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WO2024214883A1 - Nouveau variant de pyruvate déshydrogénase et procédé de production d'acide 5'-inosinique l'utilisant - Google Patents

Nouveau variant de pyruvate déshydrogénase et procédé de production d'acide 5'-inosinique l'utilisant Download PDF

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WO2024214883A1
WO2024214883A1 PCT/KR2023/012978 KR2023012978W WO2024214883A1 WO 2024214883 A1 WO2024214883 A1 WO 2024214883A1 KR 2023012978 W KR2023012978 W KR 2023012978W WO 2024214883 A1 WO2024214883 A1 WO 2024214883A1
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pyruvate dehydrogenase
inosinic acid
corynebacterium
transformant
present
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Korean (ko)
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이선희
최태열
김현호
김동현
김현숙
신종환
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Daesang Corp
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
    • C12Y102/04001—Pyruvate dehydrogenase (acetyl-transferring) (1.2.4.1)

Definitions

  • the present invention relates to a novel pyruvate dehydrogenase mutant and a method for producing 5'-inosinic acid using the same.
  • 5'-inosinic acid (or inosine monophosphate, IMP) is an intermediate in the nucleic acid biosynthesis metabolic chain that not only plays an important physiological role in the bodies of plants and animals, but is also used in various fields such as food, medicine, and various medical purposes.
  • IMP inosine monophosphate
  • it is one of the nucleic acid seasonings that is gaining attention as a palatable seasoning due to its great taste synergy effect when used with monosodium glutamate (MSG).
  • Methods for producing 5'-inosinic acid include a method of enzymatically decomposing ribonucleic acid extracted from yeast cells, a method of chemically phosphorylating inosine produced by fermentation, etc. Recently, a method of culturing a microorganism that produces 5'-inosinic acid and recovering the 5'-inosinic acid accumulated in the medium has been mainly used.
  • the present invention aims to provide a novel pyruvate dehydrogenase 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 5'-inosinic acid using the transformant.
  • One aspect of the present invention provides a pyruvate dehydrogenase mutant comprising an amino acid sequence of SEQ ID NO: 2, wherein proline at position 277 in the amino acid sequence of SEQ ID NO: 4 is substituted with leucine.
  • pyruvate dehydrogenase used in the present invention catalyzes the reaction of pyruvate and lipoamide to produce acetylated dehydrolipoamide and carbon dioxide, and may be a polypeptide or protein having pyruvate dehydrogenase activity and composed of an amino acid sequence of sequence number 4.
  • Nucleic acid and protein sequence information of the above pyruvate dehydrogenase can be obtained through known sequence databases (e.g., GenBank, UniProt).
  • the pyruvate dehydrogenase may be encoded by the base sequence of SEQ ID NO: 3.
  • the amino acid sequence of pyruvate dehydrogenase according to the present invention or the base sequence encoding it 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 pyruvate dehydrogenase may be derived from wild type Corynebacterium stationis.
  • variant means a variant in which one or more amino acids are conservatively substituted and/or modified at the N-terminus, C-terminus and/or internally of an amino acid sequence due to a change in the base sequence of a gene encoding a protein, so that the amino acid sequence is different from the amino acid sequence before the mutation, but functions or properties are maintained.
  • conservative substitution means replacing 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 a protein or polypeptide.
  • modification means substitution, insertion, deletion, etc. of an amino acid.
  • 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 protein activity level 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 protein activity level 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 pyruvate dehydrogenase 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 compared to the amino acid sequence of SEQ ID NO: 2.
  • Another aspect of the present invention provides a polynucleotide encoding the pyruvate dehydrogenase 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 more specifically, a polynucleotide fragment encoding the variant.
  • the polynucleotide may include a base sequence encoding the amino acid sequence of sequence number 2.
  • the polynucleotide may include a base sequence of SEQ ID NO: 1, in which the 830th base c is substituted with t in the base sequence of SEQ ID NO: 3 encoding pyruvate dehydrogenase.
  • Another aspect of the present invention provides a vector comprising a polynucleotide encoding the pyruvate dehydrogenase variant.
  • another aspect of the present invention provides a transformant comprising the pyruvate dehydrogenase 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 includes essential regulatory elements that are 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 allows gene expression, and “regulatory elements” include a promoter for performing transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that regulates 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 replicated independently of the genome of the host cell after being transformed into a suitable host cell, or can be incorporated into the genome itself.
  • the “suitable host cell” can include an origin of replication, which is a specific base sequence where replication is initiated as the vector is replicable.
  • the vector used is an expression vector and a prokaryotic cell is used as the host, it generally includes a strong promoter capable of promoting 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 a 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 capable of stably and continuously cloning or expressing the expression vector.
  • the host cells that can be used include, but are not limited to, various strains of Escherichia 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.
  • Escherichia 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.
  • Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis
  • Corynebacterium strains such as Corynebacterium glutamicum and Corynebacterium stationenis
  • various enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas species.
  • 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 refers to a phenomenon in which external DNA is introduced into a host cell to artificially cause a genetic change
  • 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.
  • Genes inserted into the recombinant vector of the present invention can be substituted into a host cell such as a Corynebacterium strain due to homologous recombination crossing over.
  • the transformant may be a microorganism of the genus Corynebacterium .
  • Corynebacterium genus microorganisms 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 ammoniage
  • the transformant in the present invention may be, but is not limited to, a strain comprising the above-described pyruvate dehydrogenase variant or a polynucleotide encoding the same, or a vector comprising the same, a strain expressing the above-described pyruvate dehydrogenase variant or polynucleotide, or a strain having activity against the above-described pyruvate dehydrogenase variant.
  • the transformant in the present invention may include other protein variants or genetic mutations in addition to the pyruvate dehydrogenase variant.
  • the transformant may have the ability to produce 5'-inosinic acid.
  • the above 5'-inosinic acid is a nucleic acid compound that provides flavor to foods, especially savory taste, and is used with the same meaning as inosine monophosphate (IMP).
  • IMP inosine monophosphate
  • the above transformant may have a natural ability to produce 5'-inosinic acid, or may have the ability to produce 5'-inosinic acid artificially added.
  • the transformant may have improved 5'-inosinic acid production ability due to a change in pyruvate dehydrogenase activity.
  • improved productivity means increased productivity of 5'-inosinic acid compared to the parent strain.
  • 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 transformed with a recombinant vector, etc.
  • the parent strain may be a wild type Corynebacterium strain or a Corynebacterium strain that is mutated from the wild type.
  • the transformant according to the present invention exhibits increased 5'-inosinic acid production ability compared to the parent strain due to changes in the activity of pyruvate dehydrogenase by introduction of a pyruvate dehydrogenase mutant. More specifically, the transformant may have an increase in 5'-inosinic 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 may have an increase in 5'-inosinic acid production by at least 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, or 10-fold, but is not limited thereto.
  • Another aspect of the present invention provides a method for producing 5'-inosinic acid, comprising the steps of culturing the transformant in a medium; and recovering 5'-inosinic 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 Escherichia 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 5'-inosinic acid from the cultured transformant or the medium in which the transformant is cultured can collect or recover the 5'-inosinic 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 for example, 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 the 5'-inosinic 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 step of recovering 5'-inosinic acid may include a process of purifying 5'-inosinic acid.
  • the pyruvate dehydrogenase variant according to the present invention has a changed protein activity by substituting one or more amino acids in the amino acid sequence constituting pyruvate dehydrogenase, and a recombinant microorganism containing the same can efficiently produce 5'-inosinic acid.
  • Figure 1 is the structure of a pK19msb plasmid according to one embodiment of the present invention.
  • PCR was performed with the primer pairs of primers 1 and 2 and primer pairs of primers 3 and 4, respectively. Afterwards, the two PCR products were connected into one fragment by performing overlapping PCR with the primer pairs of primers 1 and 4, respectively, using each of the two PCR products as a template.
  • the PCR fragment and the pK19msb plasmid (SEQ ID NO: 5) were treated with the restriction enzyme smaI (NEB) and cloned using T4 ligase.
  • the constructed plasmid was named pK_PH.
  • pfu premix bioneer
  • pfu premix bioneer
  • the reaction was repeated 30 times at 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
  • the primer sequences used for plasmid construction are shown in Table 1 below.
  • Primer name Sequence number Primer sequence (5'-3') Primer 1 6 GGACAGTTCTTCCAAGAGACTCACC Primer 2 7 GTCGGCATAAAGGAAATCCGT Primer 3 8 ACGGATTTCCTTTATGCCGAC Primer 4 9 GTCAGCAGCTCACCCAGCA
  • the seed culture was prepared by primary culturing Corynebacterium stationarynis KCCM13339P in 10 mL of 2YT medium (containing 16 g/L tryptone, 10 g/L yeast extract, and 5 g/L sodium chloride) supplemented with 2% glucose. Isonicotinic acid hydrazine at a concentration of 1 mg/mL and 2.5% glycine were added to 100 mL of 2YT medium excluding glucose. Then, the seed culture was inoculated such that the OD 610 value was 0.3, and the culture was cultured at 30°C and 180 rpm for 5 to 8 hours until the OD 610 value became 0.6 to 0.7.
  • 2YT medium containing 16 g/L tryptone, 10 g/L yeast extract, and 5 g/L sodium chloride
  • Isonicotinic acid hydrazine at a concentration of 1 mg/mL and 2.5% glycine were added to 100 mL of 2YT medium excluding
  • RG medium containing 18.5 g/l Brain Heart infusion and 0.5 M sorbitol
  • RG medium containing 18.5 g/l Brain Heart infusion and 0.5 M sorbitol
  • the medium was transferred to a 15 ml cap tube, cultured at 30°C for 2 hours, and streaked on selective medium (containing 5 g/l tryptone, 5 g/l NaCl, 2.5 g/l yeast extract, 18.5 g/l Brain Heart infusion powder, 15 g/l agar, 91 g/l sorbitol, and 20 ⁇ g/l kanamycin).
  • the colonies generated by culturing at 30°C for 72 hours were cultured in a medium until stationary phase to induce secondary recombination, and the cultures were diluted to 10 -5 to 10 -7 and plated on antibiotic-free plate medium (containing 10% sucrose).
  • antibiotic-free plate medium containing 10% sucrose.
  • the strain that was not resistant to kanamycin and could grow on a medium containing 10% sucrose was selected, and this was named IPH-1.
  • Each strain (parent strain or mutant) was inoculated at 1% volume by volume into a 100 mL flask containing 10 mL of the medium for producing 5'-inosinic acid in Table 2 below, and cultured with shaking at 34°C and 200 rpm for 45 hours. After completion of the culture, the concentration of 5'-inosinic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.
  • the mutant strain into which the pyruvate dehydrogenase mutant was introduced was confirmed to have approximately 19% improved 5'-inosinic acid production compared to the parent strain due to the substitution of proline at position 277 with leucine.

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Abstract

La présente invention concerne un nouveau variant de pyruvate déshydrogénase et un procédé de production d'acide 5'-inosinique l'utilisant, le variant de pyruvate déshydrogénase ayant une activité protéique modifiée due à la substitution d'un ou de plusieurs acides aminés dans la séquence d'acides aminés constituant la pyruvate déshydrogénase, ce qui permet à des micro-organismes recombinants comprenant le variant de produire efficacement de l'acide 5'-inosinique.
PCT/KR2023/012978 2023-04-11 2023-08-31 Nouveau variant de pyruvate déshydrogénase et procédé de production d'acide 5'-inosinique l'utilisant Pending WO2024214883A1 (fr)

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KR1020230047748A KR20240151932A (ko) 2023-04-11 2023-04-11 피루베이트 탈수소효소 신규 변이체 및 이를 이용한 5’-이노신산 생산 방법
KR10-2023-0047748 2023-04-11

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