WO2022143761A1 - 一种改造基因bbd29_04920的产l-谷氨酸的重组菌株及其构建方法与应用 - Google Patents

一种改造基因bbd29_04920的产l-谷氨酸的重组菌株及其构建方法与应用 Download PDF

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WO2022143761A1
WO2022143761A1 PCT/CN2021/142438 CN2021142438W WO2022143761A1 WO 2022143761 A1 WO2022143761 A1 WO 2022143761A1 CN 2021142438 W CN2021142438 W CN 2021142438W WO 2022143761 A1 WO2022143761 A1 WO 2022143761A1
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seq
sequence
bbd29
amino acid
glutamic acid
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French (fr)
Inventor
孟刚
贾慧萍
魏爱英
赵春光
苏厚波
杨立鹏
马风勇
周晓群
郭小炜
田斌
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Inner Mongolia Eppen Biotech Co Ltd
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Inner Mongolia Eppen Biotech Co Ltd
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Priority to JP2023540779A priority Critical patent/JP7845754B2/ja
Priority to KR1020237024680A priority patent/KR20230122116A/ko
Priority to EP21914493.8A priority patent/EP4273227A4/en
Priority to US18/270,490 priority patent/US20240294866A1/en
Publication of WO2022143761A1 publication Critical patent/WO2022143761A1/zh
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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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • 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 particularly relates to a recombinant strain producing L-glutamic acid and a construction method and application thereof.
  • L-glutamic acid is an important amino acid and is used in food, clinical medicine and other aspects.
  • L-glutamic acid is mainly produced by fermentation with L-glutamic acid-producing bacteria or mutants thereof belonging to genera such as Brevibacterium, Corynebacterium, or Microbacterium.
  • L-glutamate is biosynthesized from ⁇ -ketoglutarate, an intermediate product of the citric acid cycle in microbial cells.
  • One of the pathways is the synthesis of L-glutamate through the catalysis of glutamate dehydrogenase (GDH) in the presence of high concentrations of ammonium ions.
  • GDH glutamate dehydrogenase
  • Another pathway (GS/GOGAT pathway) is the synthesis of L-glutamic acid by glutamine synthase and glutamine-ketoglutarate aminotransferase.
  • Glutamine synthetase catalyzes the conversion of L-glutamic acid and ammonium ions into glutamine; glutamine-oxoglutaric acid amino transferase (also known as “glutamine-oxoglutaric acid amino transferase”) Glutamate synthase (GOGAT) catalyzes the L-glutamate synthesis reaction in which one molecule of glutamine and one molecule of ⁇ -ketoglutarate have been synthesized from GS Two molecules of L-glutamic acid.
  • Improvements to the production of L-amino acids by fermentation may involve fermentation techniques such as stirring and supplying oxygen; or the composition of the nutrient medium, such as the sugar concentration during fermentation; or the processing of the fermentation broth into a suitable product form, such as by drying and pelleted fermentation broth or ion exchange chromatography; or may involve inherent performance properties of the relevant microorganism itself.
  • Methods for improving the performance properties of these microorganisms include mutagenesis, selection and screening of mutants. Strains obtained in this way are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and produce L-amino acids and the like.
  • 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 L-glutamic acid production capacity of bacteria can be improved by modifying the gene BBD29_04920 or its homologous gene in bacteria 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_04920 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 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 asparagine at position 520 of the amino acid sequence of SEQ ID NO:3 is replaced by a different amino acid .
  • the asparagine at position 520 is replaced by lysine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 520th asparagine is replaced by lysine is shown in SEQ ID NO:4.
  • the polynucleotide sequence with point mutation is formed by mutating the 1560th base of the polynucleotide sequence shown in SEQ ID NO: 1.
  • the mutation includes the mutation of the 1560th base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) 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_04920 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. 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, Corynebacteriumammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, Brevibacterium rose roseum), Brevibacterium thiogenitalis, etc.
  • Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum
  • Corynebacterium callunae Corynebacterium glutamicum glutamicum
  • Brevibacterium flavum Brevibacterium lactofermentum
  • Corynebacteriumammoniagenes Corynebacterium pekinense
  • Brevibacterium saccharolyticum Brevibacterium rose roseum
  • the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC 13869.
  • 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 asparagine at position 520 of the sequence is substituted with a different amino acid.
  • the asparagine at position 520 is replaced by lysine.
  • amino acid sequence shown in SEQ ID NO:3 wherein the amino acid sequence after the 520th asparagine is replaced by lysine 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 1560th 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 1560th base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) 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 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_04920 gene as shown in SEQ ID NO: 1 in the modified host strain was mutated to obtain a recombinant strain comprising the mutated BBD29_04920 encoding gene.
  • the transformation includes at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to that the 1560th base in SEQ ID NO: 1 is changed from cytosine (C) to adenine (A); specifically, the polynucleotide comprising the gene encoding the mutation BBD29_04920 The acid sequence is shown in SEQ ID NO:2.
  • construction method comprises the steps:
  • the step (1) comprises: constructing the BBD29_04920 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_04920 gene fragment, and using PCR A point mutation was introduced into the wild-type BBD29_04920 gene SEQ ID NO: 1 by site-directed mutagenesis, and the nucleotide sequence of the point-mutated BBD29_04920 gene SEQ ID NO: 2 was obtained, which was denoted as BBD29_04920 C1560A .
  • 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:
  • P3 5' AACAACTTGC AAAAAGTCAC CATCGTGGCT 3' (SEQ ID NO: 7)
  • 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 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), overrun at 72°C Extend for 10min.
  • the step (2) includes the construction of recombinant plasmids, including: assembling the isolated and purified BBD29_04920 C1560A 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_04920 Amplify the upstream and downstream homology arm fragments of BBD29_04920, the coding region of BBD29_04920 gene and its promoter region sequence, and introduce BBD29_04920 or BBD29_04920 C1560A gene into the genome of the host strain by homologous recombination, so as to realize that the strain overexpresses BBD29_04920 or BBD29_04920 C1560A 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 the three fragment mixtures of the sequence fragments of the promoter region are used as the primers.
  • 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_04920 or BBD29_04920 C1560A gene is introduced into the genome of the host strain by 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:
  • P18 5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTATCGACGCTTCCCCGCGC3' (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 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_04920 or BBD29_04920 C1560A 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 present invention also provides a protein named as protein BBD29_04920 N520K , 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_04920 C1560A , and the nucleic acid molecule BBD29_04920 C1560A can be any of the following:
  • B1 a nucleic acid molecule encoding the protein BBD29_04920 N520K ;
  • 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_04920 C1560A gene described in the present invention.
  • the DNA molecule shown in SEQ ID No. 2 encodes the protein BBD29_04920 N520K shown in SEQ ID No. 4.
  • the protein BBD29_04920 N520K amino acid sequence (SEQ ID No. 4) is obtained by changing the asparagine (N) at position 520 in SEQ ID No. 3 to lysine (K).
  • 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_04920 C1560A or a recombinant vector containing the expression cassette described in C1);
  • C3 a recombinant microorganism containing the nucleic acid molecule BBD29_04920 C1560A , 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_04920 gene of the present invention.
  • the DNA molecule shown in SEQ ID No. 1 (BBD29_04920 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 or nucleotide 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 yield 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_04920 C1560A 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 asparagine residue at position 520 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 asparagine at position 520 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 to lysine to obtain a mutein with the amino acid sequence of SEQ ID No.4.
  • BBD29_04920 N520K the point mutation can be the mutation of asparagine at position 520 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 to lysine to obtain a mutein with the amino acid sequence of SEQ ID No.4.
  • 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 cytosine (C) at position 1560 of the BBD29_04920 gene (SEQ ID No. 1).
  • the point mutation can be to change the cytosine (G) at position 1560 of the BBD29_04920 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_04920C1560A , PK18mobsacB-BBD29_04920, PK18mobsacB- BBD29_04920C1560A , pXMJ19-BBD29_04920 or pXMJ19- BBD29_04920C1560A .
  • the recombinant vector pK18-BBD29_04920 C1560A contains the DNA molecule shown in positions 846-1788 of the mutated gene BBD29_04920 C1560A shown in SEQ ID No. 2.
  • the recombinant vector PK18mobsacB-BBD29_04920 is used to integrate the foreign gene BBD29_04920 into the host chromosome, and overexpress the wild-type BBD29_04920 gene in the production bacteria.
  • the recombinant vector PK18mobsacB-BBD29_04920 C1560A is used to integrate the exogenous gene BBD29_04920 C1560A into the host chromosome, and overexpress the mutant gene BBD29_04920 C1560A in the production bacteria.
  • the recombinant vector pXMJ19-BBD29_04920 is used to express the foreign gene BBD29_04920 extrachromosomally through a plasmid, and then overexpress the wild-type BBD29_04920 gene in the production bacteria.
  • the recombinant vector pXMJ19-BBD29_04920 C1560A is used to express the exogenous gene BBD29_04920 C1560A extrachromosomally through a plasmid, and then overexpress the mutant gene BBD29_04920 C1560A in the production bacteria.
  • the recombinant vectors pK18- BBD29_04920C1560A , PK18mobsacB-BBD29_04920, PK18mobsacB- BBD29_04920C1560A , pXMJ19-BBD29_04920 and pXMJ19- BBD29_04920C1560A are all within the protection scope of the present invention.
  • the recombinant microorganism may specifically be recombinant bacteria YPG-001, YPG-002, YPG-003, YPG-004 or YPG-005.
  • the recombinant bacteria YPG-001 is the recombinant bacteria obtained by transforming the recombinant vector pK18-BBD29_04920 C1560A into Corynebacterium glutamicum CGMCC No.21220, and the recombinant bacteria YPG-001 contains SEQ ID No.2
  • the mutated gene BBD29_04920C1560A is shown .
  • the recombinant bacteria YPG-002 contains double copies of the BBD29_04920 gene shown in SEQ ID No. 1; the recombinant bacteria containing double copies of the BBD29_04920 gene can significantly and stably increase the expression of the BBD29_04920 gene.
  • the recombinant strain YPG-002 is an engineered strain that overexpresses the wild-type BBD29_04920 gene on its genome.
  • the recombinant strain YPG-003 contains the mutated BBD29_04920 C1560A gene shown in SEQ ID No. 2; the recombinant strain YPG-003 is an engineering strain that overexpresses the mutant BBD29_04920 C1560A gene on the genome.
  • Recombinant bacterium YPG-004 contains the BBD29_04920 gene shown in double-copy SEQ ID No.1; recombinant bacterium YPG-004 is an engineering bacterium that overexpresses wild-type BBD29_04920 gene on a plasmid, that is, extrachromosomally overexpressed by plasmid pXMJ19-BBD29_04920 .
  • Recombinant bacteria YPG-005 contains the mutant BBD29_04920 C1560A gene shown in SEQ ID No. 2; recombinant bacteria YPG-005 is an engineering bacteria that overexpresses the mutant BBD29_04920 C1560A gene on a plasmid, that is, extrachromosomally derived from plasmid pXMJ19-BBD29_04920 C1560A overexpressed.
  • YPG-001, YPG-002, YPG-003, YPG-004 or YPG-005 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 using any one 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.
  • SEQ ID NO: 1 BBD29_04920 wild-type coding region sequence
  • SEQ ID NO: 2 BBD29_04920 C1560A coding region sequence
  • SEQ ID NO: 3 BBD29_04920 wild-type encoded protein amino acid sequence
  • SEQ ID NO:4 BBD29_04920 N520K- encoded protein amino acid sequence
  • Preservation information strain name: Corynebacterium glutamicum; Latin name: Corynebacterium glutamicum; Strain number: YPGLU001; Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee; Preservation Institution Abbreviation: CGMCC; Address: Chaoyang District, Beijing No. 3, No. 1 Courtyard, Beichen West Road; Preservation Date: November 23, 2020; Registration Number: CGMCC No.21220.
  • 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.
  • the composition of the basal medium is as follows. The above components are dissolved in water to obtain a 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 Administration Commission (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.
  • Cytosine (C) at position 1560 is changed to adenine (A) (SEQ ID NO: 2: BBD29_04920 C1560A ), and asparagine (N) at position 520 of the amino acid sequence of the encoded protein is changed to lysine (K) (SEQ ID NO:29_04920 C1560A ) ID NO: 4: BBD29_04920N520K ).
  • the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • P2 5'AGCCACGATGGTGACTTTTTGCAAGTTGTT_3' (SEQ ID NO:6)
  • P3 5' AACAACTTGC AAAAAGTCAC CATCGTGGCT 3' (SEQ ID NO: 7)
  • 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, the balance is Water, total volume 50 ⁇ L.
  • the above 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, to obtain two sizes of 766bp and 778bp, respectively, DNA fragments containing the coding region of the BBD29_04920 C1560A gene (BBD29_04920Up and BBD29_04920Down).
  • the above-mentioned two DNA fragments are separated and purified by agarose gel electrophoresis, the above-mentioned two DNA fragments are used as templates, and P1 and P4 are used as primers to amplify a fragment of 1514bp by overlapping PCR (that is, BBD29_04920C1560A- up -down), the nucleotide sequence of which is SEQ ID No. 2, positions 846-1788.
  • 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, the balance is Water, total volume 50 ⁇ L.
  • the above overlapping 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 90s, 30 cycles, and overextension at 72°C for 10 min.
  • This DNA fragment causes the cytosine (C) at position 1560 of the coding region of the BBD29_04920 gene in Corynebacterium glutamicum CGMCC No. 21220 to be changed to adenine (A), and finally the amino acid at position 520 of the encoded protein is changed from asparagine ( N) becomes lysine (K).
  • the BBD29_04920 C1560A and the linearized pK18mobsacB plasmid were separated and purified by agarose gel electrophoresis, and then assembled by the NEBuider recombination system (NEB E5520S) to obtain the vector pK18-BBD29_04920 C1560A , the plasmid contains the kanamycin resistance marker.
  • the vector pK18-BBD29_04920 C1560A was sent to a sequencing company for sequencing identification, and the vector pK18-BBD29_04920 C1560A containing the correct point mutation (CA) was saved for future use.
  • pK18-BBD29_04920 C1560A is the DNA fragment BBD29_04920 C1560A -up-down shown in SEQ ID No. 2 in the sequence listing, inserted into the XbaI recognition site of the pK18mobsacB vector, and other sequences of the pK18mobsacB vector are kept unchanged to obtain the recombinant vector.
  • allelic replacement plasmid pK18-BBD29_04920 C1560A was transformed into Corynebacterium glutamicum CGMCC No. 21220 by electroporation, and the single colony produced by culture was subjected to primer P1 and universal primer M13R (5'CAG GAA ACA GCT ATG ACC3' ) for identification, and the strain that can amplify a band of about 1521 bp in size (the sequence is shown in SEQ ID No. 29) is the positive strain.
  • the positive strains were cultured on the medium containing 15% sucrose, and 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):
  • the above-mentioned PCR amplification product (sequence shown in SEQ ID No. 30) was subjected to SSCP electrophoresis after high temperature denaturation and ice bath (with plasmid pK18-BBD29_04920 C1560A amplified fragment as positive control, ATCC13869 amplified fragment as negative control, and water as Blank control), due to different fragment structures and different electrophoresis positions, the strains whose electrophoresis positions are inconsistent with those of the negative control fragment and consistent with the position of the positive control fragment are the strains with successful allelic replacement. Using primers P5 and P6 to amplify the successful allelic replacement of the target fragment of the strain again by PCR, and connect it to the PMD19-T vector for sequencing. Designated as YPG-001.
  • Recombinant bacteria YPG-001 introduced point mutation C1560A in the coding region (SEQ ID No. 1) of BBD29_04920 gene of Corynebacterium glutamicum CGMCC No. 21220 by allele replacement, making the gene No. 1560 C mutation A at position, the other sequences of the gene remain unchanged, and the genetic engineering bacteria YPG-001 containing point mutation (C-A) is obtained.
  • Corynebacterium glutamicum YPG-001 Compared with Corynebacterium glutamicum CGMCC21220, the only difference of Corynebacterium glutamicum YPG-001 is: the BBD29_04920 gene shown in SEQ ID No.1 in the genome of Corynebacterium glutamicum CGMCC21220 is replaced with SEQ ID No.2 The BBD29_04920 C1560A gene shown. There is only one nucleotide difference between SEQ ID No. 1 and SEQ ID No. 2, located at position 1560.
  • the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • Construction method Take Corynebacterium glutamicum ATCC13869 or YPG-001 as the template, respectively use primers P7/P8, P9/P10, P11/P12 to carry out PCR amplification to obtain an upstream homology arm fragment of about 806bp (sequence as SEQ ID No.31), BBD29_04920 or BBD29_04920 C1560A gene coding region and promoter region fragment of about 1987bp (sequence shown in SEQ ID No.32 or SEQ ID No.33) and downstream homology arm fragment of about 788bp (sequence shown in shown in SEQ ID No. 34).
  • primers P7/P8, P9/P10, P11/P12 to carry out PCR amplification to obtain an upstream homology arm fragment of about 806bp (sequence as SEQ ID No.31), BBD29_04920 or BBD29_04920 C1560A gene coding region and promoter region fragment of about 1987bp (sequence
  • the plasmids PK18mobsacB were connected to obtain the integrated plasmid PK18mobsacB-BBD29_04920 or PK18mobsacB-BBD29_04920 C1560A .
  • the plasmid contained the kanamycin resistance marker, and the recombinant plasmid integrated into the genome could be obtained by kanamycin screening.
  • PK18mobsacB-BBD29_04920 is a recombinant vector obtained by inserting the upstream-BBD29_04920-downstream of the integrated homology arm fragment into the Xba I restriction site of the shuttle plasmid pk18mobsacB.
  • PK18mobsacB-BBD29_04920 C1560A is a recombinant vector obtained by inserting the upstream-BBD29_04920 C1560A -downstream of the integrated homology arm fragment 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 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 2 integrated plasmids were electro-transformed into the strain Corynebacterium glutamicum CGMCC No.21220 respectively, and the single colony produced by the culture was identified by PCR through the P13/P14 primers, and the PCR amplification contained a size of about 1674bp (sequence such as SEQ ID No. 2). .37) are the positive strains, and those that fail to amplify the fragments are the original strains. Positive strains were screened with 15% sucrose and cultured on media containing kanamycin and without kanamycin, respectively.
  • the strains that do not grow on the base were further identified by PCR using the P15/P16 primers, and the bacteria with a size of about 1943bp (sequence shown in SEQ ID No. 38) were amplified as BBD29_04920 or BBD29_04920 C1560A gene was integrated into Corynebacterium glutamicum CGMCC No.
  • the strains on the .21220 genome were named YPG-002 (without mutation) and YPG-003 (with mutation).
  • Recombinant bacterium YPG-002 is to integrate the upstream-BBD29_04920-downstream of the integrated homology arm fragment into the genome of strain Corynebacterium glutamicum YPGLU001 to obtain a recombinant bacterium containing the BBD29_04920 gene shown in double-copy SEQ ID No.1, containing double-copy
  • the recombinant bacteria of BBD29_04920 gene can significantly and stably increase the expression level of BBD29_04920.
  • the recombinant bacterium YPG-003 is a recombinant bacterium containing the BBD29_04920 C1560A mutant gene shown in SEQ ID No. 2 by integrating the upstream of the integrated homology arm fragment- BBD29_04920C1560A -downstream into the genome of the strain Corynebacterium glutamicum YPGLU001.
  • Construction method respectively take YPG-002 or YPG-001 as template, carry out PCR amplification with primer P17/P18, obtain the DNA fragment 2017bp containing BBD29_04920 or BBD29_04920 C1560A gene and its promoter (sequence such as SEQ ID No. ID No.40), the amplified product was recovered by electrophoresis, and the required 1947bp DNA fragment was recovered using a column DNA gel recovery kit.
  • the NEBuider recombination system and the shuttle plasmid recovered by EcoR I digestion were used pXMJ19 was ligated to obtain overexpression plasmid pXMJ19-BBD29_04920 or pXMJ19- BBD29_04920C1560A .
  • the plasmid contains a chloramphenicol resistance marker, and the plasmid can be transformed into the strain by screening with chloramphenicol.
  • 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 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 90s (30 cycles), and overextension at 72°C for 10 min.
  • the overexpression plasmid pXMJ19-BBD29_04920 is the recombinant vector obtained by inserting the DNA fragment (sequence shown in SEQ ID No. 39) containing the BBD29_04920 gene and its promoter into the EcoR I restriction site of the shuttle plasmid pXMJ19.
  • Overexpression plasmid pXMJ19-BBD29_04920 C1560A A recombinant vector obtained by inserting the DNA fragment (sequence shown in SEQ ID No. 40) containing the BBD29_04920 C1560A gene and its promoter into the EcoR I restriction site of the shuttle plasmid pXMJ19.
  • the two plasmids were electrotransformed into the strain Corynebacterium glutamicum CGMCC No. 21220 respectively, and the single colony produced by the culture was identified by M13R(-48) (5'AGCGGATAAC AATTTCACAC AGGA3') and P18 primers, and PCR amplification was carried out.
  • the fragment containing a size of about 2056bp (the sequence without point mutation is shown in SEQ ID No.41, the 1794th position of the sequence containing point mutation is A, and the rest are shown in SEQ ID No.41) is the transformed strain, which were named YPG-004 (without point mutation) and YPG-005 (with point mutation).
  • Recombinant bacterium YPG-004 contains the BBD29_04920 gene shown in double-copy SEQ ID No.1; recombinant bacterium YPG-004 is an engineering bacterium that overexpresses wild-type BBD29_04920 gene on a plasmid, that is, extrachromosomally overexpressed by plasmid pXMJ19-BBD29_04920 .
  • the recombinant bacteria YPG-005 contains the mutant BBD29_04920 C1560A gene shown in SEQ ID No. 2; the recombinant bacteria YPG-05 is an engineering bacteria that overexpresses the mutant BBD29_04920 C1560A gene on a plasmid, that is, extrachromosomally derived from the plasmid pXMJ19-BBD29_04920 C1560A overexpressed.
  • P20 5' GTATCAGGGGTTAAAAATTGCTTAATTTTCC CTGGCAGAA 3' (SEQ ID NO: 24)
  • Construction method Take Corynebacterium glutamicum ATCC13869 as the template, carry out PCR amplification with primers P19/P20 and P21/P22 respectively, and obtain the upstream homology arm fragment of 733bp (sequence shown in SEQ ID No. 42) and downstream homology Arm fragment 814bp (sequence is shown in SEQ ID No. 43). Then use primer P19/P22 to carry out overlapping PCR to obtain the entire homology arm fragment of 1507bp (the sequence is shown in SEQ ID No. 44).
  • the amplified product was recovered by electrophoresis, and the required 1507 bp DNA fragment was recovered by a column 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 the strain Corynebacterium glutamicum CGMCC No.21220, and the single colonies produced by the culture were identified by PCR with the following primers (synthesized by Shanghai Yingjun Company):
  • PCR amplifies the strain of the band of size 1433bp (sequence as shown in SEQ ID No.45) and 3380bp (sequence as shown in SEQ ID No.46) is a positive strain, and only the bacterial strain of amplifying the 3380bp band is original bacteria.
  • the recombinant strain YPG-006 is a strain obtained by knocking out the coding region of the BBD29_04920 gene on the genome of Corynebacterium glutamicum CGMCC No. 21220.
  • the fermentation tank purchased from Shanghai Bailun Biotechnology Co., Ltd.
  • the medium shown in Table 1 the solute shown in Table 1 was dissolved in water to obtain the medium.
  • 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.
  • 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 product encoded by the gene has an impact on the L-glutamic acid production capacity, and the recombinant strain is obtained by introducing point mutation in the coding sequence, or increasing the copy number or overexpression of the gene , the obtained strain is favorable for the production of high concentration of glutamate compared with the unmodified strain.
  • the present invention first introduced a point mutation in the BBD29_04920 gene coding region (SEQ ID No. 1) of Corynebacterium glutamicum CGMCC No. 21220 by allele replacement, and constructed a structure containing the point mutation ( CA) genetically engineered bacteria YPG-001.
  • the exogenous genes were integrated into the host chromosome or expressed extrachromosomally by plasmids, respectively.
  • Point mutation in the coding region of the BBD29_04920 gene or overexpression of the BBD29_04920 gene or its mutant gene BBD29_04920 C1560A 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_04920 gene does not Conducive to the accumulation of L-glutamic acid.
  • BBD29_04920 gene and its variant such as BBD29_04920 C1560A gene

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Abstract

提供了对棒杆菌中BBD29_04920基因编码序列引入点突变或改进其表达的方法，该点突变是使BBD29_04920基因序列第1560位碱基由胞嘧啶(C)突变为腺嘌呤(A)，使编码的相应氨基酸序列第520位天冬酰胺被赖氨酸取代。该方法可以使带有所述突变的菌株提高谷氨酸的发酵产量。还提供了该生成L-谷氨酸的细菌，含有该点突变的核酸、蛋白质，含有该核酸的重组载体、重组菌株，以及这些生物材料在调控微生物的L-谷氨酸的产量中的应用。

Description

一种改造基因BBD29_04920的产L-谷氨酸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，具体涉及一种产L-谷氨酸的重组菌株及其构建方法与应用。
背景技术
L-谷氨酸是一种重要的氨基酸，被应用于食品、临床药物及其它方面。
传统上，L-谷氨酸主要是通过发酵，用属于短杆菌属、棒状杆菌属或微菌属等菌属的生产L-谷氨酸的细菌或其突变体进行生产。
L-谷氨酸是由微生物细胞内柠檬酸循环的中间产物α-酮戊二酸通过生物合成而来的。由α-酮戊二酸通过铵离子的同化作用来形成L-谷氨酸的生物合成途径有两条。其中一条途径是在有高浓度的铵离子存在的情况下，通过谷氨酸脱氢酶(glutamate dehydrogenase，GDH)的催化来合成L-谷氨酸。另一条途径(GS/GOGAT途径)是由谷氨酰胺合成酶及谷氨酰胺-酮戊二酸氨基转移酶来合成L-谷氨酸。谷氨酰胺合成酶(glutamine synthetase，GS)催化L-谷氨酸和铵离子转化为谷氨酰胺的反应；谷氨酰胺-酮戊二酸氨基转移酶(glutamine-oxoglutaric acid amino transferase，也称“谷氨酸合成酶”(glutamate synthase，GOGAT)催化L-谷氨酸合成反应，在该反应中，由已经由GS合成的一分子的谷氨酰胺和一分子的α-酮戊二酸分子合成两分子的L-谷氨酸。
对发酵法生产L-氨基酸的改进可以涉及发酵技术如搅拌和供应氧气；或涉及营养培养基的组成，例如发酵过程中的糖浓度；或涉及将发酵液加工成合适的产品形式，例如通过干燥和造粒发酵液或离子交换色谱；或可以涉及相关微生物本身的固有性能性质。
用于改善这些微生物的性能性质的方法包括诱变、突变体的选择和筛选。以这种方式获得的菌株对抗代谢物具有抗性或对于具有调节重要性的代谢物是营养缺陷型并产生L-氨基酸等。
虽然已有大量方法可以提高L-谷氨酸的生产能力，但是为了满足日益增加的需求，仍需要发展生产L-谷氨酸的方法。
发明公开
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，对细菌中的基因BBD29_04920或其同源基因，可以通过修饰所述基因或改善其表达，能够改善细菌的L-谷氨酸生产能力。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_04920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，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520位天冬酰胺被不同的氨基酸所取代。
根据本发明，优选第520位天冬酰胺被赖氨酸所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第520位天冬酰胺被赖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1560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1560位碱基由胞嘧啶(C)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_04920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。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(Corynebacteriumammoniagenes)、北京棒杆菌(Corynebacteriumpekinense)、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ATCC 13869。
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，所述序列的第520位天冬酰胺被不同的氨基酸所取代。
根据本发明，优选第520位天冬酰胺被赖氨酸所取代。
根据本发明，SEQ ID NO:3所示的氨基酸序列，其中第520位天冬酰胺被赖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1560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1560位碱基由胞嘧啶(C)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_04920基因的多核苷酸 序列，使其第1560位碱基发生突变，得到包含突变BBD29_04920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1560位碱基由胞嘧啶(C)变为腺嘌呤(A)；具体地，所述包含突变BBD29_04920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_04920基因的核苷酸序列，使其第1560位碱基发生突变，得到突变的BBD29_04920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_04920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_04920基因构建：根据未修饰菌株的基因组序列，合成两对扩增BBD29_04920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_04920基因SEQ ID NO:1中引入点突变，得到点突变的BBD29_04920基因核苷酸序列SEQ ID NO:2，记为BBD29_04920 C1560A。
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：
P1:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG  TGTTTCTGTC  TTGACCTTGG(SEQ ID NO:5)
P2:5'AGCCACGATG GTGACTTTTT GCAAGTTGTT 3'(SEQ ID NO:6)
P3:5'AACAACTTGC AAAAAGTCAC CATCGTGGCT 3'(SEQ ID NO:7)
P4:5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC ACAGATTGGG CAGGTGCC3'(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_04920 C1560A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_04920的上下游同源臂片段、BBD29_04920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_04920或BBD29_04920 C1560A基因，以实现所述菌株过表达BBD29_04920或BBD29_04920 C1560A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'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GACCCGCTTG CCATACGAAG3'(SEQ ID NO:11)
P8:5'GCATCACAAT GACATAACGA ATCTACTCAT CTGAAGAATC 3'(SEQ ID NO:12)
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'GCGCGGGGAA GCGTCGATAG TTCGTGGGCA CTCTGGTTTG 3'(SEQ ID NO:15)
P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC CATAAGAAAC AACCACTTCC 3'(SEQ ID NO:16)
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 TCGT TATGTCATTG TGATGC 3'(SEQ ID NO:13)
P10:5'CAAACCAGAG TGCCCACGAA CTATCGACGC TTCCCCGCGC 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_04920或BBD29_04920 C1560A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_04920基因编码区及启动子区序列，或BBD29_04920 C1560A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_04920或BBD29_04920 C1560A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'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTCGTTATGTCATTGTGATGC 3'(SEQ ID NO:21)
P18:5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTATCGACGCTTCCCCGCGC3'(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(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_04920或BBD29_04920 C1560A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。
本发明还提供了蛋白质，名称为蛋白质BBD29_04920 N520K，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_04920 C1560A，所述核酸分子BBD29_04920 C1560A可为下述任一种：
B1)编码所述蛋白质BBD29_04920 N520K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_04920 C1560A基因。
SEQ ID No.2所示的DNA分子(BBD29_04920 C1560A基因)编码SEQ ID No.4所示的蛋白质BBD29_04920 N520K。
所述蛋白质BBD29_04920 N520K氨基酸序列(SEQ ID No.4)为将SEQ ID No.3中的第520位天冬酰胺(N)变为赖氨酸(K)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_04920 C1560A的表达盒；
C2)含有所述核酸分子BBD29_04920 C1560A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_04920 C1560A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_04920 N520K；
D2)所述核酸分子BBD29_04920 C1560A；
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_04920基因。
SEQ ID No.1所示的DNA分子(BBD29_04920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或核苷酸序列的同一性，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_04920 C1560A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520位的天冬酰胺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520位的天冬酰胺突变为赖氨酸，得到氨基酸序列为SEQ ID No.4的突变蛋白质BBD29_04920 N520K。
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_04920基因(SEQ ID No.1)的第1560位胞嘧啶(C)进行核酸改造。
具体地，所述点突变可为将BBD29_04920基因(SEQ ID No.1)的第1560位胞嘧啶(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_04920 C1560A、PK18mobsacB-BBD29_04920、PK18mobsacB-BBD29_04920 C1560A、pXMJ19-BBD29_04920或pXMJ19-BBD29_04920 C1560A。
所述重组载体pK18-BBD29_04920 C1560A含有SEQ ID No.2所示的突变的基因BBD29_04920 C1560A的第846-1788位所示的DNA分子。
所述重组载体PK18mobsacB-BBD29_04920用于将外源基因BBD29_04920整合到宿主染色体中，在生产菌中过表达野生型BBD29_04920基因。
所述重组载体PK18mobsacB-BBD29_04920 C1560A用于将外源基因 BBD29_04920 C1560A整合到宿主染色体中，在生产菌中过表达突变型基因BBD29_04920 C1560A。
所述重组载体pXMJ19-BBD29_04920用于将外源基因BBD29_04920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_04920基因。
所述重组载体pXMJ19-BBD29_04920 C1560A用于将外源基因BBD29_04920 C1560A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_04920 C1560A。
所述重组载体pK18-BBD29_04920 C1560A、PK18mobsacB-BBD29_04920、PK18mobsacB-BBD29_04920 C1560A、pXMJ19-BBD29_04920和pXMJ19-BBD29_04920 C1560A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-001、YPG-002、YPG-003、YPG-004或YPG-005。
所述重组菌YPG-001是将所述重组载体pK18-BBD29_04920 C1560A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-001含有SEQ ID No.2所示的突变的基因BBD29_04920 C1560A。
所述重组菌YPG-002含有双拷贝的SEQ ID No.1所示的BBD29_04920基因；含有双拷贝BBD29_04920基因的重组菌可以显著和稳定地提高BBD29_04920基因的表达量。重组菌YPG-002为在基因组上过表达野生型BBD29_04920基因的工程菌。
所述重组菌YPG-003含有SEQ ID No.2所示的突变的BBD29_04920 C1560A基因；重组菌YPG-003为在基因组上过表达突变型BBD29_04920 C1560A基因的工程菌。
重组菌YPG-004含有双拷贝SEQ ID No.1所示的BBD29_04920基因；重组菌YPG-004为在质粒上过表达野生型BBD29_04920基因的工程菌，即由质粒pXMJ19-BBD29_04920在染色体外进行过表达。
重组菌YPG-005含有SEQ ID No.2所示的突变的BBD29_04920 C1560A基因；重组菌YPG-005为在质粒上过表达突变型BBD29_04920 C1560A基因的工程菌，即由质粒pXMJ19-BBD29_04920 C1560A在染色体外进行过表达。
所述重组菌YPG-001、YPG-002、YPG-003、YPG-004或YPG-005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_04920 C1560A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。
在本发明中：
SEQ ID NO:1：BBD29_04920野生型编码区序列
Figure PCTCN2021142438-appb-000001
Figure PCTCN2021142438-appb-000002
SEQ ID NO:2：BBD29_04920 C1560A编码区序列
Figure PCTCN2021142438-appb-000003
Figure PCTCN2021142438-appb-000004
SEQ ID NO:3：BBD29_04920野生型编码蛋白氨基酸序列
Figure PCTCN2021142438-appb-000005
Figure PCTCN2021142438-appb-000006
SEQ ID NO:4：BBD29_04920 N520K编码蛋白氨基酸序列
Figure PCTCN2021142438-appb-000007
保藏信息：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。
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；所进行的操作都是本领域已知的，或者按照市售商品的用户手册进行。
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，基础培养基中的成分组成如下，将下述成分溶于水中，得到基础培养基：
Figure PCTCN2021142438-appb-000008
Figure PCTCN2021142438-appb-000009
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_04920基因编码区的转化载体pK18-BBD29_04920 C1560A
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_04920基因编码区序列(SEQ ID NO:1：BBD29_04920)的引物，以等位基因置换的方式在菌株谷氨酸棒杆菌CGMCC No.21220中(经测序确认该菌株染色体上的BBD29_04920基因编码区与ATCC13869是一致的)引入点突变，对应编码蛋白的氨基酸序列为SEQ ID NO:3，BBD29_04920基因的核苷酸序列第1560位胞嘧啶(C)变为腺嘌呤(A)(SEQ ID NO:2：BBD29_04920 C1560A)，对应编码蛋白的氨基酸序列第520位天冬酰胺(N)变为赖氨酸(K)(SEQ ID NO:4：BBD29_04920 N520K)。引物设计如下(上海invitrogen公司合成)：
P1:5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG  TGTTTCTGTC  TTGACCTTGG(SEQ ID NO:5)
P2:5'AGCCACGATG GTGACTTTTT GCAAGTTGTT_3'(SEQ ID NO:6)
P3:5'AACAACTTGC AAAAAGTCAC CATCGTGGCT 3'(SEQ ID NO:7)
P4:5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC ACAGATTGGG CAGGTGCC 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，获得两条大小分别为766bp和778bp，含有BBD29_04920 C1560A基因编码区的DNA片段(BBD29_04920Up和BBD29_04920Down)。
将上述两条DNA片段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扩增长为1514bp的片段(即为BBD29_04920 C1560A-up-down)，其核苷酸序列为SEQ ID No.2第846-1788位。
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1μL，余量为水，总体积50μL.
上述重叠PCR扩增按如下方式进行：94℃预变性5min，94℃变性30s、52℃退火30s、72℃延伸90s，30个循环，72℃过度延伸10min。
此DNA片段导致谷氨酸棒杆菌CGMCC No.21220中BBD29_04920基因编码区的第1560位的胞嘧啶(C)变为腺嘌呤(A)，最终导致编码蛋白的第520位氨基酸由天冬酰胺(N)变为赖氨酸(K)。
将pK18mobsacB质粒(购自Addgene公司)用Xba I酶切后，用琼脂糖凝胶电泳分离纯化BBD29_04920 C1560A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_04920 C1560A，该质粒上含有卡那霉素抗性标记。并将载体pK18-BBD29_04920 C1560A送测序公司测序鉴定，将含有正确点突变(C-A)的载体pK18-BBD29_04920 C1560A保存备用。
pK18-BBD29_04920 C1560A是将序列表中SEQ ID No.2第846-1488位所示的DNA片段BBD29_04920 C1560A-up-down插入pK18mobsacB载体的XbaI识别位点之后，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_04920 C1560A的工程菌株
构建方法：将等位替换质粒pK18-BBD29_04920 C1560A通过电击转化入谷氨酸棒杆菌CGMCC No.21220中，对培养产生的单菌落分别通过引物P1和通用引物M13R(5’CAG GAA ACA GCT ATG ACC3’)进行鉴定，能扩增出大小约1521bp条带(序列如SEQ ID No.29所示)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'CTATTGCTTT CTGGTGGTG 3'(SEQ ID NO:9)
P6:5'TCGCCTTACG CTCCCTGCGT 3'(SEQ ID NO:10)
上述PCR扩增产物(序列如SEQ ID No.30所示)通过高温变性、冰浴后进行SSCP电泳(以质粒pK18-BBD29_04920 C1560A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载体进行测序，通过序列比对，碱基序列发生突变的序列验证菌株的等位替换成功，并被命名为YPG-001。
重组菌YPG-001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_04920基因编码区(SEQ ID No.1)中引入点突变C1560A，使该基因第1560位的C突变A，该基因其他序列不变，得到包含点突变(C-A)的基因工程菌YPG-001。
与谷氨酸棒杆菌CGMCC21220相比，谷氨酸棒杆菌YPG-001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_04920基因取代为SEQ ID No.2所示的BBD29_04920 C1560A基因。SEQ ID No.1和SEQ ID No.2仅存在一个核苷酸差异，位于第1560位。
SSCP电泳PAGE的制备及条件如下：
Figure PCTCN2021142438-appb-000010
实施例3 构建基因组上过表达BBD29_04920或BBD29_04920 C1560A基因的工程菌株
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组序列，设计并合成三对扩增上下游同源臂片段及BBD29_04920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CGMCC No.21220中引入BBD29_04920或BBD29_04920 C1560A基因。
引物设计如下(上海invitrogen公司合成)：
P7:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GACCCGCTTG CCATACGAAG 3'(SEQ ID NO:11)
P8:5'GCATCACAAT GACATAACGA ATCTACTCAT CTGAAGAATC 3'(SEQ ID NO:12)
P9:5'GATTCTTCAG ATGAGTAGAT TCGT TATGTCATTG TGATGC 3'(SEQ ID NO:13)
P10:5'CAAACCAGAG TGCCCACGAA CTATCGACGC TTCCCCGCGC 3'(SEQ ID NO:14)
P11:5'GCGCGGGGAA GCGTCGATAG TTCGTGGGCA CTCTGGTTTG 3'(SEQ ID NO:15)
P12:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC CATAAGAAAC AACCACTTCC 3'(SEQ ID NO:16)
构建方法：分别以谷氨酸棒杆菌ATCC13869或YPG-001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(序列如SEQ ID No.31所示)，BBD29_04920或BBD29_04920 C1560A基因编码区及启动子区 片段约1987bp(序列如SEQ ID No.32或SEQ ID No.33所示)及下游同源臂片段约788bp(序列如SEQ ID No.34所示)。再以P7/P12为引物，以以上扩增的三种片段混合为模板进行扩增，获得整合同源臂片段上游-BBD29_04920-下游(序列如SEQ ID No.35所示)或整合同源臂片段上游-BBD29_04920 C1560A-下游(序列如SEQ ID No.36所示)。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(TIANGEN)进行回收所需要的约3501bp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PK18mobsacB-BBD29_04920或PK18mobsacB-BBD29_04920 C1560A，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_04920为将整合同源臂片段上游-BBD29_04920-下游插入穿梭质粒pk18mobsacB的Xba I酶切位点之后，得到的重组载体。
PK18mobsacB-BBD29_04920 C1560A为将整合同源臂片段上游-BBD29_04920 C1560A-下游插入穿梭质粒pk18mobsacB的Xba I酶切位点之后，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。
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120s(30个循环)，72℃过度延伸10min。
将2个整合质粒分别电转化入菌株谷氨酸棒杆菌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1674bp(序列如SEQ ID No.37所示)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1943bp(序列如SEQ ID No.38所示)的菌为BBD29_04920或BBD29_04920 C1560A基因整合到谷氨酸棒杆菌CGMCC No.21220基因组上的菌株，其被命名为YPG-002(不含突变点)和YPG-003(含突变点)。
P13:5'GTCCAAGGTG ACGGCCGCAC 3'(SEQ ID NO:17)
P14:5'CTTTTTCACC AATGAGTGGC 3'(SEQ ID NO:18)
P15:5'CCGTAAACAG CTTCTAAGCT 3'(SEQ ID NO:19)
P16:5'ATATTCGGCC CAGCAGCAGC 3'(SEQ ID NO:20)
重组菌YPG-002为将整合同源臂片段上游-BBD29_04920-下游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_04920基因的重组菌，含有双拷贝BBD29_04920基因的重组菌可以显著和稳定地提高BBD29_04920的表达量。
重组菌YPG-003为将整合同源臂片段上游-BBD29_04920 C1560A-下游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_04920 C1560A突变基因的重组菌。
实施例4 构建质粒上过表达BBD29_04920或BBD29_04920 C1560A基因的工程菌株
依据NCBI公布的谷氨酸棒杆菌ATCC13869基因组序列，设计并合成一对扩增BBD29_04920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'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC TCGTTATGTCATTGTGATGC 3'(SEQ ID NO:21)
P18:5' ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTATCGACGCTTCCCCGCGC3'(SEQ ID NO:22)
构建方法：分别以YPG-002或YPG-001为模板，以引物P17/P18进行PCR扩增，获得含有BBD29_04920或BBD29_04920 C1560A基因及其启动子的DNA片段2017bp(序列如SEQ ID No.39或SEQ ID No.40所示)，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1947bp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相连接，获得过表达质粒pXMJ19-BBD29_04920或pXMJ19-BBD29_04920 C1560A。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。
过表达质粒pXMJ19-BBD29_04920将含有BBD29_04920基因及其启动子的DNA片段(序列如SEQ ID No.39所示)插入穿梭质粒pXMJ19的EcoR I酶切位点之后，得到的重组载体。
过表达质粒pXMJ19-BBD29_04920 C1560A将含有BBD29_04920 C1560A基因及其启动子的DNA片段(序列如SEQ ID No.40所示)插入穿梭质粒pXMJ19的EcoR I酶切位点之后，得到的重组载体。
将2个质粒分别电转化入菌株谷氨酸棒杆菌CGMCC No.21220中，对培养产生的单菌落通过M13R(-48)(5'AGCGGATAAC AATTTCACAC AGGA3')和P18引物进行PCR鉴定，PCR扩增出含有大小约2056bp的片段(不含点突变的序列如SEQ ID No.41所示，含点突变序列第1794位为A，其余如SEQ ID No.41所示)的为转入菌株，其被命名为YPG-004(不含点突变)和YPG-005(含点突变)。
重组菌YPG-004含有双拷贝SEQ ID No.1所示的BBD29_04920基因；重组菌YPG-004为在质粒上过表达野生型BBD29_04920基因的工程菌，即由质粒pXMJ19-BBD29_04920在染色体外进行过表达。
重组菌YPG-005含有SEQ ID No.2所示的突变的BBD29_04920 C1560A基因；重组菌YPG-05为在质粒上过表达突变型BBD29_04920 C1560A基因的工程菌，即由质粒pXMJ19-BBD29_04920 C1560A在染色体外进行过表达。
实施例5 构建基因组上缺失BBD29_04920基因的工程菌株
根据NCBI公布的谷氨酸棒杆菌ATCC13869的基因组序列，合成两对扩增BBD29_04920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'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTAAGGGGCAG TTGGTCTCG 3'(SEQ ID NO:23)
P20:5'GTATCAGGGGTTAAAAATTGCTTAATTTTCC CTGGCAGAA 3'(SEQ ID NO:24)
P21:5'TTCTGCCAGGGAAAATTAAGCAATTTTTAACCCCTGATAC 3'(SEQ ID NO:25)
P22:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATATCGCGCG  ACATTGCGCG 3'(SEQ ID NO:26)
构建方法：以谷氨酸棒杆菌ATCC13869为模板，分别以引物P19/P20和P21/P22进行PCR扩增，获得上游同源臂片段733bp(序列如SEQ ID No.42所示)及下游同源臂片段814bp(序列如SEQ ID No.43所示)。再用引物P19/P22进行重叠PCR得到整个同源臂片段1507bp(序列如SEQ ID No.44所示)。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1507bp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菌株谷氨酸棒杆菌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'TAAGGGGCAG TTGGTCTCG 3'(SEQ ID NO:27)
P24:5'ATATCGCGCG ACATTGCGCG 3'(SEQ ID NO:28)
上述PCR扩增出大小1433bp(序列如SEQ ID No.45所示)及3380bp(序列如SEQ ID No.46所示)的条带的菌株为阳性菌株，只扩增出3380bp条带的菌株为原菌。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1433bp条带的菌株为BBD29_04920基因编码区被敲除的基因工程菌株，其被命名为YPG-006。
重组菌YPG-006为将谷氨酸棒杆菌CGMCC No.21220上的基因组上的BBD29_04920基因编码区敲除获得的菌株。
实施例6 L-谷氨酸发酵实验
将实施例构建的菌株YPG-001、YPG-002、YPG-03、YPG-004、YPG-005、YPG-006和原始菌株谷氨酸棒杆菌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1所示的培养基(将表1所示的溶质溶于水中，得到培养基。)和表2所示的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3所示。
表1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2发酵控制工艺
Figure PCTCN2021142438-appb-000011
表3 L-谷氨酸发酵实验结果
Figure PCTCN2021142438-appb-000012
Figure PCTCN2021142438-appb-000013
结果如表3所示，在谷氨酸棒杆菌中对BBD29_04920基因过表达，或者对BBD29_04920基因编码区进行点突变BBD29_04920 C1560A和/或过表达，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_04920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。
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_04920基因编码区(SEQ ID No.1)中引入点突变，构建了包含点突变(C-A)的基因工程菌YPG-001。为进一步研究验证在生产菌中过表达野生型BBD29_04920基因或其突变基因BBD29_04920 C1560A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_04920基因或BBD29_04920 C1560A基因的工程菌YPG-002、YPG-003、YPG-004和YPG-005。实验表明，BBD29_04920基因及其变体参与了L-谷氨酸的生物合成，通过对BBD29_04920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_04920基因编码区进行点突变或在生产菌中过表达BBD29_04920基因或其突变基因BBD29_04920 C1560A，有助于L-谷氨酸产量及转化率的提高，而对BBD29_04920基因进行敲除或弱化，不利于L-谷氨酸的积累。可利用BBD29_04920基因及其变体(如BBD29_04920 C1560A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520位天冬酰胺被不同的氨基酸所取代；优选第520位天冬酰胺被赖氨酸所取代。
  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1560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1560位碱基由胞嘧啶(C)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520位天冬酰胺被不同的氨基酸所取代；优选第520位天冬酰胺被赖氨酸所取代；
    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1560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1560位碱基由胞嘧啶(C)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. 一种氨基酸序列，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. 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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