时可以收集谷氨酸。
所述细菌可为自然采集的野生型细菌也可为修饰后的细菌。
“修饰后的细菌”指的是将自然采集的野生型细菌进行人工突变和/或诱变得到的改造后的细菌。
具体的，所述谷氨酸棒杆菌可为谷氨酸棒杆菌CGMCC21260。
谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC21220，菌株名称为YPGLU001，简称为谷氨酸棒杆菌CGMCC21220，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 21220。
以上任一所述谷氨酸的含义为广义的谷氨酸，包括游离形式谷氨酸、谷氨酸的盐或两者的混合物。
具体的，所述谷氨酸为L-谷氨酸。
以上任何方法或应用还可用于谷氨酸的下游产品的制备。
谷氨酸棒杆菌中的BBD29_14900蛋白如SEQ ID NO:3所示，其编码基因如SEQ ID NO:1所示。本发明中通过引入点突变，得到了SEQ ID NO:4所示的BBD29_14900 D372N蛋白，BBD29_14900 D372N蛋白的编码基因如SEQ ID NO:2所示。与BBD29_14900基因相比，BBD29_14900 G1114A基因的差异在于第1114位由鸟嘌呤脱氧核糖核苷酸(G)变为腺嘌呤脱氧核糖核苷酸(A)。与BBD29_14900蛋白相比，BBD29_14900 D372N蛋白的差异在于第372位氨基酸残基由天冬氨酸(D)变为天冬酰胺(N)。
本发明发现BBD29_14900蛋白对细菌的谷氨酸产量存在正调控，即BBD29_14900蛋白含量增高、谷氨酸产量增高，BBD29_14900蛋白含量降低、谷氨酸产量降低。抑制BBD29_14900基因表达可以降低谷氨酸产量，过表达BBD29_14900基因提高谷氨酸产量。进一步的，本发明通过点突变，得到了BBD29_14900 D372N蛋白，其功能优于BBD29_14900蛋白。本发明对于谷氨酸工业化生产，具有重大的应用价值。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
以下实施例中培养菌株使用的培养基均是在基础培养基的基础上添加其他成分得到的。其他成分为蔗糖、卡那霉素或氯霉素等。固体培养基中含有琼脂糖。基础培养基组成见表1。如无特殊说明，实施例中的菌株培养温度均为32℃。
表1
成分 配方
蔗糖 10g/L
多聚蛋白胨 10g/L
牛肉膏 10g/L
酵母粉 5g/L
尿素 2g/L
氯化钠 2.5g/L
琼脂粉 20g/L
pH 7.0
sscp电泳中的PAGE的配方见表2。电泳条件：将电泳槽置入冰中，使用1×TBE缓冲液，电压120V，电泳时间10h。
表2
成分 用量(配置丙烯酰胺终浓度为8％)
40％丙烯酰胺 8mL
ddH 2O 26mL
甘油 4mL
10×TBE 2mL
TEMED 40μL
10％APS 600μL
谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC21220，菌株名称为YPGLU001，简称为谷氨酸棒杆菌CGMCC21220，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 21220。谷氨酸棒杆菌ATCC 13869，即谷氨酸棒杆菌CICC 20216；CICC全称为中国工业微生物菌种保藏管理中心。
实施例1、构建包含点突变的BBD29_14900基因编码区的转化载体pK18-BBD29_14900 G1114A
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组序列，设计并合成两对扩增BBD29_14900基因编码区序列的引物，以等位基因置换的方式在谷氨酸棒杆菌CGMCC21220中(经测序确认谷氨酸棒杆菌CGMCC21220染色体上的BBD29_14900基因编码区与谷氨酸棒杆菌ATCC13869染色体上的BBD29_14900基因编码区是一致的)引入点突变。突变前对应编码蛋白的氨基酸序列为SEQ ID NO:3，突变前BBD29_14900基因的核苷酸序列为SEQ ID NO:1。基因中引入了单点突变，即编码区第1114位由鸟嘌呤脱氧核糖核苷酸(G)变为腺嘌呤脱氧核糖核苷酸(A)。相应的，引起蛋白质的单点突变，即第372位氨基酸残基由天冬氨酸(D)变为天冬酰胺(N)。突变后基因称为BBD29_14900 G1114A基因，如SEQ ID NO:2所示。突变后蛋白称为BBD29_14900 D372N蛋白， 如SEQ ID NO:4所示。
引物如下(上海invitrogen公司合成)：
P1(SEQ ID NO:5):
5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGCTGTGGTTATCCTCGCTG-3'
P2(SEQ ID NO:6):5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3'
P3(SEQ ID NO:7):5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3'
P4(SEQ ID NO:8):
5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGTGTCACTAGGCTAGTC-3'
1、以谷氨酸棒杆菌ATCC 13869为模板，分别采用P1和P2组成的引物对或者采用P3和P4组成的引物对，进行PCR扩增。
PCR体系(50μL)：模板、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，余量为水。
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。
2、分别回收步骤1的两个PCR体系的的扩增产物，同时作为模板，采用P1和P4组成的引物对进行PCR扩增。
PCR体系(50μL)：模板、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，余量为水。
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90s，30个循环，72℃过度延伸10min。
3、回收步骤2的PCR扩增得到的约1253bp的扩增产物，该回收的DNA片段即为BBD29_14900 G1114A片段(经测序，该DNA片段如SEQ ID NO:29所示)。
BBD29_14900 G1114A片段可以通过同源重组整合至谷氨酸棒杆菌CGMCC21220中，从而导致BBD29_14900基因编码区的第1114位核苷酸由鸟嘌呤脱氧核糖核苷酸(G)突变为腺嘌呤脱氧核糖核苷酸(A)，最终导致编码的蛋白质的第372位氨基酸残基由天冬氨酸(D)突变为天冬酰胺(N)。
4、采用限制性内切酶Xba I酶切pK18mobsacB质粒(Addgene公司产品)，回收线性化质粒。
5、将步骤3获得的BBD29_14900 G1114A片段和步骤4获得的线性化质粒通过NEBuider重组系统组装，获得载体pK18-BBD29_14900 G1114A。载体pK18-BBD29_14900 G1114A已进行测序验证，其中具有SEQ ID NO:29所示的BBD29_14900 G1114A片段。载体pK18-BBD29_14900 G1114A中具有卡那霉素抗性标记。
实施例2、构建包含点突变的BBD29_14900 G1114A的工程菌株
将实施例1构建的载体pK18-BBD29_14900 G1114A通过电击转化导入谷氨酸棒杆菌CGMCC21220中，培养产生的单菌落分别通过引物P1和通用引物M13R(M13R:5’-CAGGAAACAGCTATGACC-3’)组成的引动对进行PCR鉴定，能扩增出约 1260bp条带的菌株为阳性菌株。阳性菌株在含15％蔗糖的培养基平板上培养，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平板上培养，在不含卡那霉素的培养基上生长且而在含卡那霉素的培养基上不生长的菌株进一步采用P5和P6组成的引物对(上海invitrogen公司合成)进行PCR鉴定。回收PCR鉴定中的PCR扩增产物，通过高温变性、冰浴后进行sscp电泳(以载体pK18-BBD29_14900 G1114A的扩增片段为阳性对照，谷氨酸棒杆菌ATCC13869的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。将筛选的等位替换成功的菌株，采用引物P5和P6组成的引物对进行PCR扩增，回收扩增产物并连接到PMD19-T载体上进行测序，通过序列比验证等位替换成功的菌株命名为谷氨酸棒杆菌YPG-019。
P5(SEQ ID NO:9):5'-GCAGCCAAGGCCAAGAAGAT-3'；
P6(SEQ ID NO:10):5'-TCCCTGTTTAAGACTGCATT-3'。
与谷氨酸棒杆菌CGMCC21220相比，谷氨酸棒杆菌YPG-019的差异仅在于：将谷氨酸棒杆菌CGMCC21220基因组中的序列表的序列1所示的BBD29_14900基因取代为了序列表的序列2所示的BBD29_14900 G1114A基因。序列1和序列2仅存在一个核苷酸差异，位于第1114位。
实施例3、构建基因组上过表达BBD29_14900或BBD29_14900 G1114A基因的工程菌株
一、构建整合质粒
依据NCBI公布的谷氨酸棒杆菌ATCC 13869基因组序列，设计并合成三对引物，分别扩增上游同源臂片段、BBD29_14900基因编码区(或BBD29_14900 G1114A基因编码区)、下游同源臂片段，以同源重组的方式在谷氨酸棒杆菌CGMCC21220基因组DNA中整合入BBD29_14900基因或BBD29_14900 G1114A基因。
引物如下(上海invitrogen公司合成)：
P7(SEQ ID NO:11):
5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG-3'
P8(SEQ ID NO:12):5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-3'
P9(SEQ ID NO:13):5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3'
P10(SEQ ID NO:14):5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3'
P11(SEQ ID NO:15):5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3'
P12(SEQ ID NO:16):
5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCATAAGAAACAACCACTTCC-3'
以谷氨酸棒杆菌YPG-019为模板，分别以P7/P8组成的引物对、P9/P10组成的引物对、P11/P12组成的引物对，进行PCR扩增，获得上游同源臂片段约806bp、BBD29_14900 G1114A基因片段约1494bp、下游同源臂片段约788bp，再采用P7/P12组成的引物对，以以上扩增的三个片段混合为模板进行扩增，获得约3008bp的整合同源 臂片段(经测序，如SEQ ID NO:30所示)。采用NEBuider重组系统将整合同源臂片段与经Xba I酶切回收的穿梭质粒PK18mobsacB相连接，获得整合质粒PK18mobsacB-BBD29_14900 G1114A。整合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。PCR体系(50μL)：模板、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，余量为水。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120s，30个循环，72℃过度延伸10min。
用谷氨酸棒杆菌ATCC13869代替谷氨酸棒杆菌YPG-019进行上述步骤，得到整合质粒PK18mobsacB-BBD29_14900。
二、制备重组菌
将整合质粒电转化导入谷氨酸棒杆菌CGMCC21220中，培养产生的单菌落通过P13/P14组成的引物对进行PCR鉴定，PCR扩增出含有大小约1827bp的片段的为转化成功的阳性菌株，扩增不到片段的为未转化成功的菌株。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平板上培养，在不含卡那霉素的培养基上生长且在含卡那霉素的培养基上不生长的菌株进一步采用P15/P16组成的引物对进行PCR鉴定，扩增出大小约1517bp的菌为目的基因整合到谷氨酸棒杆菌CGMCC21220基因组上的菌株。
P13(SEQ ID NO:17):5'-GTCCAAGGTGACGGCCGCAC-3'
P14(SEQ ID NO:18):5'-GCAGCCTTAACTGGGGAAAG-3'
P15(SEQ ID NO:19):5'-GGAGCGCCGCCTCATCGAGC-3'
P16(SEQ ID NO:20):5'-ATATTCGGCCCAGCAGCAGC-3'
整合质粒PK18mobsacB-BBD29_14900进行上述步骤，得到的重组菌为谷氨酸棒杆菌YPG-020。谷氨酸棒杆菌YPG-020为基因组DNA中过表达BBD29_14900基因的重组菌。
整合质粒PK18mobsacB-BBD29_14900 G1114A进行上述步骤，得到的重组菌为谷氨酸棒杆菌YPG-021。谷氨酸棒杆菌YPG-021为基因组DNA中过表达BBD29_14900 G1114A基因的重组菌。
实施例4、构建质粒上过表达BBD29_14900或BBD29_14900 G1114A基因的工程菌株
一、构建重组质粒
依据NCBI公布的野生型谷氨酸棒杆菌ATCC 13869基因组序列，设计并合成一对扩增BBD29_14900基因编码区及启动子区序列的引物，P17和P18(上海invitrogen公司合成)。
P17(SEQ ID NO:21):
5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCCCAAATTCAGCCCACT-3'
P18(SEQ ID NO:22):
5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAAGCGTTTTGCGCTTCGG-3'
以谷氨酸棒杆菌YPG-019为模板，以引物P17/P18组成的引物对进行PCR扩增，获得1524bp的BBD29_14900 G1114A基因片段(经测序，BBD29_14900 G1114A基因片段如SEQ ID NO:31所示)。采用NEBuider重组系统将BBD29_14900 G1114A基因片段与经EcoR I酶切回收的穿梭质粒pXMJ19相连接，获得过表达质粒pXMJ19-BBD29_14900 G1114A。过表达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(50μL)：模板、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，余量为水。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90s，30个循环，72℃过度延伸10min。
用谷氨酸棒杆菌YPG-020代替谷氨酸棒杆菌YPG-019进行上述步骤，得到过表达质粒pXMJ19-BBD29_14900。
二、制备重组菌
将过表达质粒电转化导入谷氨酸棒杆菌CGMCC21220中，对培养产生的单菌落采用M13R(-48)和P18组成的引物对进行PCR鉴定，PCR扩增出约1563bp的片段的为重组菌。M13R(-48):5'-AGCGGATAACAATTTCACACAGGA-3'。
过表达质粒pXMJ19-BBD29_14900进行上述步骤，得到的重组菌为谷氨酸棒杆菌YPG-022。谷氨酸棒杆菌YPG-022为质粒过表达BBD29_14900基因的重组菌。
过表达质粒pXMJ19-BBD29_14900 G1114A进行上述步骤，得到的重组菌为谷氨酸棒杆菌YPG-023。谷氨酸棒杆菌YPG-023为质粒过表达BBD29_14900 G1114A基因的重组菌。
实施例5、构建基因组上缺失BBD29_14900基因的工程菌株
一、构建敲除质粒
根据NCBI公布的谷氨酸棒杆菌ATCC 13869的基因组序列，合成两对扩增BBD29_14900基因编码区两端片段的引物，用于扩增上、下游同源臂片段。
引物如下(上海英俊公司合成)：
P19(SEQ ID NO:23):
5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCGCTGAAACAGCAGGGGAC-3'
P20(SEQ ID NO:24):5'-AGGCGTCGATAAGCAAATTTATCATTTAGCCTTGTTAATC-3'
P21(SEQ ID NO:25):5'-GATTAACAAGGCTAAATGATAAATTTGCTTATCGACGCCT-3'
P22(SEQ ID NO:26):
5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAACTTGCCATGAGTCGTCTT-3'
以谷氨酸棒杆菌ATCC13869为模板，分别采用P19/P20组成的引物对或P21/P22组成的引物对进行PCR扩增，分别获得上游同源臂片段(约804bp)和下游同源臂片段(约791bp)。同时将上游同源臂片段和下游同源臂片段作为模板，采用引物P19/P22组成的引物对进行PCR扩增，获得整个同源臂片段(约1555bp，经测序如SEQ ID NO:32所示)。通过NEBuider重组系统将整个同源臂片段与经Xba I酶切回收的穿梭质粒pk18mobsacB质粒相连接，获得敲除质粒。敲除质粒上含有卡那霉素抗性 标记。
二、制备重组菌
将敲除质粒电转化导入谷氨酸棒杆菌CGMCC21220中，培养产生的单菌落分别通过P23和P24组成的引物对(上海英俊公司合成)进行PCR鉴定。PCR扩增出大小1481bp及2660bp的条带的菌株为转化成功的阳性菌株，只扩增出1481bp条带的菌株为未转化成功的菌株。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平板上培养，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和P24组成的引物对进行PCR鉴定，扩增产物只有一种且为1481bp的菌株为BBD29_14900基因编码区被敲除的基因工程菌株，其被命名为谷氨酸棒杆菌YPG-024。
P23(SEQ ID NO:27):5'-TCGCTGAAACAGCAGGGGAC-3'
P24(SEQ ID NO:28):5'-AACTTGCCATGAGTCGTCTT-3'
实施例6、L-谷氨酸发酵实验
将以上实施例构建的各个重组菌以及谷氨酸棒杆菌CGMCC21220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)中进行发酵。
发酵培养基配方见表3(余量为水)，dl的含义为0.1L。
控制工艺见表4。完成接种的初始时刻，体系中的菌浓度为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)。
均设置三个重复处理，结果见表5。
表3发酵培养基配方
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
表4发酵控制工艺
Figure PCTCN2021142439-appb-000001
表5 L-谷氨酸发酵实验结果
菌株 L-谷氨酸氨酸产量(g/L) OD(562nm)
谷氨酸棒杆菌CGMCC21220 102.1 46.1
YPG-019 102.3 45.6
YPG-020 105.1 46.6
YPG-021 106.8 45.4
YPG-022 107.5 45.1
YPG-023 108.8 45.5
YPG-024 96.5 46.3
结果如表5所示，在谷氨酸棒杆菌中对BBD29_14900基因编码区进行点突变BBD29_14900 G1114A及过表达，有助于L-谷氨酸产量的提高，而对基因进行弱化或敲除，不利于L-谷氨酸的积累。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明公开了的具有如下作用：本发明通过对BBD29_14900基因的弱化或敲除，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谷氨酸。

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的氨基酸序列或其同源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的氨基酸序列或其同源序列的多核苷酸的表达增强，或者编码SEQ ID NO:3的氨基酸序列或其同源序列的多核苷酸具有点突变，或者编码SEQ ID NO:3的氨基酸序列或其同源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372位天冬氨酸被不同的氨基酸所取代；优选第372位天冬氨酸被天冬酰胺所取代。
  3. 如权利要求1-2任一项所述的细菌，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1114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1114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)；更优选为谷氨酸棒杆菌CGMCC No.21220或ATCC 13869。
  6. 一种多核苷酸序列，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372位天冬氨酸被不同的氨基酸所取代；优选第372位天冬氨酸被天冬酰胺所取代；
    优选所述多核苷酸序列包括编码含有SEQ ID NO:4所示的氨基酸序列的多核苷酸；
    优选所述多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第1114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1114位碱基由鸟嘌呤(G)突变为腺嘌呤(A)；
    优选所述多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
  7. 一种蛋白质，其特征在于，所述序列如SEQ ID NO:4所示。
  8. 一种重组载体，其特征在于，包含权利要求6所述的多核苷酸序列。
  9. 一种重组菌株，其特征在于，包含权利要求6所述的多核苷酸序列。
  10. 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。
  11. 一种蛋白质，命名为BBD29_14900 D372N蛋白，是将BBD29_14900蛋白的第372位氨基酸残基由天冬氨酸突变为天冬酰胺得到的；
    所述BBD29_14900蛋白蛋白为如下(a1)或(a2)或(a3)：
    (a1)序列表的序列3所示的蛋白质；
    (a2)来源于细菌且与(a1)具有95％以上同一性且与细菌产谷氨酸相关的蛋白质；
    (a3)将(a1)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且与细菌产谷氨酸相关的由(a1)衍生的蛋白质。
  12. 权利要求11所述BBD29_14900 D372N蛋白的编码基因。
  13. 具有权利要求12所述编码基因的表达盒或重组载体或重组菌。
  14. 权利要求11所述蛋白质、权利要求12所述编码基因、权利要求13所述表达盒、权利要求13所述重组载体或权利要求13所述重组菌的应用；
    所述应用为如下(Ⅰ)或(Ⅱ)：
    (Ⅰ)在提高细菌谷氨酸产量中的应用；
    (Ⅱ)在生产谷氨酸中的应用。
  15. 特定物质的应用；
    所述应用为如下(Ⅰ)或(Ⅱ)：
    (Ⅰ)在提高细菌谷氨酸产量中的应用；
    (Ⅱ)在生产谷氨酸中的应用；
    所述特定物质为如下(d1)、(d2)、(d3)、(d4)、(d5)或(d6)：
    (d1)用于提高BBD29_14900 G1114A基因表达的物质；
    (d2)用于提高BBD29_14900 D372N蛋白丰度的物质；
    (d3)用于提高BBD29_14900 D372N蛋白活性的物质；
    (d4)用于提高BBD29_14900基因表达的物质；
    (d5)用于提高BBD29_14900蛋白丰度的物质；
    (d6)用于提高BBD29_14900蛋白活性的物质；
    所述BBD29_14900 D372N蛋白为权利要求11中所述的BBD29_14900 D372N蛋白；
    所述BBD29_14900 G1114A基因为编码所述BBD29_14900 D372N蛋白的基因；
    所述BBD29_14900蛋白为权利要求11中所述的BBD29_14900蛋白；
    所述BBD29_14900基因为编码所述BBD29_14900蛋白的基因。
  16. 一种重组菌，是在细菌中过表达BBD29_14900 G1114A基因或BBD29_14900基因得到的；所述BBD29_14900 G1114A基因为权利要求15中所述的BBD29_14900 G1114A基因；所述BBD29_14900基因为权利要求15中所述的BBD29_14900基因。
  17. 权利要求16所述重组菌在制备谷氨酸中的应用。
  18. 一种提高细菌的谷氨酸产量的方法，包括如下步骤：将细菌基因组中的BBD29_14900基因替换为BBD29_14900 G1114A基因；
    所述BBD29_14900 G1114A基因为权利要求15中所述的BBD29_14900 G1114A基因；所述BBD29_14900基因为权利要求15中所述的BBD29_14900基因。
  19. 一种提高细菌的谷氨酸产量的方法，包括如下步骤：在细菌中过表达BBD29_14900 G1114A基因或在细菌中过表达BBD29_14900基因或提高细菌中BBD29_14900 D372N蛋白的丰度或提高细菌中BBD29_14900蛋白的丰度或提高细菌中BBD29_14900 D372N蛋白的活性或提高细菌中BBD29_14900蛋白的活性；
    所述BBD29_14900 D372N蛋白为权利要求11中所述的BBD29_14900 D372N蛋白；
    所述BBD29_14900 G1114A基因为编码所述BBD29_14900 D372N蛋白的基因；
    所述BBD29_14900蛋白为权利要求11中所述的BBD29_14900蛋白；
    所述BBD29_14900基因为编码所述BBD29_14900蛋白的基因。
  20. BBD29_14900 D372N蛋白或BBD29_14900蛋白在调控细菌的谷氨酸产量中的应用；所述BBD29_14900 D372N蛋白为权利要求11中所述的BBD29_14900 D372N蛋白；所述BBD29_14900蛋白为权利要求11中所述的BBD29_14900蛋白。
PCT/CN2021/142439 2020-12-30 2021-12-29 一种改造基因bbd29_14900的重组菌株及其构建方法与应用 WO2022143762A1 (zh)

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