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WO2024258199A1 - Nouveau variant de la n-acétyltransférase de la famille gnat et procédé de production d'acide l-glutamique l'utilisant - Google Patents

Nouveau variant de la n-acétyltransférase de la famille gnat et procédé de production d'acide l-glutamique l'utilisant Download PDF

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WO2024258199A1
WO2024258199A1 PCT/KR2024/008116 KR2024008116W WO2024258199A1 WO 2024258199 A1 WO2024258199 A1 WO 2024258199A1 KR 2024008116 W KR2024008116 W KR 2024008116W WO 2024258199 A1 WO2024258199 A1 WO 2024258199A1
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glutamic acid
acetyltransferase
transformant
present
corynebacterium
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Korean (ko)
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최원우
이자경
이선희
김석수
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Daesang Corp
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Daesang Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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.)
    • 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

Definitions

  • the present invention relates to a novel mutant of a GNAT family N-acetyltransferase and a method for producing L-glutamic acid using the same.
  • L-glutamic acid is a representative amino acid produced by microbial fermentation, and its salt form, monosodium L-glutamate (MSG), adds balance and harmony to the overall taste of food, increasing the preference for foods such as meat, fish, chicken, vegetables, sauces, soups, and seasonings, and can enhance the taste of low-salt foods with up to 30% less salt, so it is widely used as a seasoning for household and processed food production.
  • MSG monosodium L-glutamate
  • glucose mainly goes through the glycolytic pathway, but some goes through the pentose phosphate pathway and is metabolized into two molecules of pyruvate. One of them fixes CO2 to become oxaloacetic acid, and the other molecule combines with acetyl CoA from pyruvate to become citric acid. Oxaloacetic acid and citric acid then enter the citric acid cycle (TCA cycle) to become ⁇ -ketoglutaric acid.
  • TCA cycle citric acid cycle
  • the reductive amino acid oxidation reaction of alpha-ketoglutarate proceeds efficiently to produce L-glutamic acid because the oxidative metabolic pathway from alpha-ketoglutarate to succinic acid is absent and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved.
  • L-glutamic acid can be produced using a wild-type strain obtained in nature or a mutant strain modified to enhance its glutamic acid production ability.
  • various recombinant strains or mutant strains with excellent L-glutamic acid production ability and methods for producing L-glutamic acid using the same have been developed by applying genetic recombination technology to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as amino acids and nucleic acids.
  • the present invention aims to provide a novel GNAT family N-acetyltransferase variant.
  • the present invention aims to provide a polynucleotide encoding the mutant.
  • the present invention aims to provide a transformant comprising the mutant or polynucleotide.
  • the present invention aims to provide a method for producing L-glutamic acid using the transformant.
  • One aspect of the present invention provides a GNAT family N-acetyltransferase mutant having an amino acid sequence of SEQ ID NO: 4, wherein the 174th valine (V) in the amino acid sequence of SEQ ID NO: 2 is substituted with methionine (M).
  • the “GNAT family N-acetyltransferase” used in the present invention transfers an acetyl group to glutamic acid to biosynthesize N-acetyl-L-glutamate.
  • the GNAT family N-acetyltransferase in the present invention may be a polypeptide having GNAT family N-acetyltransferase activity encoded by a gene located at locus BBD29_RS13390 of Corynebacterium glutamicum ATCC13869 or the NCgl2644 gene, but is not limited thereto.
  • Nucleic acid and protein sequence information for the above GNAT family N-acetyltransferases can be obtained through known sequence databases (e.g., GenBank, UniProt).
  • the GNAT family N-acetyltransferase may be encoded by the base sequence of SEQ ID NO: 1 and may be composed of the amino acid sequence of SEQ ID NO: 2.
  • the base sequence or amino acid sequence of the GNAT family N-acetyltransferase according to the present invention may include a base sequence or amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity compared to each sequence.
  • “homology” or “identity” means the rate of correspondence (%) between a reference base sequence or amino acid sequence and any other base sequence or amino acid sequence when they are aligned and analyzed so as to correspond as much as possible.
  • the GNAT family N-acetyltransferase or the gene encoding it may be derived from wild type Corynebacterium glutamicum .
  • variant used in the present invention means a protein whose original amino acid sequence is different due to a change in the base sequence of the gene encoding the protein. Specifically, one or more bases or nucleotides in the gene sequence are different due to substitution, insertion, deletion, etc., and the resulting translated polypeptide or protein is a protein variant in which one or more amino acids in the N-terminus, C-terminus, and/or internal portion of the amino acid sequence are conservatively substituted and/or modified so as to be different from the amino acid sequence before the mutation, but the functions or properties are maintained.
  • a “conservative substitution” means substituting one amino acid with another amino acid having similar structural and/or chemical properties, and may have little or no effect on the activity of the protein or polypeptide.
  • the above amino acids are selected from alanine (Ala, A), isoleucine (Ile, I), valine (Val, V), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), cysteine (Cys, C), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K), glycine (Gly, G), and proline (Pro, P).
  • variants include those in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted, or portions are deleted from the N- and/or C-terminus of the mature protein.
  • Such mutants may have their abilities increased (enhanced), unchanged, or decreased (weakened) compared to the protein before the mutation.
  • "increased or strengthened” includes cases where the activity of the protein itself is increased compared to the protein before the mutation, cases where the overall level of enzyme activity in the cell is higher than that of the wild-type strain or the strain expressing the protein before the mutation due to increased expression or increased translation of the gene encoding the protein, and combinations thereof.
  • “decreased or weakened” includes cases where the activity of the protein itself is decreased compared to the protein before the mutation, cases where the overall level of enzyme activity in the cell is lower than that of the wild-type strain or the strain expressing the protein before the mutation due to inhibition of expression or inhibition of translation of the gene encoding the protein, and combinations thereof.
  • the term “variant” may be used interchangeably with “variant,” “modification,” “mutant polypeptide,” “mutated protein,” “mutant,” etc.
  • the GNAT family N-acetyltransferase variant according to the present invention may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 4, excluding the mutation position (amino acid residue at position 174).
  • Such a GNAT family N-acetyltransferase variant may comprise, without limitation, an amino acid sequence that maintains the function or property of the GNAT family N-acetyltransferase variant.
  • Another aspect of the present invention provides a polynucleotide encoding the GNAT family N-acetyltransferase variant.
  • polynucleotide used in the present invention means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and specifically, a polynucleotide fragment encoding the GNAT family N-acetyltransferase variant.
  • the polynucleotide comprises a base sequence encoding an amino acid sequence of SEQ ID NO: 4, for example, may comprise a base sequence of SEQ ID NO: 3.
  • Another aspect of the present invention provides a vector comprising a polynucleotide encoding the GNAT family N-acetyltransferase variant.
  • another aspect of the present invention provides a transformant comprising the GNAT family N-acetyltransferase variant or polynucleotide.
  • vector refers to any type of nucleic acid sequence carrier structure used as a means for delivering a target gene to a host cell to cause expression. Unless otherwise specified, the vector may mean one that allows the carried nucleic acid sequence to be inserted into the host cell genome and expressed and/or to be expressed independently.
  • Such a vector contains essential regulatory elements operably linked to allow the gene insert to be expressed, and "operably linked” means that the target gene and its regulatory sequence are functionally linked to each other in a manner that enables gene expression, and a "regulatory sequence” includes a promoter sequence for performing transcription, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation.
  • the vector used in the present invention is not particularly limited as long as it is replicable in a host cell, and any vector known in the art can be used.
  • the vector include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state.
  • phage vectors or cosmid vectors include pWE15, M13, ⁇ MBL3, ⁇ MBL4, ⁇ IXII, ⁇ ASHII, ⁇ APII, ⁇ t10, ⁇ t11, Charon4A, Charon21A, etc.
  • plasmid vectors include, but are not limited to, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series.
  • the above vector can typically be constructed as a vector for cloning or as a vector for expression.
  • the vector for expression can be a conventional one used in the art to express foreign genes or proteins in plants, animals or microorganisms, and can be constructed through various methods known in the art.
  • the "recombinant vector" used in the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host, and can be replicated independently of the genome of the host cell or can be incorporated into the genome itself.
  • the host cell can include an origin of replication, which is a specific base sequence where replication is initiated, as long as the vector is replicable.
  • the vector used is an expression vector and a prokaryotic cell is used as a host, it generally includes a strong promoter capable of driving transcription (e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence.
  • the replication origin operating in a eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, and the BBV replication origin.
  • a promoter derived from the genome of a mammalian cell e.g., a metallothionein promoter
  • a promoter derived from a mammalian virus e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus promoter, the tk promoter of HSV
  • a promoter derived from the genome of a mammalian cell e.g., a metallothionein promoter
  • a promoter derived from a mammalian virus e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus promoter, the tk promoter of HSV
  • the above recombinant vector may include a selection marker, and the selection marker is used to select transformants (host cells) transformed with the vector. Since only cells expressing the selection marker can survive in a medium treated with the selection marker, selection of transformed cells is possible.
  • Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.
  • a transformant can be created by inserting the above recombinant vector into a host cell, and the transformant can be obtained by introducing the recombinant vector into an appropriate host cell.
  • Any host cell known in the art can be used as the host cell, as long as it is a cell capable of stably and continuously cloning or expressing the above expression vector.
  • the host cells that can be used include, but are not limited to, E. coli such as E. coli DH5 ⁇ , E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392 , E. coli B, E. coli X 1776, E. coli W3110, and E. coli XL1-Blue; Corynebacterium genus, Bacillus genus such as Bacillus subtilis and Bacillus thuringiensis; and various enterobacteria such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas genus.
  • E. coli such as E. coli DH5 ⁇ , E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392 , E. coli B, E. coli X 1776, E. coli W3110, and E. coli XL1-Blue
  • yeast e.g., Saccharomyces cerevisiae
  • insect cells plant cells
  • animal cells such as Sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines
  • Sp2/0 e.g., Saccharomyces cerevisiae
  • insect cells plant cells
  • animal cells such as Sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines
  • transformation used in the present invention refers to a phenomenon in which external DNA is introduced into a host cell to artificially cause a genetic change
  • a “transformant” refers to a host cell into which external DNA is introduced and which stably maintains the expression of a target gene.
  • the above transformation can be performed by selecting a suitable vector introduction technique depending on the host cell, so that the target gene or the recombinant vector containing it can be expressed in the host cell.
  • vector introduction can be performed by electroporation, heat-shock, calcium phosphate ( CaPO4 ) precipitation, calcium chloride ( CaCl2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto.
  • the transformed gene can be included without limitation, whether it is inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell.
  • the above transformant includes a cell transfected, transformed, or infected with a recombinant vector according to the present invention in vivo or in vitro, and may be used as the same term as recombinant host cell, recombinant cell, or recombinant microorganism.
  • the transformant may be a strain of the genus Escherichia or a strain of the genus Corynebacterium .
  • Escherichia genus strains may include, but are not limited to, Escherichia coli , Escherichia albertii , Escherichia blattae , Escherichia fergusonii , Escherichia hermannii , and Escherichia vulneris .
  • the above-mentioned strains of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium crudilactis , Corynebacterium deserti, Corynebacterium callunae , Corynebacterium suranareeae , Corynebacterium lubricantis, Corynebacterium doosanense , Corynebacterium efficiens , Corynebacterium uterequi , Corynebacterium stationis , Corynebacterium pacaense, Corynebacterium singulare , Corynebacterium humireducens , Corynebacterium marinum , Corynebacterium halotolerans , Corynebacterium spheniscorum , Corynebacterium grisburgense , Corynebacterium striatum , Corynebacterium canis , Corynebacterium ammoniagenes
  • the transformant may be a strain of the genus Corynebacterium, specifically Corynebacterium glutamicum.
  • the transformant in the present invention may be, but is not limited to, a strain comprising the above-described GNAT family N-acetyltransferase variant or a polynucleotide encoding the same, or a vector comprising the same, a strain expressing the above-described GNAT family N-acetyltransferase variant or polynucleotide, or a strain having activity against the above-described GNAT family N-acetyltransferase variant.
  • the transformant may be transformed to express the GNAT family N-acetyltransferase variant, or may be transformed by introducing a polynucleotide encoding the GNAT family N-acetyltransferase variant.
  • the transformant of the present invention may include other protein variants or genetic mutations in addition to the GNAT family N-acetyltransferase variants.
  • the transformant may have the ability to produce L-glutamic acid.
  • the above transformant may have a natural ability to produce L-glutamic acid, or may have L-glutamic acid production ability artificially added to it.
  • the transformant may have improved L-glutamic acid production ability due to a change in the activity of a GNAT family N-acetyltransferase.
  • the transformant according to the present invention will have a reduced level of conversion of glutamic acid to N-acetyl-L-glutamic acid due to a mutation in the GNAT family N-acetyltransferase, resulting in weakened glutamic acid consumption within the strain and improved L-glutamic acid production ability.
  • the parent strain refers to a wild type or mutant strain that is the target of mutation, and includes a subject that is directly the target of mutation or a subject transformed with a recombinant vector, etc.
  • the parent strain may be a wild type Escherichia spp. strain or a Corynebacterium spp. strain, or an Escherichia spp. strain or a Corynebacterium spp. strain that is mutated from the wild type.
  • the transformant according to the present invention exhibits increased L-glutamic acid production ability compared to a strain (parent strain) containing the protein before the mutation, due to changes in the activity of the GNAT family N-acetyltransferase by introduction of the GNAT family N-acetyltransferase mutant.
  • the transformant has an increase in L-glutamic acid production by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the parent strain, or 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 20-fold, 30-fold, 40-fold, This may be increased by, but is not limited to, 50x, 60x, 70x, 80x, 90x, or 100x.
  • a composition comprising a transformant according to the present invention can be used as a composition for producing L-glutamic acid.
  • Another aspect of the present invention provides a method for producing L-glutamic acid, comprising the steps of culturing the transformant in a medium; and recovering L-glutamic acid from the transformant or the medium in which the transformant is cultured.
  • the above culture can be carried out according to appropriate media and culture conditions known in the art, and those skilled in the art can easily adjust the media and culture conditions and use them.
  • the media may be a liquid media, but is not limited thereto.
  • the culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.
  • the medium should meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by a person skilled in the art.
  • the culture medium for Bacillus spp. strains can be referred to a known document (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited thereto.
  • the medium may include various carbon sources, nitrogen sources, and trace element components.
  • Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid.
  • sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose
  • oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil
  • fatty acids such as palmitic acid, stearic acid, and linoleic acid
  • alcohols such as glycerol and ethanol
  • organic acids such as acetic acid.
  • Nitrogen sources that can be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture, but are not limited thereto. Sources of phosphorus that can be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. In addition, the culture medium may contain, but are not limited to, metal salts such as magnesium sulfate or iron sulfate required for growth.
  • essential growth substances such as amino acids and vitamins may be included.
  • suitable precursors may be used in the culture medium.
  • the medium or individual components may be added to the culture solution in a suitable manner during the culturing process, either batchwise or continuously, but are not limited thereto.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be appropriately added to the microbial culture solution during culturing to adjust the pH of the culture solution.
  • an antifoaming agent such as fatty acid polyglycol ester may be used during culturing to suppress bubble formation.
  • oxygen or an oxygen-containing gas e.g., air
  • the temperature of the culture solution may be typically 20 to 45°C, for example, 25 to 40°C. The culturing period may continue until a desired amount of useful substances is obtained, for example, 10 to 160 hours.
  • the step of recovering L-glutamic acid from the cultured transformant or the medium in which the transformant is cultured can collect or recover the L-glutamic acid produced from the medium using a suitable method known in the art depending on the culturing method.
  • a suitable method known in the art depending on the culturing method.
  • centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but the present invention is not limited thereto.
  • the step of recovering L-glutamic acid can be performed by removing biomass by low-speed centrifugation of the culture medium and separating the obtained supernatant through ion exchange chromatography.
  • the GNAT family N-acetyltransferase variant according to the present invention has a protein activity changed by substituting one or more amino acids in the amino acid sequence constituting the GNAT family N-acetyltransferase, so that L-glutamic acid can be efficiently produced from a recombinant microorganism expressing the variant.
  • the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template, and each PCR was performed using the primer pairs of primers 1 and 2 and primer pairs of primers 3 and 4. Two PCR products of about 0.5 kb and 0.6 kb in size amplified through PCR were mixed and used as templates, and overlapping PCR was performed with the primer pairs of primers 1 and 4 to link them into a single fragment.
  • the pK19msb vector SEQ ID NO: 5
  • the restriction enzyme smaI NEB
  • the vector constructed in this way was named pK_BBD29_RS13390 (V174M).
  • PCRs used pfu premix (bioneer), and were denatured at 95°C for 5 minutes, then cycled 30 times at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and then reacted at 72°C for 5 minutes.
  • the primer sequences used for vector construction are shown in Table 1 below.
  • the culture was transferred to a 15 ml cap tube, cultured at 30°C for 2 hours, and plated on a selection medium (containing 5 g / l of tryptone, 5 g / l of NaCl, 2.5 g / l of yeast extract, 18.5 g / l of Brain Heart infusion powder, and 15 g / l of agar) containing 20 mg / l of kanamycin.
  • a selection medium containing 5 g / l of tryptone, 5 g / l of NaCl, 2.5 g / l of yeast extract, 18.5 g / l of Brain Heart infusion powder, and 15 g / l of agar
  • the colonies generated after culturing at 30°C for 72 hours were cultured in BHI medium (18.5 g / l of Brain Heart infusion powder) for 15 hours to induce secondary recombination, diluted to 10 -2 ⁇ 10 -3, and plated on a selection medium supplemented with 10% sucrose to isolate the colonies.
  • the isolated colonies were cultured on two types of selection media supplemented with kanamycin and sucrose, respectively, to select strains that were not resistant to kanamycin and could grow on a medium containing sucrose.
  • the selected strain was named BBD29_RS13390 (V174M).
  • Each strain was inoculated at 1% of the volume into a 100 mL flask containing 10 mL of the medium for producing glutamic acid in Table 2 below, and cultured with shaking at 30°C, 200 rpm, for 48 hours. After completion of the culture, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.
  • strain BBD29_RS13390 (V174M) expressing a GNAT family N-acetyltransferase mutant was confirmed to have an approximately 11.4% increase in L-glutamic acid production compared to the parent strain by substituting valine, the 174th amino acid in the amino acid sequence of the GNAT family N-acetyltransferase, with methionine.

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Abstract

La présente invention concerne un nouveau variant de la N-acétyltransférase de la famille GNAT et un procédé de production d'acide L-glutamique l'utilisant. Dans le variant de la N-acétyltransférase de la famille GNAT, un ou plusieurs acides aminés dans la séquence d'acides aminés constituant la N-acétyltransférase de la famille GNAT sont substitués, ce qui permet d'obtenir un changement d'activité protéique. Par conséquent, l'acide L-glutamique peut être efficacement produit à partir d'un micro-organisme recombiné exprimant le variant.
PCT/KR2024/008116 2023-06-16 2024-06-13 Nouveau variant de la n-acétyltransférase de la famille gnat et procédé de production d'acide l-glutamique l'utilisant Pending WO2024258199A1 (fr)

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KR20230077733 2023-06-16
KR10-2023-0077733 2023-06-16
KR10-2024-0075095 2024-06-10
KR1020240075095A KR20240176939A (ko) 2023-06-16 2024-06-10 Gnat 패밀리 n-아세틸기전이효소 신규 변이체 및 이를 이용한 l-글루탐산의 생산 방법

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WO2024258199A1 true WO2024258199A1 (fr) 2024-12-19

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