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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
• • 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
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/07—Nucleotidyltransferases (2.7.7)
  • C12Y207/0701—UTP--hexose-1-phosphate uridylyltransferase (2.7.7.10)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • 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 relates to a galactose-1-phosphate uridyl transferase mutant and application thereof in preparing L-lysine in the field of biotechnology.
• A2 A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A1).
• identity refers to sequence similarity with a natural nucleic acid sequence. “Identity” includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
• the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1mM EDTA, and washing in 50°C, 2 ⁇ SSC, 0.1% SDS; or: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse in 50°C, 1 ⁇ SSC, 0.1% SDS; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse in 50°C, 0.5 ⁇ SSC, 0.1% SDS; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse in 50°C, 0.1 ⁇ SSC, 0.1% SDS; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM
• B2 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule described in B1).
• the expression cassette is a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.
• a plant expression vector can be used to construct a recombinant vector containing the gene expression cassette.
• the substance may be a substance that mutates the 414th glutamic acid residue codon of SEQ ID No. 2 into a terminator;
• the biological cell can be yeast, bacteria, algae, fungi, plant cells or animal cells that can synthesize L-lysine.
• the bacteria of the present invention include but are not limited to Corynebacterium glutamicum. Any gene containing SEQ ID No. 1 in the sequence table can be mutated or knocked out to produce L-lysine.
• the bacteria can be Corynebacterium glutamicum, Escherichia coli, Pantoea ananatis, Bacillus brevis or Brevis lactobacillus.
• the recombinant biological cells can be used to produce a variety of products, including but not limited to lysine in the examples, and the products produced can also be glutamic acid, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha-ketoglutaric acid, citric acid, ornithine, citrulline, etc.
• the present invention also provides a biomaterial, which is the following b1) or b2) or b3) or b4) or b5):
• b2) a DNA molecule that has 75% or more identity with the DNA molecule sequence defined in b1) and encodes the protein shown in SEQ ID No.6;
• NCgl2002 G1240T gene The results of sequencing the NCgl2002 gene by extracting plasmids from Corynebacterium glutamicum YP097158 mutant strain 3 confirmed that the mutation site of NCgl2002 was the mutation of guanine (G) at position 1240 in the coding region of the gene to thymine (T) (the gene containing this mutation was recorded as NCgl2002 G1240T gene), and the glutamic acid (E) at position 414 in the amino acid sequence of its mutant protein NCgl2002 was mutated to a terminator (*) (the protein containing this mutation was recorded as NCgl2002 G1240T protein).
• the positive strain NCgl2002 G1240T gene fragment was amplified again by primers P6/P7PCR and connected to the PMD19-T vector for sequencing. Through sequence alignment, the strain with base sequence mutation (GT) was the positive strain with successful allelic replacement.
• the positive strains obtained from Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum strain ATCC13032 were named YPL-NCgl2002-1, L2002-1.
• SEQ ID No.3 pXMJ19-NCgl2002 sequence in which NCgl2002 gene and its promoter are integrated into pXMJ19 (nucleotide sequence 1441 bp)

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• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

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• 2022
  • 2022-12-29 CN CN202211706015.0A patent/CN115820706B/zh active Active
• 2023
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  • 2023-03-30 WO PCT/CN2023/084971 patent/WO2024138928A1/zh not_active Ceased
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