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WO2024214884A1 - Novel variant of aldehyde dehydrogenase and method for producing 5'-inosinic acid using same - Google Patents

Novel variant of aldehyde dehydrogenase and method for producing 5'-inosinic acid using same Download PDF

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WO2024214884A1
WO2024214884A1 PCT/KR2023/012980 KR2023012980W WO2024214884A1 WO 2024214884 A1 WO2024214884 A1 WO 2024214884A1 KR 2023012980 W KR2023012980 W KR 2023012980W WO 2024214884 A1 WO2024214884 A1 WO 2024214884A1
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aldehyde dehydrogenase
inosinic acid
corynebacterium
transformant
present
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PCT/KR2023/012980
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French (fr)
Korean (ko)
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이선희
최태열
김현호
김동현
김현숙
신종환
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대상 주식회사
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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

Definitions

  • the present invention relates to a novel mutant of aldehyde dehydrogenase 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 aldehyde 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 an aldehyde dehydrogenase variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the 167th glycine in the amino acid sequence of SEQ ID NO: 4 is substituted with aspartic acid.
  • aldehyde dehydrogenase used in the present invention is an enzyme that catalyzes the oxidation of aldehyde, converts aldehyde into carboxylic acid, and may be a polypeptide or protein having aldehyde dehydrogenase activity and composed of an amino acid sequence of sequence number 4.
  • Nucleic acid and protein sequence information of the above aldehyde dehydrogenase can be obtained through known sequence databases (e.g., GenBank, UniProt).
  • the aldehyde dehydrogenase may be encoded by the base sequence of SEQ ID NO: 3.
  • the amino acid sequence of the aldehyde 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 aldehyde 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 aldehyde 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 aldehyde 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 500th base g is substituted with a in the base sequence of SEQ ID NO: 3 encoding aldehyde dehydrogenase.
  • Another aspect of the present invention provides a vector comprising a polynucleotide encoding the aldehyde dehydrogenase variant.
  • another aspect of the present invention provides a transformant comprising the aldehyde 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 aldehyde dehydrogenase variant or a polynucleotide encoding the same, or a vector comprising the same, a strain expressing the above-described aldehyde dehydrogenase variant or polynucleotide, or a strain having activity against the above-described aldehyde dehydrogenase variant.
  • the transformant in the present invention may include other protein variants or genetic mutations in addition to the above aldehyde dehydrogenase variants.
  • 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 aldehyde 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 aldehyde dehydrogenase by introduction of an aldehyde 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 aldehyde dehydrogenase 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 aldehyde dehydrogenase, and a recombinant microorganism containing the variant 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_AD.
  • 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 ATGTCCACTATCGAGTACCAGAACT Primer 2 7 CGATCGTATTGTCGGTAATCGC Primer 3 8 GCGATTACCGACAATACGATCG Primer 4 9 CGATCGTATTGTCGGTAATCGC
  • 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 the medium until stationary phase to induce secondary recombination, and the diluted to 10 -5 to 10 -7 were streaked on antibiotic-free plate medium (containing 10% sucrose) to select a strain that was not resistant to kanamycin and could grow on the medium containing 10% sucrose, and this was named IAD-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 aldehyde dehydrogenase mutant was introduced was confirmed to have an approximately 18% increase in 5'-inosinic acid production compared to the parent strain due to the substitution of the 167th glycine with aspartic acid.

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Abstract

The present invention relates to a novel variant of aldehyde dehydrogenase and a method for producing 5'-inosinic acid using same, the aldehyde dehydrogenase variant having altered protein activity due to substitution of one or more amino acids in the amino acid sequence constituting aldehyde dehydrogenase, thereby allowing recombinant microorganisms comprising the variant to efficiently produce 5'-inosinic acid.

Description

알데히드 탈수소효소 신규 변이체 및 이를 이용한 5'-이노신산 생산 방법Novel variant of aldehyde dehydrogenase and method for producing 5'-inosinic acid using the same
본 발명은 알데히드 탈수소효소 신규 변이체 및 이를 이용한 5'-이노신산 생산 방법에 관한 것이다.The present invention relates to a novel mutant of aldehyde dehydrogenase and a method for producing 5'-inosinic acid using the same.
5'-이노신산(5'-inosinic acid 또는 inosine monophosphate, IMP)은 핵산 생합성 대사계의 중간물질로서 동식물의 체내에서 생리적으로 중요한 역할을 수행할 뿐 아니라 식품, 의약품 및 각종 의료적 이용 등 다방면에 이용되고 있으며. 특히 글루탐산 나트륨(monosodium glutamate, MSG)과 같이 사용하면 맛의 상승효과가 커서 정미성 조미료로 각광을 받고 있는 핵산계 조미료 중 하나이다.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. In particular, 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).
5'-이노신산의 생산 방법으로는 효모 세포로부터 추출한 리보핵산을 효소적으로 분해하는 방법, 발효에 의해 생산된 이노신을 화학적으로 인산화하는 방법 등이 있으며, 최근에는 5'-이노신산을 생산하는 미생물을 배양하여 배지에 축적된 5'-이노신산을 회수하는 방법이 주로 이용되고 있다. 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.
미생물을 이용한 5'-이노신산 생산에서는 5'-이노신산의 생산 효율을 개선하기 위해 핵산이나 L-아미노산 등 유용물질 생산에 많이 이용되는 대장균, 코리네박테리움 등의 미생물을 대상으로 유전자 재조합 기술을 적용하여 우수한 5'-이노신산 생산능을 갖는 다양한 재조합 균주 또는 변이주 및 이를 이용한 5'-이노신산 생산 방법이 개발되고 있다. 특히 5'-이노신산의 생합성 경로에 관여하는 효소, 전사인자, 수송 단백질 등의 유전자를 대상으로 하거나, 또는 이들의 발현을 조절하는 프로모터에 변이를 유도하여 5'-이노신산의 생산량을 증대시키려는 시도가 있었다. 그러나 5'-이노신산 생산에 직간접적으로 연관된 효소, 전사인자, 수송 단백질 등 단백질의 종류가 수십 ~ 수백여 종에 이르기 때문에 이러한 단백질의 활성 변화에 따른 5'-이노신산 생산능 증가 여부에 관해 여전히 많은 연구가 필요한 실정이다.In the production of 5'-inosinic acid using microorganisms, genetic recombination technology is applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as nucleic acids and L-amino acids, in order to improve the production efficiency of 5'-inosinic acid. Various recombinant strains or mutants with excellent 5'-inosinic acid production ability and methods for producing 5'-inosinic acid using them are being developed. In particular, there have been attempts to increase the production of 5'-inosinic acid by targeting genes such as enzymes, transcription factors, and transport proteins involved in the biosynthetic pathway of 5'-inosinic acid, or by inducing mutations in promoters that control their expression. However, since there are tens to hundreds of types of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to 5'-inosinic acid production, much research is still needed on whether 5'-inosinic acid production increases according to changes in the activity of these proteins.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Document]
한국등록특허 제10-116602호Korean Patent No. 10-116602
본 발명은 신규한 알데히드 탈수소효소 변이체를 제공하는 것을 목적으로 한다.The present invention aims to provide a novel aldehyde dehydrogenase variant.
또한, 본 발명은 상기 변이체를 암호화하는 폴리뉴클레오티드를 제공하는 것을 목적으로 한다.In addition, the present invention aims to provide a polynucleotide encoding the mutant.
또한, 본 발명은 상기 변이체 또는 폴리뉴클레오티드를 포함하는 형질전환체를 제공하는 것을 목적으로 한다.In addition, the present invention aims to provide a transformant comprising the mutant or polynucleotide.
또한, 본 발명은 상기 형질전환체를 이용한 5'-이노신산의 생산 방법을 제공하는 것을 목적으로 한다.In addition, the present invention aims to provide a method for producing 5'-inosinic acid using the transformant.
본 발명의 일 양상은 서열번호 4의 아미노산 서열에서 167번째 글리신이 아스파르트산으로 치환된, 서열번호 2의 아미노산 서열로 구성된 알데히드 탈수소효소 변이체를 제공한다.One aspect of the present invention provides an aldehyde dehydrogenase variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the 167th glycine in the amino acid sequence of SEQ ID NO: 4 is substituted with aspartic acid.
본 발명에서 사용된 “알데히드 탈수소효소(aldehyde dehydrogenase)”는 알데히드의 산화를 촉매하는 효소로, 알데히드를 카르복실산으로 전환하며, 서열번호 4의 아미노산 서열로 구성되어 알데히드 탈수소효소 활성을 가지는 폴리펩티드 또는 단백질일 수 있다.The “aldehyde dehydrogenase” used in the present invention is an enzyme that catalyzes the oxidation of aldehyde, converts aldehyde into carboxylic acid, and may be a polypeptide or protein having aldehyde dehydrogenase activity and composed of an amino acid sequence of sequence number 4.
상기 알데히드 탈수소효소의 핵산 및 단백질 서열 정보는 공지된 서열 데이터베이스 (예컨대, GenBank, UniProt)를 통해 얻을 수 있다.Nucleic acid and protein sequence information of the above aldehyde dehydrogenase can be obtained through known sequence databases (e.g., GenBank, UniProt).
본 발명의 일 구체예에 따르면, 상기 알데히드 탈수소효소는 서열번호 3의 염기서열에 의해 암호화된 것일 수 있다.According to one specific example of the present invention, the aldehyde dehydrogenase may be encoded by the base sequence of SEQ ID NO: 3.
본 발명에 따른 알데히드 탈수소효소의 아미노산 서열 또는 이를 암호화하는 염기서열은 각 서열과 비교하여 적어도 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 또는 100%의 상동성 또는 동일성을 가지는 염기서열 또는 아미노산 서열을 포함할 수 있다. 여기서 "상동성" 또는 "동일성"이란 기준이 되는 염기서열 또는 아미노산 서열과 임의의 다른 염기서열 또는 아미노산 서열을 최대한 대응되도록 정렬하여 분석하였을 때 두 서열 간의 일치율(%)을 의미한다.The amino acid sequence of the aldehyde 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. Here, "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.
본 발명의 일 구체예에 따르면, 상기 알데히드 탈수소효소는 야생형 코리네박테리움 스테셔니스(Corynebacterium stationis)에서 유래한 것일 수 있다.According to one specific example of the present invention, the aldehyde dehydrogenase may be derived from wild type Corynebacterium stationis.
본 발명에서 사용된 “변이체”는 단백질을 암호화하는 유전자의 염기서열 상 변이로 인해 아미노산 서열 중 N-말단, C-말단 및/또는 내부에서 하나 이상의 아미노산이 보존적 치환(conservative substitution) 및/또는 변형(modification)되어 변이 전 아미노산 서열과 상이하나, 기능(functions) 또는 특성(properties)이 유지되는 것을 의미한다. 여기서 "보존적 치환"이란 하나의 아미노산을 구조적 및/또는 화학적 성질이 유사한 다른 아미노산으로 치환시키는 것을 의미하며, 단백질 또는 폴리펩티드의 활성에 거의 영향을 미치지 않거나, 또는 전혀 영향을 미치지 않을 수 있다. 또한, "변형"이란 아미노산의 치환(substitution), 삽입(insertion), 결실(deletion) 등을 의미한다. 상기 아미노산으로는 알라닌(Ala, A), 이소류신(Ile, I), 발린(Val, V), 류신(Leu, L), 메티오닌(Met, M), 아스파라긴(Asn, N), 시스테인(Cys, C), 글루타민(Gln, Q), 세린(Ser, S), 트레오닌(Thr, T), 페닐알라닌(Phe, F), 트립토판(Trp, W), 티로신(Tyr, Y), 아스파트산(Asp, D), 글루탐산(Glu, E), 아르기닌(Arg, R), 히스티딘(His, H), 라이신(Lys, K), 글리신(Gly, G) 및 프롤린(Pro, P)으로부터 선택된 것이다.The term “variant” as used in the present invention 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. Here, “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. In addition, “modification” means substitution, insertion, deletion, etc. of an amino acid. 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).
또한, 변이체는 N-말단 리더 서열 또는 막전이 도메인(transmembrane domain)과 같은 하나 이상의 부분이 제거되거나, 또는 성숙 단백질(mature protein)의 N- 및/또는 C-말단으로부터 일부분이 제거된 것을 포함한다. Additionally, 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.
이러한 변이체는 그 능력이 변이 전 단백질에 비하여 증가 (강화)되거나, 변하지 않거나, 또는 감소 (약화)될 수 있다. 여기서 "증가 또는 강화"는 단백질 자체의 활성이 변이 전 단백질에 비하여 증가한 경우, 단백질을 암호화하는 유전자의 발현 증가 또는 번역 증가 등으로 세포 내에서 전체적인 단백질 활성 정도가 야생형 균주 또는 변이 전 단백질을 발현하는 균주에 비하여 높은 경우, 및 이들의 조합을 포함한다. 또한 "감소 또는 약화"는 단백질 자체의 활성이 변이 전 단백질에 비해 감소한 경우, 단백질을 암호화하는 유전자의 발현 저해 또는 번역 저해 등으로 세포 내에서 전체적인 단백질 활성 정도가 야생형 균주 또는 변이 전 단백질을 발현하는 균주에 비하여 낮은 경우, 및 이들의 조합을 포함한다. 본 발명에서는 변이체가 변이형, 변형, 변이형 폴리펩티드, 변이된 단백질, 변이 등과 혼용될 수 있다.Such mutants may have their abilities increased (enhanced), unchanged, or decreased (weakened) compared to the protein before the mutation. Here, "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. In addition, "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. In the present invention, the term "variant" may be used interchangeably with "variant," "modification," "mutant polypeptide," "mutated protein," "mutant," etc.
본 발명에 따른 알데히드 탈수소효소 변이체는 서열번호 2의 아미노산 서열과 비교하여 적어도 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 또는 100%의 상동성 또는 동일성을 가지는 아미노산 서열을 포함할 수 있다. The aldehyde 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.
본 발명의 다른 양상은 상기 알데히드 탈수소효소 변이체를 암호화하는 폴리뉴클레오티드를 제공한다.Another aspect of the present invention provides a polynucleotide encoding the aldehyde dehydrogenase variant.
본 발명에서 사용된 “폴리뉴클레오티드(polynucleotide)”는 뉴클레오티드 단위체(monomer)가 공유결합에 의해 길게 사슬모양으로 이어진 뉴클레오티드의 중합체(polymer)로 일정한 길이 이상의 DNA 또는 RNA 가닥으로서, 보다 구체적으로는 상기 변이체를 암호화하는 폴리뉴클레오티드 단편을 의미한다.The “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.
본 발명의 일 구체예에 따르면, 상기 폴리뉴클레오티드는 서열번호 2의 아미노산 서열을 암호화하는 염기서열을 포함하는 것일 수 있다.According to one specific example of the present invention, the polynucleotide may include a base sequence encoding the amino acid sequence of sequence number 2.
보다 구체적으로, 상기 폴리뉴클레오티드는 알데히드 탈수소효소를 암호화하는 서열번호 3의 염기서열에서 500번째 염기 g가 a로 치환된, 서열번호 1의 염기서열을 포함할 수 있다.More specifically, the polynucleotide may include a base sequence of SEQ ID NO: 1, in which the 500th base g is substituted with a in the base sequence of SEQ ID NO: 3 encoding aldehyde dehydrogenase.
본 발명의 다른 양상은 상기 알데히드 탈수소효소 변이체를 암호화하는 폴리뉴클레오티드를 포함하는 벡터를 제공한다.Another aspect of the present invention provides a vector comprising a polynucleotide encoding the aldehyde dehydrogenase variant.
또한, 본 발명의 다른 양상은 상기 알데히드 탈수소효소 변이체 또는 폴리뉴클레오티드를 포함하는 형질전환체를 제공한다.In addition, another aspect of the present invention provides a transformant comprising the aldehyde dehydrogenase variant or polynucleotide.
본 발명에서 사용된 “벡터(vector)”는 숙주세포에 목적 유전자를 전달하여 발현시키기 위한 수단으로 사용되는 모든 유형의 핵산 서열 운반 구조체를 의미한다. 상기 벡터는 특별한 언급이 없는 한, 담지된 핵산 서열이 숙주세포 유전체 내 삽입되어 발현되도록 하는 것 및/또는 독자적으로 발현되도록 하는 것을 의미할 수 있다. 이러한 벡터는 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절요소를 포함하며, “작동가능하게 연결된(operably linked)”이란 목적 유전자와 이의 조절 서열이 서로 기능적으로 결합되어 유전자 발현을 가능케 하는 방식으로 연결된 것을 의미하고, “조절요소”는 전사를 수행하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 암호화하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함한다.The term “vector” as used in the present invention 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.
본 발명에서 사용되는 벡터는 숙주세포 내에서 복제 가능한 것이라면 특별히 한정되지 않으며, 당업계에 알려진 임의의 벡터를 이용할 수 있다. 상기 벡터의 일례로는 천연 상태이거나 재조합된 상태의 플라스미드, 코스미드, 바이러스 및 박테리오파지를 들 수 있다. 예를 들면, 파지 벡터 또는 코스미드 벡터로는 pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A 등이 있으며, 플라스미드 벡터로는 pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등이 있으나, 이에 한정되는 것은 아니다.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. Examples of the vector include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, phage vectors or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., and plasmid vectors include, but are not limited to, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series.
상기 벡터는 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 발현을 위한 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 유전자 또는 단백질을 발현하는데 사용되는 통상의 것을 사용할 수 있으며, 당업계에 공지된 다양한 방법을 통해 구축될 수 있다.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.
본 발명에서 사용된 “재조합 벡터”는 적합한 숙주세포 내로 형질전환된 후, 숙주세포의 게놈과 무관하게 복제 가능하거나 게놈 그 자체에 봉합될 수 있다. 이때, 상기 "적합한 숙주세포"는 벡터가 복제 가능한 것으로서 복제가 개시되는 특정 염기서열인 복제 원점을 포함할 수 있다. 예를 들어, 사용되는 벡터가 발현 벡터이고 원핵세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터 (예컨대, pLλ 프로모터, CMV 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 진핵세포를 숙주로 하는 경우에는, 벡터에 포함되는 진핵세포에서 작동하는 복제원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 및 BBV 복제원점 등을 포함하나, 이에 한정되는 것은 아니다. 또한, 포유동물 세포의 게놈으로부터 유래된 프로모터 (예컨대, 메탈로 티오닌 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터 (예컨대, 아데노 바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토 메갈로 바이러스 프로모터, HSV의 tk 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 가진다.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. At this time, 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. For example, when 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., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. In the case where a eukaryotic cell is used as a host, 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. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or 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) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.
상기 재조합 벡터는 선택 마커(selection marker)를 포함할 수 있는데, 상기 선택 마커는 벡터로 형질전환된 형질전환체 (숙주세포)를 선별하기 위한 것으로 상기 선택 마커가 처리된 배지에서 선택 마커를 발현하는 세포만 생존할 수 있기 때문에, 형질전환된 세포의 선별이 가능하다. 상기 선택 마커는 대표적인 예로 카나마이신, 스트렙토마이신, 클로람페니콜 등이 있으나, 이에 한정되는 것은 아니다.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.
재조합 벡터를 숙주세포에 삽입함으로써 형질전환체를 만들 수 있으며, 상기 형질전환체는 재조합 벡터를 적절한 숙주세포에 도입시킴으로써 얻어진 것일 수 있다. 숙주세포는 상기 발현벡터를 안정되면서 연속적으로 클로닝 또는 발현시킬 수 있는 세포로서 당업계에 공지된 어떠한 숙주세포도 이용할 수 있다.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.
재조합 미생물을 제작하기 위하여 원핵세포에 형질전환시키는 경우에는 숙주세포로서 E. coli DH5α, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli XL1-Blue와 같은 대장균 속 균주, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 코리네박테리움 글루타미쿰, 코리네박테리움 스테셔니스와 같은 코리네박테리움 속 균주, 살모넬라 티피무리움, 세라티아 마르세슨스 및 슈도모나스 종과 같은 다양한 장내균과 균주 등이 이용되는 것일 수 있으나, 이에 한정되는 것은 아니다.When transforming prokaryotic cells to produce recombinant microorganisms, the host cells that can be used include, but are not limited to, various strains of Escherichia coli such as E. coli DH5α, 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, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Corynebacterium strains such as Corynebacterium glutamicum and Corynebacterium stationenis, and various enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas species.
재조합 미생물을 제작하기 위하여 진핵세포에 형질전환을 하는 경우에는 숙주세포로서 효모 (예컨대, 사카로마이세스 세레비지에), 곤충 세포, 식물 세포 및 동물 세포, 예를 들어, Sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK 세포주 등이 이용될 수 있으나, 이에 한정되는 것은 아니다.When transforming eukaryotic cells to produce recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, can be used as host cells, but are not limited thereto.
본 발명에서 사용된 “형질전환(transformation)”은 외부 DNA를 숙주세포 내로 도입하여 인위적으로 유전적인 변화를 일으키는 현상을 의미하며, “형질전환체(transformat)”는 외부 DNA가 도입되어 목적 유전자의 발현을 안정적으로 유지하는 숙주세포를 의미한다.“Transformation” as 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, and “transformant” refers to a host cell into which external DNA is introduced and which stably maintains the expression of a target gene.
상기 형질전환은 숙주세포에 따라 적합한 벡터 도입 기술이 선택되어 목적 유전자 또는 이를 포함하는 재조합 벡터를 숙주세포 내에서 발현시킬 수 있다. 예를 들면, 벡터 도입은 전기천공법(electroporation), 열 충격(heat-shock), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 초산 리튬-DMSO법, 또는 이들의 조합에 의해 수행될 수 있으나, 이에 한정되는 것은 아니다. 형질전환된 유전자는 숙주세포 내에서 발현될 수 있으면 숙주세포의 염색체 내 삽입 또는 염색체 외에 위치하고 있는 것이든 제한하지 않고 포함될 수 있다.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. For example, 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.
상기 형질전환체는 생체내 또는 시험관내에서 본 발명에 따른 재조합 벡터로 형질감염, 형질전환, 또는 감염된 세포를 포함하며, 재조합 숙주세포, 재조합 세포 또는 재조합 미생물과 동일한 용어로 사용될 수 있다.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.
본 발명의 재조합 벡터 내에 삽입된 유전자들은 상동 재조합 교차로 인하여 코리네박테리움 속 균주와 같은 숙주세포 내로 치환될 수 있다.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.
본 발명의 일 구체예에 따르면, 상기 형질전환체는 코리네박테리움(Corynebacterium) 속 미생물인 것일 수 있다.According to one specific example of the present invention, the transformant may be a microorganism of the genus Corynebacterium .
상기 코리네박테리움 속 미생물로는 코리네박테리움 글루타미쿰(Corynebacterium glutamicum), 코리네박테리움 크루디락티스(Corynebacterium crudilactis), 코리네박테리움 데저티(Corynebacterium deserti), 코리네박테리움 칼루나에(Corynebacterium callunae), 코리네박테리움 수라나래에(Corynebacterium suranareeae), 코리네박테리움 루브리칸티스(Corynebacterium lubricantis), 코리네박테리움 두사넨세(Corynebacterium doosanense), 코리네박테리움 이피시엔스(Corynebacterium efficiens), 코리네박테리움 우테레키(Corynebacterium uterequi), 코리네박테리움 스테셔니스(Corynebacterium stationis), 코리네박테리움 파캔세(Corynebacterium pacaense), 코리네박테리움 싱굴라레(Corynebacterium singulare), 코리네박테리움 휴미레듀센스(Corynebacterium humireducens), 코리네박테리움 마리눔(Corynebacterium marinum), 코리네박테리움 할로톨레란스(Corynebacterium halotolerans), 코리네박테리움 스페니스코룸(Corynebacterium spheniscorum), 코리네박테리움 프레이부르겐세(Corynebacterium freiburgense), 코리네박테리움 스트리아툼(Corynebacterium striatum), 코리네박테리움 카니스(Corynebacterium canis), 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes), 코리네박테리움 레날레(Corynebacterium renale), 코리네박테리움 폴루티솔리(Corynebacterium pollutisoli), 코리네박테리움 이미탄스(Corynebacterium imitans), 코리네박테리움 카스피움(Corynebacterium caspium), 코리네박테리움 테스투디노리스(Corynebacterium testudinoris), 코리네박테리움 슈도펠라지(Corynebacaterium pseudopelargi) 또는 코리네박테리움 플라베스센스(Corynebacterium flavescens)일 수 있으며, 이에 한정되는 것은 아니다.The above-mentioned 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 freiburgense , Corynebacterium striatum , Corynebacterium canis , Corynebacterium ammoniagenes , Corynebacterium renale The spore-forming bacterium may be, but is not limited to, Corynebacterium renale, Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium caspium , Corynebacterium testudinoris , Corynebacterium pseudopelargi, or Corynebacterium flavescens .
본 발명에서의 형질전환체는 전술한 알데히드 탈수소효소 변이체 또는 이를 암호화하는 폴리뉴클레오티드를 포함하거나, 또는 이를 포함하는 벡터를 포함하는 균주, 상기 알데히드 탈수소효소 변이체 또는 폴리뉴클레오티드를 발현하는 균주, 또는 상기 알데히드 탈수소효소 변이체에 대한 활성을 가지는 균주일 수 있으나, 이에 한정되는 것은 아니다.The transformant in the present invention may be, but is not limited to, a strain comprising the above-described aldehyde dehydrogenase variant or a polynucleotide encoding the same, or a vector comprising the same, a strain expressing the above-described aldehyde dehydrogenase variant or polynucleotide, or a strain having activity against the above-described aldehyde dehydrogenase variant.
본 발명에서의 형질전환체는 상기 알데히드 탈수소효소 변이체 외에도 다른 단백질 변이체 또는 유전자 변이를 포함할 수 있다.The transformant in the present invention may include other protein variants or genetic mutations in addition to the above aldehyde dehydrogenase variants.
본 발명의 일 구체예에 따르면, 상기 형질전환체는 5'-이노신산 생산능을 가지는 것일 수 있다.According to one specific example of the present invention, the transformant may have the ability to produce 5'-inosinic acid.
상기 5'-이노신산(5'-inosinic acid)은 식품의 향미, 특히 감칠맛을 내는 핵산계 화합물로, 이노신 모노포스페이트(inosine monophosphate, IMP)와 동일한 의미로 사용된다.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).
상기 형질전환체는 자연적으로 5'-이노신산 생산능을 가지고 있거나, 또는 인위적으로 5'-이노신산 생산능이 부여된 것일 수 있다.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.
본 발명의 일 구체예에 따르면, 상기 형질전환체는 알데히드 탈수소효소 활성이 변화되어 5'-이노신산 생산능이 향상된 것일 수 있다.According to one specific example of the present invention, the transformant may have improved 5'-inosinic acid production ability due to a change in aldehyde dehydrogenase activity.
본 발명에서 사용된 “생산능이 향상된”은 모균주에 비해 5'-이노신산의 생산성이 증가된 것을 의미한다. 상기 모균주는 변이의 대상이 되는 야생형 또는 변이주를 의미하며, 직접 변이의 대상이 되거나 재조합된 벡터 등으로 형질전환되는 대상을 포함한다. 본 발명에 있어서, 모균주는 야생형 코리네박테리움 속 균주이거나 야생형으로부터 변이된 코리네박테리움 속 균주일 수 있다.As used herein, “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. In the present invention, the parent strain may be a wild type Corynebacterium strain or a Corynebacterium strain that is mutated from the wild type.
본 발명에 따른 형질전환체는 알데히드 탈수소효소 변이체가 도입됨으로써 알데히드 탈수소효소의 활성이 변화하여 모균주에 비해 증가된 5'-이노신산 생산능을 나타낸다. 보다 구체적으로, 상기 형질전환체는 모균주에 비해 5'-이노신산 생산량이 적어도 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 또는 100% 증가하거나, 또는 1.1배, 1.5배, 2배, 2.5배, 3배, 3.5배, 4배, 4.5배, 5배, 5.5배, 6배, 6.5배, 7배, 7.5배, 8배, 8.5배, 9배, 9.5배 또는 10배 증가된 것일 수 있으나, 이에 한정되는 것은 아니다. 일례로, 상기 알데히드 탈수소효소 변이체를 포함한 형질전환체는 모균주에 비해 5'-이노신산 생산량이 5% 이상, 구체적으로는 5 내지 50% (바람직하게는 10 내지 40%) 증가된 것일 수 있다.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 aldehyde dehydrogenase by introduction of an aldehyde 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. For example, a transformant including the above aldehyde dehydrogenase mutant may have an increased 5'-inosinic acid production of 5% or more, specifically 5 to 50% (preferably 10 to 40%) compared to the parent strain.
본 발명의 다른 양상은 상기 형질전환체를 배지에서 배양하는 단계; 및 상기 형질전환체 또는 형질전환체가 배양된 배지로부터 5'-이노신산을 회수하는 단계를 포함하는 5'-이노신산의 생산 방법을 제공한다.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.
상기 배양은 당업계에 알려진 적절한 배지와 배양 조건에 따라 이루어질 수 있으며, 통상의 기술자라면 배지 및 배양 조건을 용이하게 조정하여 사용할 수 있다. 구체적으로, 상기 배지는 액체 배지일 수 있으나, 이에 한정되는 것은 아니다. 배양 방법은 예를 들면, 회분식 배양(batch culture), 연속식 배양(continuous culture), 유가식 배양(fed-batch culture) 또는 이들의 조합 배양을 포함할 수 있으나, 이에 한정되는 것은 아니다.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. Specifically, 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.
본 발명의 일 구체예에 따르면, 상기 배지는 적절한 방식으로 특정 균주의 요건을 충족해야 하며, 통상의 기술자에 의해 적절하게 변형될 수 있다. 에스케리치아 속 균주에 대한 배양 배지는 공지된 문헌 (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981)을 참조할 수 있으나, 이에 한정되는 것은 아니다.According to one specific embodiment of the present invention, 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.
본 발명의 일 구체예에 따르면, 배지에 다양한 탄소원, 질소원 및 미량원소 성분을 포함할 수 있다. 사용될 수 있는 탄소원으로는 글루코스, 수크로스, 락토스, 프락토스, 말토스, 전분, 셀룰로스와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함된다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있으나, 이에 한정되는 것은 아니다. 사용될 수 있는 질소원으로는 펩톤, 효모 추출물, 육즙, 맥아 추출물, 옥수수 침지액, 대두밀 및 요소 또는 무기 화합물, 예를 들면 황산 암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄이 포함될 수 있다. 질소원 또한 개별적으로 또는 혼합물로서 사용할 수 있으나 이에 한정되는 것은 아니다. 사용될 수 있는 인의 공급원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함될 수 있으며, 이에 한정되는 것은 아니다. 또한, 배양 배지는 성장에 필요한 황산마그네슘 또는 황산철과 같은 금속염을 함유할 수 있으며, 이에 한정되는 것은 아니다. 그 외에, 아미노산 및 비타민과 같은 필수 성장 물질이 포함될 수 있다. 또한 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기 배지 또는 개별 성분은 배양과정에서 배양액에 적절한 방식에 의해 회분식으로 또는 연속식으로 첨가될 수 있으나, 이에 한정되는 것은 아니다.According to one specific embodiment of the present invention, 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. These materials may be used individually or as a mixture, but are not limited thereto. 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. In addition, essential growth substances such as amino acids and vitamins may be included. Also, 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.
본 발명의 일 구체예에 따르면, 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산 및 황산과 같은 화합물을 미생물 배양액에 적절한 방식으로 첨가하여 배양액의 pH를 조정할 수 있다. 또한, 배양 중에 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 추가적으로, 배양액의 호기 상태를 유지하기 위하여, 배양액 내로 산소 또는 산소-함유 기체 (예, 공기)를 주입할 수 있다. 배양액의 온도는 통상 20 내지 45℃, 예를 들면 25 내지 40℃일 수 있다. 배양기간은 유용물질이 원하는 생산량으로 수득될 때까지 계속될 수 있으며, 예를 들면 10 내지 160시간일 수 있다.According to one specific example of the present invention, 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. In addition, an antifoaming agent such as fatty acid polyglycol ester may be used during culturing to suppress bubble formation. Additionally, in order to maintain the aerobic state of the culture solution, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture solution. 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.
본 발명의 일 구체예에 따르면, 상기 배양된 형질전환체 또는 형질전환체가 배양된 배지에서 5'-이노신산을 회수하는 단계는 배양 방법에 따라 당해 분야에 공지된 적합한 방법을 이용하여 배지로부터 생산된 5'-이노신산을 수집 또는 회수할 수 있다. 예를 들면 원심분리, 여과, 추출, 분무, 건조, 증발, 침전, 결정화, 전기영동, 분별용해 (예를 들면, 암모늄 설페이트 침전), 크로마토그래피 (예를 들면, 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용할 수 있으나, 이에 한정되는 것은 않는다.According to one specific example of the present invention, 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. 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.
본 발명의 일 구체예에 따르면, 상기 5'-이노신산을 회수하는 단계는 배양 배지를 저속 원심분리하여 바이오매스를 제거하고 얻어진 상등액을 이온교환 크로마토그래피를 통하여 분리할 수 있다.According to one specific example of the present invention, 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.
본 발명의 일 구체예에 따르면, 상기 5'-이노신산을 회수하는 단계는 5'-이노신산을 정제하는 공정을 포함할 수 있다.According to one specific example of the present invention, the step of recovering 5'-inosinic acid may include a process of purifying 5'-inosinic acid.
본 발명에 따른 알데히드 탈수소효소 변이체는 알데히드 탈수소효소를 구성하는 아미노산 서열 중 하나 이상의 아미노산이 치환됨으로써 단백질 활성이 변화되어, 이를 포함하는 재조합 미생물은 5'-이노신산을 효율적으로 생산할 수 있다.The aldehyde dehydrogenase 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 aldehyde dehydrogenase, and a recombinant microorganism containing the variant can efficiently produce 5'-inosinic acid.
도 1은 본 발명의 일 실시예에 따른 pK19msb 플라스미드의 구조이다.Figure 1 is the structure of a pK19msb plasmid according to one embodiment of the present invention.
이하, 본 발명을 보다 상세하게 설명한다. 그러나, 이러한 설명은 본 발명의 이해를 돕기 위하여 예시적으로 제시된 것일 뿐, 본 발명의 범위가 이러한 예시적인 설명에 의하여 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail. However, this description is provided only as an example to help understand the present invention, and the scope of the present invention is not limited by this exemplary description.
실시예 1. 알데히드 탈수소효소 변이체를 발현하는 균주 제작Example 1. Production of strain expressing aldehyde dehydrogenase mutant
알데히드 탈수소효소의 아미노산 서열 (서열번호 4)에서 167번째 글리신(G)이 아스파르트산(D)으로 치환된 변이체 (서열번호 2)가 5'-이노신산의 생산에 미치는 영향을 확인하기 위해, 상기 알데히드 탈수소효소 변이체를 발현하는 벡터 및 상기 벡터가 도입된 균주를 제작하였다.In order to confirm the effect of a mutant (SEQ ID NO: 2) in which glycine (G) at position 167 in the amino acid sequence of aldehyde dehydrogenase (SEQ ID NO: 4) is substituted with aspartic acid (D) on the production of 5'-inosinic acid, a vector expressing the aldehyde dehydrogenase mutant and a strain into which the vector was introduced were constructed.
1-1. 알데히드 탈수소효소 변이체 발현을 위한 벡터 제작1-1. Production of vector for expression of aldehyde dehydrogenase mutants
야생형 코리네박테리움 스테셔니스 ATCC6872의 genomic DNA를 주형으로 프라이머 1 및 2의 프라이머 쌍과 프라이머 3 및 4의 프라이머 쌍을 이용하여 각각 PCR을 수행하였다. 이후, 두 개의 PCR 산물을 각각 주형으로 사용하여 프라이머 1 및 4의 프라이머 쌍으로 오버랩핑(overlapping) PCR을 수행하여 하나의 단편으로 연결시켰다. PCR 단편과 pK19msb 플라스미드 (서열번호 5)에 제한효소 smaI (NEB)을 처리하고 T4 리가아제(ligase)를 사용하여 클로닝하였다. 구축된 플라스미드를 pK_AD라 명명하였다.Using the genomic DNA of wild type Corynebacterium stationaryus ATCC6872 as a template, 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_AD.
PCR 증폭에서는 pfu premix (bioneer)를 사용하였고, 95℃에서 5분 변성한 후 95℃에서 30초, 58℃에서 30초, 72℃에서 1분 30초를 30회 반복하고, 72℃에서 5분간 반응시켰다.For PCR amplification, pfu premix (bioneer) was used, and after denaturation at 95°C for 5 minutes, 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.
플라스미드 제작에 사용된 프라이머 서열은 하기 표 1과 같다.The primer sequences used for plasmid construction are shown in Table 1 below.
프라이머 명칭Primer name 서열번호Sequence number 프라이머 서열 (5'-3')Primer sequence (5'-3')
프라이머 1Primer 1 66 ATGTCCACTATCGAGTACCAGAACTATGTCCACTATCGAGTACCAGAACT
프라이머 2Primer 2 77 CGATCGTATTGTCGGTAATCGCCGATCGTATTGTCGGTAATCGC
프라이머 3Primer 3 88 GCGATTACCGACAATACGATCGGCGATTACCGACAATACGATCG
프라이머 4Primer 4 99 CGATCGTATTGTCGGTAATCGCCGATCGTATTGTCGGTAATCGC
1-2. 알데히드 탈수소효소 변이체가 도입된 변이주 제작1-2. Production of mutant strains with aldehyde dehydrogenase mutants
코리네박테리움 스테셔니스 KCCM13339P의 형질전환을 위한 방법으로 van der Rest 등의 방법을 기본으로 수식한 일렉트로컴피턴트 셀(electrocompetent cell) 제조법을 사용하였다. For the transformation of Corynebacterium stationaryus KCCM13339P, an electrocompetent cell production method based on the method of van der Rest et al. was used.
먼저, 2% 포도당이 첨가된 2YT 배지 (트립톤 16 g/ℓ, 효모추출물 10 g/ℓ 및 염화나트륨 5 g/ℓ 함유) 10 ㎖에서 코리네박테리움 스테셔니스 KCCM13339P를 1차 배양하여 종배양액을 준비하였다, 포도당을 제외한 2YT 배지 100 ㎖에 1 mg/㎖ 농도의 이소니코틴산 히드라진(isonicotinic acid hydrazine) 및 2.5% 글라이신(glycine)을 첨가하였다. 그 다음, OD610 값이 0.3이 되도록 종배양액을 접종한 후, 30℃, 180 rpm으로 5 ~ 8시간 배양하여 OD610 값이 0.6 ~ 0.7가 되도록 하였다. 배양액을 얼음에서 30분간 방치한 후, 4℃, 3500 rpm으로 10분간 원심분리하였다. 그 뒤 상등액을 버리고 침전된 코리네박테리움 스테셔니스 KCCM13339P를 10% 글리세롤 용액으로 4회 세척하고, 최종적으로 10% 글리세롤 용액 0.5 ㎖에 재현탁하여 컴피턴트 셀(competent cell)을 준비하였다. 전기천공(Electroporation)은 바이오-라드(Bio-Rad)사의 전기천공기(electroporator)를 사용하였다. 전기천공 큐벳 (0.2 mm)에 준비된 컴피턴트 셀과 제조된 pK_AD 벡터를 첨가한 후, 2.5 kV, 200 Ω 및 12.5 ㎌의 조건으로 전기충격을 가하였다. 전기충격이 끝난 즉시 RG 배지(Brain Heart infusion 18.5 g/ℓ 및 소비톨 0.5 M 함유) 1 ㎖을 첨가하고 46℃에서 6분간 열처리하였다. 그 후 실온에서 식힌 뒤 15 ㎖ 캡 튜브로 옮겨 30℃에서 2시간 배양하고 선별 배지 (트립톤 5 g/ℓ, NaCl 5 g/ℓ, 효모추출물 2.5 g/ℓ, Brain Heart infusion powder 18.5 g/ℓ, 아가(agar) 15 g/ℓ, 소비톨 91 g/ℓ 및 카나마이신 (kanamycine) 20 ㎍/ℓ 함유)에 도말하였다. 30℃에서 72시간 배양해 생성된 콜로니는 배지에서 정지기까지 배양하여 2차 재조합을 유도했으며, 10-5 ~ 10-7까지 희석하여 항생제가 없는 평판배지 (10% sucrose 함유)에 도말하여 카나마이신 내성도가 없고 10% 수크로오스가 포함된 배지에서 성장성이 있는 균주를 선별하였으며, 이를 IAD-1이라 명명하였다.First, 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. After leaving the culture on ice for 30 minutes, it was centrifuged at 4℃, 3500 rpm for 10 minutes. The supernatant was discarded, and the precipitated Corynebacterium stationarynis KCCM13339P was washed four times with 10% glycerol solution, and finally resuspended in 0.5 ㎖ of 10% glycerol solution to prepare competent cells. Electroporation was performed using an electroporator from Bio-Rad. The prepared competent cells and the manufactured pK_AD vector were added to an electroporation cuvette (0.2 mm), and then electroporation was performed under the conditions of 2.5 kV, 200 Ω, and 12.5 ㎌. Immediately after the electric shock, 1 ㎖ of RG medium (containing 18.5 g/ℓ Brain Heart infusion and 0.5 M sorbitol) was added and heat-treated at 46℃ for 6 minutes. After cooling to room temperature, the medium was transferred to a 15 ㎖ cap tube, cultured at 30℃ 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 ㎍/ℓ kanamycin). The colonies generated by culturing at 30℃ for 72 hours were cultured in the medium until stationary phase to induce secondary recombination, and the diluted to 10 -5 to 10 -7 were streaked on antibiotic-free plate medium (containing 10% sucrose) to select a strain that was not resistant to kanamycin and could grow on the medium containing 10% sucrose, and this was named IAD-1.
실험예 1. 알데히드 탈수소효소 변이체를 발현하는 균주의 5'-이노신산 생산능 평가Experimental Example 1. Evaluation of 5'-inosinic acid production ability of strain expressing aldehyde dehydrogenase mutant
모균주 KCCM13339P과 알데히드 탈수소효소 변이체가 도입된 변이주 IAD-1의 5'-이노신산 생산능을 비교하였다.The 5'-inosinic acid production ability of the parent strain KCCM13339P and the mutant strain IAD-1 with the aldehyde dehydrogenase mutant was compared.
하기 표 2의 5'-이노신산 생산용 배지 10 mL가 담긴 100 mL 플라스크에 각 균주 (모균주 또는 변이주)를 부피 기준으로 1%씩 접종하여 34℃, 200 rpm의 조건으로 45시간 진탕 배양하였다. 배양 종료 후 HPLC (Agilent)를 사용하여 배지 내 5'-이노신산의 농도를 측정하였고, 그 결과를 하기 표 3에 나타내었다.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.
성분ingredient 함량Content
GlucoseGlucose 70 g/L70 g/L
(NH4)2SO4 (NH 4 ) 2 SO 4 2 g/L2 g/L
MgSO4 MgSO 4 1 g/L1 g/L
UreaUrea 2 g/L2 g/L
Yeast extractYeast extract 20 g/L20 g/L
KH2PO4 KH 2 PO 4 2 g/L2 g/L
FeSO4 FeSO 4 10 mg/L10 mg/L
MnSO4 MnSO 4 10 mg/L10 mg/L
Thiamine_HClThiamine_HCl 5 mg/L5 mg/L
biotinbiotin 20 ug/L20 ug/L
CysteinCystein 20 mg/L20 mg/L
Bata-alanineBata-alanine 20 mg/L20 mg/L
AdenineAdenine 30 mg/L30 mg/L
균주Strain 5'-이노신산 생산량 (g/L)5'-Inosinic acid production (g/L)
KCCM13339PKCCM13339P 19.519.5
IAD-1IAD-1 23.0123.01
상기 표 3에 나타낸 바와 같이, 알데히드 탈수소효소 변이체가 도입된 변이주는 167번째 글리신이 아스파르트산으로 치환됨으로써 모균주에 비해 5'-이노신산 생산량이 약 18% 향상된 것으로 확인되었다. 이러한 결과는 알데히드 탈수소효소의 점 돌연변이 도입이 5'-이노신산 생산성에 대한 유효한 효과를 제공함을 시사한다.As shown in Table 3 above, the mutant strain into which the aldehyde dehydrogenase mutant was introduced was confirmed to have an approximately 18% increase in 5'-inosinic acid production compared to the parent strain due to the substitution of the 167th glycine with aspartic acid. These results suggest that the introduction of a point mutation in aldehyde dehydrogenase has a significant effect on 5'-inosinic acid productivity.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.
[수탁번호][Acceptance number]
기탁기관명 : 한국미생물보존센터(KCCM)Name of depositor: Korea Center for Microbiological Conservation (KCCM)
수탁번호 : KCCM13339PAccession number: KCCM13339P
수탁일자 : 20230329Date of acceptance: 20230329
Figure PCTKR2023012980-appb-img-000001
Figure PCTKR2023012980-appb-img-000001

Claims (6)

  1. 서열번호 4의 아미노산 서열에서 167번째 글리신이 아스파르트산으로 치환된, 서열번호 2의 아미노산 서열로 구성된 알데히드 탈수소효소 변이체.An aldehyde dehydrogenase mutant having an amino acid sequence of sequence number 2, in which the 167th glycine in the amino acid sequence of sequence number 4 is substituted with aspartic acid.
  2. 청구항 1의 변이체를 암호화하는 폴리뉴클레오티드.A polynucleotide encoding a variant of claim 1.
  3. 청구항 1의 변이체 또는 청구항 2의 폴리뉴클레오티드를 포함하는 형질전환체.A transformant comprising a variant of claim 1 or a polynucleotide of claim 2.
  4. 청구항 3에 있어서,In claim 3,
    상기 형질전환체는 코리네박테리움(Corynebacterium) 속 미생물인 것인 형질전환체.The above transformant is a transformant which is a microorganism of the genus Corynebacterium .
  5. 청구항 3에 있어서,In claim 3,
    상기 형질전환체는 5'-이노신산 생산능을 가지는 것인 형질전환체.The above transformant is a transformant having the ability to produce 5'-inosinic acid.
  6. 청구항 3의 형질전환체를 배지에서 배양하는 단계; 및A step of culturing the transformant of claim 3 in a medium; and
    상기 형질전환체 또는 형질전환체가 배양된 배지로부터 5'-이노신산을 회수하는 단계를 포함하는 5'-이노신산의 생산 방법.A method for producing 5'-inosinic acid, comprising a step of recovering 5'-inosinic acid from the transformant or the medium in which the transformant is cultured.
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US20090298127A1 (en) * 2006-12-22 2009-12-03 Ekaterina Aleksandrovna Kutukova Method for producing purine nucleosides and nucleotides by fermentation using a bacterium belonging to the genus escherichia or bacillus
KR101916622B1 (en) * 2018-01-04 2018-11-07 씨제이제일제당 (주) Novel polypeptide and method of producing IMP using the same
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US20090298127A1 (en) * 2006-12-22 2009-12-03 Ekaterina Aleksandrovna Kutukova Method for producing purine nucleosides and nucleotides by fermentation using a bacterium belonging to the genus escherichia or bacillus
KR101916611B1 (en) * 2017-12-15 2018-11-07 씨제이제일제당 (주) Novel polypeptide and method of producing IMP using the same
KR101916622B1 (en) * 2018-01-04 2018-11-07 씨제이제일제당 (주) Novel polypeptide and method of producing IMP using the same

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